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[The importance of primary operation from the point of view of mortality in patients with femoral neck fractures. I. Comparative study of mortality after primary and delayed operations. The part played by the patients age (author's transl)].

Authors with regard to primary or delayed surgical treatment analyse the mortality of 2,612 patients with proximal femur fractures treated in the National Institute of Traumatology during the years 1971--75. Within this material 2,055 case history of surgically treated femoral neck and pertrochanteric fractures are evaluated. An important difference in favour of primary intervention was found between the mortality of primary and delayed operations both in femoral neck and pertrochanteric fractures. In pertrochanteric fractures the mortality increased gradually starting from the day of injury. Regarding age related mortality, there was a significant difference in each age group in favour of primary operations. Thus the increased mortality encountered after delayed operations can't be explained by a selection with regard to age.

Single-strand breaks in DNA during repair of UV-induced damage in normal human and xeroderma pigmentosum cells as determined by alkaline DNA unwinding and hydroxylapatite chromatography: effects of hydroxyurea, 5-fluorodeoxyuridine and 1-beta-D-arabinofuranosylcytosine on the kinetics of repair.

A simple and sensitive technique for detection of strand breaks in DNA has been further developed. The method has been used to follow UV-induced excision-repair in human fibroblasts. It has been possible to study the kinetics of enzymic reactions in intact cells, in which strand breaks in DNA are produced and sealed again. Hydroxyurea, 5-fluorodeoxyuridine and 1-beta-D-arabinofuranosylcytosine, potent inhibitors of DNA synthesis, drastically increased the number of breaks observed during the repair process. This was probably due to a decreased polymerase activity, which will cause the strand breaks formed by endonuclease to remain open longer. The initial rate of strand-break formation did not seem to be influenced by hydroxyurea or araC, and was about 4000 breaks per minute in a diploid genome, at a dose of 20 J/m2. After 5--30 min, depending on the dose of UV, the number of breaks reached a maximum and started to decrease again. Hydroxyurea decreased the rate of polymerization in the sites under repair. However, there was no concomitant reduction of repair-induced incorporation of [3H]thymidine and no reduction of the excision of pyrimidine dimers. It therefore seems that the action of the polymerase was not a rate-limiting event, but rather an earlier step. It is likely that the endonucleolytic activity determined the rate of repair. As a consequence, the endonuclease and polymerase cannot be bound in a permanent complex. Under certain assumptions, the time for repair of a site, i.e. the time from incision to final ligase sealing, can be estimated as between 3 and 10 min. Essentially no breaks were produced in Xeroderma pigmentosum cells belonging to complementation group A, and there was no enhancement by hydroxyurea. Cells from the variant type of Xeroderma pigmentosum behaved like normal cells in this respect.

Potentiation of cataleptogenic effects of neuroleptics by prostaglandins.

Prostaglandins (PGs) E1, E2, F2alpha injected intracerebroventricularly (icv) in rats, potentiated chlorpromazine (CPZ) and pimozide (PI) catalepsy similarly. Cataleptogenic effect of haloperidol (HL) was potentiated specifically be PGE2. These phenomena were diminished by apomorphine (AP). Phenoxybenzamine (PB) diminished the potentiating effect of PGE2 and PGF2alpha on CPZ catalepsy, and strongly inhibited PGE2 the potentiating effect on HL and on PI catalepsy. Propranolol (PN) diminished the potentiating effect on HL and on PI catalepsy. Propranolol PN) diminished potentiating effect of PG on HL catalepsy and increased this effect of PGE1 on PI catalepsy. All examined PG induced catalepsy only when given in high doses (50 or 100 microgram icv). Cataleptogenic effect of PGE2 and PGF2 but not of PGE1, was evidently inhibited by AP. PGS inhibited AP stereotypy. The results suggest that the central dopaminergic receptors blockade is involved in the mechanism of potentiation of neuroleptic induced catalepsy by PGs, and that PGs are similar to neuroleptics in some aspects of central action.

Effects of food deprivation on etonitazene consumption in rats.

One group of free-feeding rats was given a 5 microgram/ml etonitazene HCl solution as their sole liquid. This group increased their drug intake by 100% when they were partially food-deprived during a 23-day period. Another group that remained food-satiated and received etonitazene for an equal number of days did not show similar increases in drug intake. However, this group drank greater volumes of the etonitazene solution than a food-satiated control group drank of water. These results are contrasted with a fourth group showing a 50% decrement in water intake during similar food-deprived conditions. The food-deprived group drinking etonitazene showed highly erratic drinking patterns compared to all the other groups. Daily liquid intake ranged from 30 to 250 ml in this group, and volumes oscillated from high to low on alternating days. When the food-deprived/food-satiated conditions were replicated in this experimental group, corresponding increases and decreases in drinking reliably occurred. However, during the second food-deprived phase, the large increases occurred almost immediately as contrasted with a gradual increase over 17 days during the first food-deprived phase. This would suggest a learning mechanism may be involved. Self-mutilation and other forms of stereotypy were noted only in food-deprived rats consuming etonitazene.

Diazepam and flurazepam: effects on conditioned fear as measured with the potentiated startle paradigm.

Diazepam (0.3, 0.6, 1.2, or 2.5 mg/kg) produced a dose-dependent reduction of the potentiated startle effect where acoustic startle amplitude is normally increased in the presence of a light previously paired with a shock. Even the lowest dose tested (0.3 mg/kg) significantly attenuated potentiated startle. The effect was selective since the same doses did not depress baseline startle amplitude measured in the same animals in the same test session. A 2 X 2 design in which rats were trained and tested under the same or different drug condition (diazepam or saline) showed the results could not be explained by state-dependent learning. The primary effect of diazepam was to block expression of rather than acquisition of fear as measured by potentiated startle. Flurazepam (2.5, 10, or 20 mg/kg) also reduced potentiated startle selectively but was 6--8 times less potent than diazepam. These and other results suggest that the potentiated startle paradigm, as a measure of classical conditioning that involves no operant, might provide a useful adjunct to behavioral methods currently being used to analyze antianxiety compounds.

The prerequisites for local lysolecithin formation in the human gallbladder. III. Demonstration of two different phospholipase A activities.

The positional specificity of the phospholipase A (EC 3.1.1.4) in human gallbladder epithelium has been studied using 14C-phosphatidylethanolamine radiolabeled either in the 1-acyl or in the 2-acyl position. After heating of homogenized epithelial cells at 70 degrees C for 2 min, their lysophospholipase activity was lost. In contrast, the ability to hydrolyze 14C-phosphatidylethanolamine in biosynthetically radiolabeled Escherichia coli was largely retained. The amounts of radioactivity found in the products of hydrolysis under different conditions suggest that there are two different phospholipase A activities in the gallbladder epithelium: one, with optimal activity at pH 7, that requires Ca2+ and is specific for the 2-acyl position, and another, with optimal activity at pH 4, that does not require Ca2+ and that, apart from the 2-acyl position, attacks the 1-acyl position as well. It is possible, therefore, that a complete deacylation of diacylphosphoglycerides in the gallbladder wall is brought about in two different ways: at neutral pH through a combined action of phospholipase A2 and lysophospholipase, the latter being able to hydrolyze the 1-acyl-lysophosphoglyceride, and, at acid pH, through the action of phospholipase A1 and A2 activity, presuming 1-acyl- and 2-acyl-lysophosphoglycerides are also attacked. Both these processes have to be considered in order to explain a turnover of diacylphosphoglycerides that physiologically would prevent the accumulation of lytic lysophosphoglycerides. The possible relevance of these findings to the pathogenesis of aseptic cholecystitis is inferred.

Acid-base balance made simple.

The control of extracellular acid-base balance is difficult to grasp unless it is realized that the pH of the blood depends upon the ratio H2CO3/NaHCO3 and that, without alteration of pulmonary ventilation, the H2CO3 neither rises nor falls. It must also be understood that the H+ ions, excreted by the kidneys, are byproducts of NaHCO3 reclamation and neogenesis and are not derived from metabolic acids.

[Caesarean section: low transverse (pfannenstiel) or midline incision? (author's transl)].

In 67 elective Caesarean sections and 70 emergency sections the effect of the duration of anaesthesia upon the condition of the newborn was examined. The induction-delivery time (IDT), the operation time (OT), and the difference between these, delta t, were correlated with the 1-, 5- and 10-minute Apgar scores and the pH's of the venous and arterial umbilical cord blood. A highly significant negative association was found for the pH in the umbilical vein and delta t in the series of primary sections. Analysis of the emergency sections showed a negative association between the operation time and the 5 minute Apgar score, and a positive association between the delta t and 1 minute Apgar score. Despite these findings we have observed that the induction-delivery time which we are able to achieve in our hospital has no negative effect upon the biochemical condition (pH) of the newborn. A comparison of 619 sections performed by low midline incision with 328 section by Pfannenstiel incision showed no difference with regard to postoperative complications such as disturbance in wound healing or haematoma formation. In conclusion, with respect to the IDT and postoperative complications we have found no contraindication to the use of the low transverse Pfannenstiel incision for Caesarean section.

The prognosis of near-drowned children.

Thirty children were treated for near-drowning in the Children's Hospital, University of Helsinki during 1971--1976. The patients were divided into 3 groups according to the prognosis: group I included 13 children (43%) with a favourable prognosis, group II four children (13%) with a less favourable prognosis who developed severe sequelae, and group III 13 children with poor prognosis and in whom the subsequent outcome proved fatal. The surviving children underwent neurological, neurophysiological and psychological examination 6--58 months after the accident. The children in group I had slight neurological or psychological signs, some children presented a lowered intellectual functioning level. The children in group II were tetraplegic, unable to speak and had convulsions. The following factors were important in affecting prognosis: the longer the immersion time, the worse the prognosis. However, prognosis could still be favourable with an immersion-time of 11-20 min. Prognosis was bad if the first pH value was less than 7.00. The arterial oxygen pressure values measured during the treatment did not correlate with the prognosis but a low rectal temperature on admission was usually associated with a bad prognosis. The degree of EEG-disturbance had a prognostic value. However, the follow-up recording correlated better with the prognosis than the recordings during the first 24 hours, after which worsening of the EEG sometimes showed a progressive brain lesion.

Skin-piercing blood-sucking moths II: Studies on a further 3 adult Calyptra [Calpe] sp. (Lepid., Noctuidae).

1. Of the scarce Calyptra minuticornis, C. orthograpta and C. labilis, 51, 24, and 7 adults, respectively, were observed during some 600 night inspections at over 100 sites in 1965--1967 and 1971--1977. 2. Hitherto biologically completely unknown, and not recorded before in S.E. Asia, the latter two species flew in or near tropical monsoon forests in hilly regions (300--600 m) of N. Thailand (C. orthograpta also N. Laos). C. minuticornis was found in these and in tropical evergreen and semi-evergreen rain forests of S. Thailand and N.W. Malaysia. 3. In N. Thailand the three species were more common at the end of the cool season/start of the hot season and at the start of the rainy season. They were active mainly during the first half of the night 4. Flight and piercing behaviour, alighting, resting, enemies, and the lack of females, were similar to virtually identical with the "classical" skin-piercing blood-sucking C. eustrigata. 5. C. labilis was seen attacking elephant, C. orthograpta also water buffalo and sambar, C. minuticornis also zebu and tapir but not sambar. C. minuticornis settled on man also but did not pierce. 6. Through no piercing of hosts' skin has actually been seen in nature, indirect evidence suggests that the 3 moths are likely to be occasional blood-suckers. They pierced and sucked blood from the author's skin in experiments. 7. Reasons for lack of direct evidence may be: less developed hematophagy, less favoured hosts, lack of easy-to-pierce injured skin (which also trigger the piercing response), different climatic and phytoecological environment, fewer specimens than in the case of C. eustrigata. 8. Field observations and experiments indicate that the closely related, fruit-piercing Oraesia emarginata is not skin-piercing blood-sucking--a habit likely to be exhibited mainly in humid equatorial regions by a few Calyptra only.

Specific alteration of Sendai virus glycoprotein subunits.

The alteration produced by chemical and physical agents in the structure and neuraminidase activity of Sendai virus glycoproteins was studied. While dissociation of glycoproteins with 1% sodium dodecyl sulphate (SDS), 2% beta-mercaptoethanol and 5 M urea for 2 min at 100 degrees C yielded the known HN and F subunits (mol. wts. 60,000 and 53,000), in the presence of 1% SDS the glycoproteins were converted into components with approximate mol. wts. of 60,000, 120, 000 and higher. Treatment of the glycoproteins with 2% beta-mercaptoethanol and 0.1% SDS favoured the formation of a single component with a mol. wt. of 75,000. The alterations in the glycoprotein structure were very likely caused by their free -SH groups content. An average value of 6 free -SH groups per glycoprotein subunit was estimated. Glycoproteins stored at 4 degrees C contained only 53,000 mol. wt. subunits. During storage a kind of conversion of 67,000 to 53,000 mol. wt. components took place, preserving about 60% of the initial neuraminidase activity.

Lower esophageal sphincter pressure during the normal menstrual cycle.

Lower esophageal sphincter pressure (LESP), basal gastric pH, and plasma levels of gastrin, estradiol, and progesterone were determined in ten women known to have normal menstrual cycles. All determinations were performed both during the follicular phase (Days 2 to 8) and during the luteal phase (Days 20 to 30). In addition, an intraluminal pH probe placed 5 cm. above the lower esophageal sphincter was used to test for the presence of acid reflux in response to three provocative procedures. LESP during the follicular phase was 19.0 +/- 1.5 mm. Hg (mean +/- S.E.M.) and during the luteal phase 16.5 +/- 1.3 mm. Hg (p less than 0.01). Basal gastric pH and plasma gastrin levels were similar at both times. Plasma estradiol in the follicular phase (76.1 +/- 7.0 pg. per milliliter) increased twofold during the luteal phase (159.0 +/- 6.0) (p less than 0.01). Plasma progesterone increased from a level of 1.5 +/- 0.8 ng. per milliliter during the follicular phase to 19.2 +/- 4.2 during the luteal phase. Coincident with these changes in LESP and increases in steroid levels, acid reflux was detected in five women during the luteal phase but was present in only one during the follicular phase.

Effect of Krebs cycle intermediates and inhibitors on toad gastric mucosa.

An attempt to increase the permeability of gastric mucosa to exogenous Krebs cycle intermediates seemed advisable for a better understanding their relationship with acid secretion. At pH 7.4, citrate, oxoglutarate, fumarate, and malate had no significant effect on oxygen uptake (QO2) nor on acid secretion (QH+) by toad gastric mucosa; succinate increased QO2 slightly and had no effect on QH+; but at pH 5.0, oxoglutarate and succinate increased QO2 by 18 and 21%, respectively. 14CO2 evolved by gastric mucosa incubated with [14C]oxoglutarate, succinate, malate, or citrate was 155, 92, 128, and 353%, respectively, greater at pH 5. Citrate, oxoglutarate, succinate, fumarate, and malate increased QH+ by theophylline-stimulated mucosa at pH 5.0 by 25, 39, 35, 17 and 28%, respectively. Oxoglutarate-dependent respiration was shown to correlate with oxoglutarate oxidation. Malonate and arsenite inhibited QO2 and QH+; malonate inhibition was reversed by washout or by succinate. Arsenite was reversed by washout and accelerated by addition of lipoate immediately after washout. The results suggest that the Krebs cycle has concomitant roles in the regulation of QH+ and oxidative metabolism in the toad gastric mucosa.

Response of patients with Hodgkin's disease to pneumococcal vaccine.

Postsplenectomy, 41 patients previously treated for Hodgkin's disease were given pneumococcal vaccine, and type-specific antibody levels were measured before and after immunization. Postimmunization antibody levels in patients with Hodgkin's disease were significantly lower than those in normal control subjects for 10 of the 12 serotypes measured. Mean postimmunization antibody level for patients (587 +/- 427 ng of antibody nitrogen/mL) was much lower than that for control subjects (1787 +/- 694). Antibody levels tended to increase with time from therapy for Hodgkin's disease, and several patients who had not received therapy for more than 3 years had normal responses to immunization. Despite vaccination, one patient developed pneumococcal meningitis and another, pneumococcal bacteremia. Both infected patients had low postimmunization mean antibody levels (282 and 137 ng/mL, respectively). Postsplenectomy sepsis in patients with Hodgkin's disease is related to a humoral immune deficiency probably induced by radiation and chemotherapy, and this immune deficiency persists for several years.

Influence of hydrogen ion concentration on bile acid induced acute gastric mucosal ulcerogenesis.

Aggressive treatment with H(2) receptor blocking agents and/or antacids has been advocated as effective prophylaxis against and treatment for "stress ulcer," based on the logical but infrequently tested assumption that the severity of the disease is critically determined by the concentration of intraluminal acid. The present study investigated this assumption in a model which employed topical acid, topical bile acid and mucosal ischemia to induce ulcerogenesis. With vascularized, chambered ex vivo wedges of canine proximal gastric wall, groups of animals were studied during three sequential periods using topical test solutions (TS) containing either 0 mM, 100 mM or 160 mM HCI. During period 1, mucosae were exposed to TS alone; during period 2, either to TS containing 1 mM sodium taurocholate (TC) or to TS and concomitant vasopressin infusion (VP); and during period 3, to TS + TC + VP. Parameters evaluated included net H(+) flux ( big up tri, openH(+)), aminopyrine clearance (AC), a measure of mucosal blood flow, net TC flux ( big up tri, openTC) and the lesion index, graded 0-5. The data indicate that in nonischemic mucosa exposed to constant [TC], AC was significantly increased, big up tri, openH(+) ("back-diffusion") increased as a linear function of [H(+)] and no lesions were observed. Under the same circumstances in ischemic mucosa, big up tri, openH(+) increased as linear function of [H(+)]. As a consequence, lesion severity was also a linear function of [H(+)]. big up tri, openTC was enhanced at low pH but bore no relation to the degree of mucosal damage induced. Assuming applicability of the model, these studies provide support for the use of H(2) receptor blocking agents and/or antacids to prevent or ameliorate "stress ulcer" disease.

Cardiorespiratory assessment of decongestant-antihistamine effects on altitude, +Gz, and fatigue tolerances.

Decongestants and antihistamines are known to produce effects capable of adversely modifying physiological function and psychomotor task performance. Because of relevance to safe pilot performance, the effects of single doses of two decongestant-antihistamine preparations (Compound A and Compound B), or a placebo on cardiorespiratory responses to two equally spaced +2 Gz tests during separate 2-h exposures at 388 m (1,274 ft MSL) ground level (GL) and 3,810 m (12,500 ft) chamber altitude were assessed. Post-altitude fatigue was assessed by cardiorespiratory responses to submaximal bicycle ergometry. Compound A and Compound B appeared to exert no significant detrimental effects on short-duration post-altitude ergometric fatigue-ability. With two exceptions, all combinations of medication, altitude, and +Gz were well tolerated. Two subjects were clearly incapacitated during the first +2 Gz test under Compound A at 3,810 m (12,500 ft) altitude. It is felt that the +Gz-intolerance resulted mainly from an adverse interactive effect of Compound A and altitude on vasomotor and/or chronotropic mechanisms.

Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase.

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7--0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.

Antitumor drug-protein interactions. Binding of 1-beta-D-arabinofuranosyl cytosine to albumin and chemically modified albumin.

Equilibrium dialysis measurements were carried out to study the binding of 1-beta-D-arabinofuranosyl cytosine (ara-C) to human and bovine serum albumin (HSA, BSA) and to chemically modified albumin. The binding of 4-phenylbutyric acid to HSA was studied, too. Binding data were presented as Scatchard plots. There are two types of binding sites of different affinity for ara-C both on HSA and BSA. The relatively small value of affinity constant indicates that the pharmacological properties of ara-C might not be influenced very strongly by the HSA interaction or by competitive binding of other drugs. Selective chemical modifications of HSA with diethylpyrocarbonate (DEP) or o-nitrophenylsulfenyl chloride (NPS-Cl) reduce significantly the affinity of the strong binding area. On the other hand, the attachment of poly-alpha-L-glutamyl or poly-DL-alanyl side-chains to BSA increase the number of the strong and secondary binding sites and also the affinity at the first group of sites. Experimental results suggest a correlation between the binding affinity and therapeutic efficacy of various cytotoxic drug-protein complexes.

Cardiovascular effects of prenalterol (H133/22) in normal man.

1 Prenalterol, (S-(-)-1-(4 hydroxyphenoxy)-3-isopropylaminopropanol-2 hydrochloride) a cardio-selective beta-adrenergic receptor agonist, was infused intravenously into six normal male volunteers to determine the cardiovascular effects of this drug. 2 On different occasions, each volunteer received a placebo infusion, an infusion of 0.5 mg prenalterol and an infusion of 1 mg prenalterol. Cardiac output (impedance cardiography), arterial pressure (sphygmomanometry), heart rate and ECG were measured throughout. 3 Prenalterol produced a statistically significant increase in cardiac output and at the end of the infusion this increase was 24% with 0.5 mg and 29% with 1 mg, mainly due to an increase in stroke volume (18% and 17%) with a lesser change in heart rate (+2 and +7 beats/min). Pulse pressure increased but mean arterial pressure showed little change. Peripheral resistance fell by 18% and 20%. As indicated by systolic time indices myocardial contractility increased. 4 Prenalterol at plasma concentrations in excess of 20 nmol l-1 produced significant inotropic effects but did not markedly increase heart rate at concentrations of 60 nmol l-1.

Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase.

Investigations have been made of the slow, tight-binding inhibition by methotrexate of the reaction catalyzed by dihydrofolate reductase from Streptococcus faecium A. Quantitative analysis has shown that progress curve data are in accord with a mechanism that involves the rapid formation of an enzyme-NADPH-methotrexate complex that subsequently undergoes a relatively slow, reversible isomerization reaction. From the Ki value for the dissociation of methotrexate from the E-NADPH-methotrexate complex (23 nM) and values of 5.1 and 0.013 min-1 for the forward and reverse rate constants of the isomerization reaction, the overall inhibition constant for methotrexate was calculated to be 58 pM. The formation of an enzyme-methotrexate complex was demonstrated by means of fluorescence quenching, and a value of 0.36 muM was determined for its dissociation constant. The same technique was used to determine dissociation constants for the reaction of methotrexate with the E-NADP and E-NADPH complexes. The results indicate that in the presence of either NADPH or NADP there is enhancement of the binding of methotrexate to the enzyme. It is proposed that methotrexate behaves as a pseudosubstrate for dihydrofolate reductase.

Isoelectric points of spinach thylakoid membrane surfaces as determined by cross partition.

The isoelectric points of unbroken chloroplast lamellae and various subchloroplast fractions, including a preparation of inside-out thylakoids, have been determined using aqueous two-phase systems containing dextran and charged polyethylene glycol. When the amounts of material in the top phase in a phase system with the positively charged trimethylamino polyethylene glycol are plotted against pH the curve intersects the corresponding curve obtained from phase systems with the negatively charged polyethylene glycol sulfonate. This cross-point can be correlated with the isoelectric point of the material. The cross-point for unbroken chloroplast lamellae was found to be around pH 4.7. Mechanical disintegration lowered the cross-point to around pH 4.4, probably because of exposure of new membrane surfaces. The disintegrated chloroplasts were fractionated by differential centrifugation to separate the grana and stroma lamellae. The stroma lamellae vesicles showed the same isoelectric point as the unbroken lamellae, while a cross-point at pH 4.3 was obtained for the grana-enriched fraction. For thylakoid membranes destacked under low salt conditions the cross-point was 0.3 pH unit lower than for membranes originating exclusively from the stroma lamellae. The most acidic crosspoint (pH 4.1) was observed for the fraction enriched in inside-out granathylakoids. It is suggested that the differences in isoelectric point between various subchloroplast fractions reflect a heterogeneous arrangement of surface charge along and across the thylakoid membrane.

Urate transport in human red blood cells. Activation by ATP.

Urate transport in human erythrocytes were measured and compared to previous observations by other authors regarding inorganic anions, especially chloride. Conclusions wwere as follows: 1. Urate influx as a function of increasing concentrations showed saturation kinetics. 2. The effects of pH and of several passive anion transport inhibitors such as dinitrofluorobenzene, sodium salicylate, sodium benzoate and phenylbutazone suggest that urate and chloride are transported by different mechanisms. 3. Urate influx seems to depend on intracellular glycolysis. The results obtained on red blood cells after glycolysis inhibition agree with those obtained on ghosts where metabolism does not take place. 4. The large drop in urate influxes into erythrocytes in the presence of a glycolysis inhibitor and of a passive ion transport inhibitor seems to argue in favour of a dual urate transport mechanism, one for passive diffusion and the other connected with glycolysis. 5. The drop in the urate influx into ghosts in the absence of ATP suggests that the latter might intervene in urate transport by human red cell membranes.

Use of immobilized antibodies in investigating acid alpha-glucosidase in urine in relation to Pompe's disease.

(1) A simple method is described for the isolation of the lysosomal enzyme, acid alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from normal human liver. Antibodies raised against the purified enzyme were immobilized by covalent coupling to Sepharose 4B. (2) Acid alpha-glucosidase can be quantitatively removed from normal urine by incubating with an excess of immobilized antibody. With p-nitrophenyl-alpha-glucoside as substrate, acid alpha-glucosidase accounts for 91 +/- 3% of the total alpha-glucosidase activity at pH 4.0 IN Normal urine. (3) In urine from a patient with the infantile form of Pompe's disease ('acid maltase deficiency'), no alpha-glucosidase activity could be removed by the immobilized antibody, in agreement with the fact that acid alpha-glucosidase is absent in these patients. (4) In urine from patients with the late-onset form of Pompe's disease, 46 +/- 11% of the alpha-glucosidase activity at pH 4.0 can be removed by incubation with immobilized antibodies, indicating that residual acid alpha-glucosidase activity is present in urine of these patients. The residual acid alpha-glucosidase activity amounts to about 5% of that in the urine of control persons. (5) If acid alpha-glucosidase is adsorbed to immobilized antibodies, the activity can still be measured with p-nitrophenyl-alpha-glucoside as substrate. The Km for p-nitrophenyl-alpha-glucoside is not significantly changed by adsorbing purified acid alpha-glucosidase to immobilized antibodies. (6) The properties of acid alpha-glucosidase from urine of patients with late-onset Pompe's disease were compared with those of acid alpha-glucosidase from normal urine, both adsorbed to immobilized antiserum. The pH-activity profile of the enzyme from urine of patients with late-onset Pompe's disease can not be distinguished from that of the normal urinary enzyme. The Km for p-nitro-phenyl-alpha-glucoside of the two enzymes is identical, both at pH 4 and 3. The titration curves of the two enzymes with immobilized antibodies are identical.

Characterization of highly purified native streptokinase and altered streptokinase after alkaline treatment.

Physical and chemical data are reported for highly purified native streptokinase (staphylokinase, EC 3.4.99.22) (Kabikinase) and streptokinase treated with an alkaline agent (altered streptokinase). The mol. wts. were similar and were determined to be 50 200 by sedimentation equilibrium methods, polyacrylamide gradient gel electrophoresis and sodium dodecylsulphate-polyacrylamide gel electrophoresis. The sedimentation coefficient so20,w of native and altered streptokinase was found to be 3.37 S. The frictional ratio and the absorptivity (A1%1cm) at 280 nm of native streptokinase was found to be 1.29 and 7.5, respectively. Native streptokinase showed essentially a single band in the isoelectro-focusing pattern (pI 5.2), while altered streptokinase showed at least two separate bands. Polyacrylamide gel electrophoresis in the presence of Triton X-100 exhibited one band for native streptokinase but altered streptokinase showd two bands. At pH 12 the biological and immunological activity of streptokinase was markedly decreased in a time-dependent reaction. The amino-terminal amino acid of the two streptokinase forms was isoleucine and the carboxyl-terminal amino acid of native streptokinase was tyrosine. Peptide analysis showed that some peptides in altered streptokinase exhibited higher mobility compared to native streptokinase. The data suggest that streptokinase undergoes a conformational change when incubated in alkaline media, but no simultaneous loss of peptides was observed.

Characterization of specific differences in protein phosphorylation of the plasma membrane and the endoplasmic reticulum of mouse fibroblasts.

Endogenous phosphorylation was studied with highly purified fractions of the plasma membrane and the endoplasmic reticulum of SV40-transformed mouse fibroblasts using [gamma-32P]ATP and [gamma-32P]GTP as precursors. With ATP maximum overall incorporation of 32P into both membrane fractions occurred at pH 7.8 in the presence of 10 mM MgCl2 after incubation for 1 min. GTP could be utilized only by the plasma membrane fraction showing maximum incorporation of 32P at pH 7.8 and 10 mM MgCl2 after incubation for 3 min. The pattern of phosphoproteins of the plasma membrane is represented by more than 15 proteins whereas the endoplasmic reticulum essentially contained only one phosphorylated component of 35 000 molecular weight. The comparison of ATP- and GTP-specific phosphorylation of the plasma membrane revealed GTP to be a less efficient precursor yielding a similar phosphoprotein pattern with one significant difference: the GTP-specific main component exhibited a molecular weight of about 100 000 and the ATP-specific main component a molecular weight of 110 000. The relative distribution of individual phosphoproteins in the pattern of the plasma membrane was dependent on pH but not on MgCl2 concentration or time of incubation. Increasing concentrations of plasma membrane protein altered the patterns of phosphoproteins dramatically: At high protein concentrations the ATP-specific main component (Mr = 110 000) was no more phosphorylated whereas with GTP the main component Mr = 100 000 was essentially the sole phosphorylated protein.

Presynaptic muscarinic and alpha-adrenergic receptor blocking effect of atropine on the noradrenergic neurones of the rabbit pulmonary artery.

The effect of atropine on the electrical-field stimulation-evoked overflow of tritium from isolated rabbit pulmonary arteries preincubated with 3H-noradrenaline was studied. Atropine (10(-4) M) and phentolamine (10(-6) M) increased stimulation-induced overfoow of tritium. Clonidine (10(-6) to 10(-5) M) and acetylcholine (10(-6) M) diminished the stimulation-evoked overflow of tritium. After the overflow had been raised by either atropine (10(-4) M) or phentolamine (10(-6) M), clonidine (10(-6) M) decreased the overflow below control values. Clonidine (10(-5) M) prevented the enhancement of tritium overflow evoked by atropine (10(-4) M). A lower concentration of clonidine (10(-6) M) only caused a partial prevention. Enhancement of the overflow by phentolamine (10(-6) and 3 X 10(-5) M) was not altered by atropine (10(-4) M). Atropine (10(-7) M), in a concentration which was without any effect on the stimulation-induced tritium overflow, prevented the reduction evoked by acetylcholine (10(-6) M). It is concluded that atropine in a low concentration blocks presynaptic inhibitory muscarinic receptors; at higher concentrations it blocks in addition presynaptic alpha-adrenoceptors.

Methylmalonic acidemia: 6 years' clinical experience with two variants unresponsive to vitamin B12 therapy.

Two infants with lethargy, vomiting, convulsions, coma and marked metabolic acidosis were found to have very high concentrations of methylmalonic acid in their serum and urine. In vitro studies of fibroblasts demonstrated that the infants had different variants of methylmalonic acidemia.Vitamin B(12) was given in two different forms at 1 month of age and at 12 months of age. Each trial continued for 4 months but neither infant showed a clinical or biochemical response.In both infants hyperglycinemia, neutropenia and thrombocytopenia developed during acute metabolic crises only. Hypoglycemia was found in patient 2. Hyperammonemia was severe in patient 2 during acute crises but never appeared in patient 1. When clinically well, both infants continued to excrete abnormal amounts of methylmalonic acid in the urine and both had persistent compensated metabolic acidosis.Marked hyperuricemia developed in patient 1 at 18 months of age and led to progressive renal failure. Allopurinol therapy was necessary to keep the uric acid concentration within the normal range. Renal function returned to normal, as indicated by a marked increase in the renal clearance of creatinine and uric acid.Patient 1 is physically and mentally retarded, and has moderate hypotonia, hepatomegaly and persistent vomiting. Patient 2 has developed normally.The urine concentrations of methylmalonic acid in the four parents were normal.

Measurement of HBDH, CK and gamma-GT activities and comparison of the precision of four instruments used in kinetic analyses.

A comparative analysis of low, normal and high activity of HBDH, CK, and gamma-GT was made using different kinetic measurement principles. The data revealed that the two-step integration technique and multi-point measurements evaluated by computer are essentially more accurate than the traditionally applied 3 x 1 minute enzyme determination.

Tyrosine hydroxylase and the conversion of L-thyroxine into 3',3,5-triiodo-L-thyronone in the rat.

We have studied the effects of alpha-methyl-p-tyrosine (alpha-MPT), an inhibitor of tyrosine hydroxylase, on the in vivo conversion of L-T4 (T4) to 3',3,5-triiodo-L-thyronine (T3), and on the biological effectiveness of T4. Thyroidectomized rats were used and were injected daily with T4 maintenance doses. Three different types of experiments were carred out. The first involved isotopic equilibration with 125I-labeled T4 and measurement of urinary 125I excretion. The second series involved the injection of a single dose of [125I]T4, with the amounts of [125I]T3 in different tissues being studied 7 or 20 h later. The third series involved daily treatment for 13 days with T4 and alpha-MPT, at the end of ehich the liver alpha-glycerophosphate dehydrogenase activity was measured as a parameter of the biological effects of the hormone. Though the experimental approaches used clearly disclosed the well known effects of 6-propyl-2-thiouracil, no clear-cut effects of alpha-MPT were observed. It is concluded that alpha-MPT neither inhibits the conversion of T4 to T3 in vivo in rats nor affects the biological potency of a given dose of T4, at least to an extent compararble to that observed when 6-propyl-2-thiouracil is used. Thus, present results do not support the hypothesis that tyrosine hydroxylase is involved in the extrathyroidal deiodination of T4 to T3.

Effect of leucine on the pyridine nucleotide contents of islets and on the insulin released--interactions in vitro with methylene blue, thiol oxidants, and p-chloromercuribenzoate.

In the presence of glucose (2 mg/ml), leucine (10 mM) noticeably increased islets' NADPH contents as well as the NADPH:NADP ratio; the changes occurred as soon as 1 min after its addition. NADH concentrations were also increased by leucine. The NADPH:NADP ratio as well as insulin release stimulated by glucose plus leucine were markedly decreased by methylene blue. The thiol oxidants diamide and tert-butyl hydroperoxide also inhibited insulin secretion in response to glucose plus leucine. Employing the perfused pancreas technique, the insulin-releasing action of p-chloromercuribenzoate was further enhanced by leucine. The combined effects were inhibited by tert-butyl hydroperoxide, however. Our data suggest that the insulin-releasing action of leucine depends on the islets' NADPH and reduced glutathione (GSH); in addition, leucine may contribute to insulin secretion by increasing the islet NADPH:NADP ratio and the NADH:NAD ratio. From the data, we assume that the observed increase of NADPH may lead via GSH to an increase in the number of such thiol groups in the beta-cell membrane, which are believed to be related to stimulation of insulin release and, thus, to increase the sensitivity of the beta-cell to stimulation by glucose and/or leucine.

Protection by histamine receptor antagonists and prostaglandin against gastric mucosal barrier disruption in the rat.

This study was undertaken to determine if the cytoprotective effect of prostaglandin and the H2 histamine receptor antagonist cimetidine involves protection against disruption of the gastric mucosal barrier. Groups of anesthetized, vagotomized rats received one of the following parenterally: saline (control), mepyramine--an H1 histamine receptor antagonist, cimetidine, cimetidine and mepyramine, or 16,16 dimethyl prostaglandin E2. Parameters of barrier disruption were then determined before and after exposure of the gastric mucosa to 40mM acetylsalicylic acid. At the end of the study, gastric lesions were scored according to size and number. Lesion score and fall in potential difference were significantly lower in rats receiving cimetidine, cimetidine and mepyramine, and prostaglandin. Other parameters of barrier disruption--H+ back diffusion, Na+ and K+ influx, and protein outpouring--exhibited the same pattern and correlated with change in potential difference. We conclude that both prostaglandin and cimetidine, but not mepyramine, protect against barrier disruption by topical aspirin, and this may be a factor in the mechanism of their cytoprotective action.

Purification and characterization of rat pepsinogens whose contents increase with developmental progress.

Two pepsinogens, the contents of which increase with developmental progress, were purified from the gastric mucosa of the adult rat by ammonium sulfate fractionation and chromatography on DEAE-cellulose and DEAE-Sepharose CL-6B columns. The purified zymogens, designated as pepsinogens I and II, were each shown to be homogeneous by polyacrylamide gel disc electrophoresis. Pepsinogen II had a greater electrophoretic mobility toward the anode at pH 8.0 than pepsinogen I. The molecular weights of both zymogens were estimated to be 38,000 by SDS-polyacrylamide gel electrophoresis. The activated enzymes, pepsins I and II, each had the same molecular weight of 32,000. The pH optima for both enzymes were found to be 2.0. The enzymes showed high stabilities at pH 8.0, while they lost their activities within 60 min at pH 10.0. The enzymes were inhibited by pepstatin and diazoacetyl-DL-norleucine methyl ester (DAN). The activities of the enzymes in hydrolyzing N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine (APDT) were about 1/8 of that of porcine pepsin. These results suggest that pepsins I and II are very similar.

The metabolic fate of 13N-labeled ammonia in rat brain.

13N-labeled ammonia was used to study the cerebral uptake and metabolism of ammonia in conscious rats. After infusion of physiological concentrations of [13N]ammonia for 10 min via one internal carotid artery, the relative specific activities of glutamate, glutamine (alpha-amino), and glutamine (amide) in brain were approximately 1:5:400, respectively. The data are consistent with the concept that ammonia, entering the brain from the blood, is metabolized in a small pool of glutamate that is both rapidly turning over and distinct from a larger tissue glutamate pool (Berl, S., Takagaki, G., Clarke, D.D., and Waelsch, H. (1962) J. Biol. Chem. 237, 2562-2569). Analysis of 13N-metabolites, after infusion of [13N]ammonia into one lateral cerebral ventricle, indicated that ammonia entering the brain from the cerebrospinal fluid is also metabolized in a small glutamate pool. Pretreatment of rats with methionine sulfoximine led to a decrease in the label present in brain glutamine (amide) following carotid artery infusion of [13N]ammonia. On the other hand, 13N activity in brain glutamate was greater than that in the alpha-amino group of glutamine, i.e. following methionine sulfoximine treatment the expected precursor-product relationship was observed, indicating that the two pools of glutamate in the brain were no longer metabolically distinct. The amount of label recovered in the right cerebral hemisphere, 5 s after a rapid bolus injection of [13N]ammonia via the right common carotid artery, was found to be independent of ammonia concentration within the bolus over a 1000-fold range. This finding indicates that ammonia enters the brain from the blood largely by diffusion. In normal rats that were killed by a freeze-blowing technique 5 s after injection of an [13N]ammonia bolus, approximately 60% of the label recovered in brain had already been incorporated into glutamine, indicating that the t1/2 for conversion of ammonia to glutamine in the small pool is in the range of 1 to 3 s or less. The data emphasize the importance of the small pool glutamine synthetase as a metabolic trap for the detoxification of blood-borne and endogenously produced brain ammonia. The possibility that the astrocytes represent the anatomical site of the small pool is considered.

Degradation of bis(monoacylglycero)phosphate by an acid phosphodiesterase in rat liver lysosomes.

Bis(monoacylglycero)phosphate was purified from the livers of chloroquine-treated rats and labeled with tritium by a nonreductive catalytic exchange procedure. The mechanism of its degradation by rat liver lysosomes has been examined. A substantial amount of bis(monoacylglycero)P is degraded to monoglyceride and lysophosphatidic acid by a lysosomal phosphodiesterase having an acid pH optimum. Some bis(monoacylglycero)P is degraded to lysophosphatidylglycerol by lysosomal phospholipase A. In contrast, other phosphoglycerides have been reported to be degraded by sequential deacylation in lysosomes. The initial rate of breakdown of bis(monoacylglycero)P is only 10% of the rate observed for dioleoylphosphatidylcholine. [3H]Lysophosphatidylglycerol conversion to [3H]bis(monoacylglycero)P is stimulated by unlabeled bis(monoacylglycero)P, resulting in a futile cycle which allows the resynthesis of bis(monoacylglycero)P from its breakdown product, lysophosphatidylglycerol. This futile cycle and the unusual sn-1-glycerophospho-sn-1'-glycerol stereoconfiguration of the water-soluble backbone (Joutti, A., Brotherus, J., Renkonen, O., Laine, R., and Fischer, W. (1976) Biochim. Biophys. Acta 450, 206-209) may be important factors in the marked resistance of bis(monoacylglycero)P to degradation by lysosomal acid hydrolases.

Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions.

The observed equilibrium constants (Kobs) of the creatine kinase (EC 2.7.3.2), myokinase (EC 2.7.4.3), glucose-6-phosphatase (EC 3.1.3.9), and fructose-1,6-diphosphatase (EC 3.1.3.11) reactions have been determined at 38 degrees C, pH 7.0, ionic strength 0.25, and varying free magnesium concentrations. The equilibrium constant (KCK) for the creatine kinase reaction defined as: KCK = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] [H+]) was measured at 0.25 ionic strength and 38 degrees C and was shown to vary with free [Mg2+]. The value was found to be 3.78 x 10(8) M-1 at free [Mg2+] = 0 and 1.66 x 10(9) M-1 at free [Mg2+] = 10(-3) M. Therefore, at pH 7.0, the value of Kobs, defined as Kobs = KCK[H+] = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] was 37.8 at free [Mg2+] = 0 and 166 at free [Mg2+] = 10(-3) M. The Kobs value for the myokinase reaction, 2 sigma ADP equilibrium sigma AMP + sigma ATP, was found to vary with free [Mg2+], being 0.391 at free [Mg2+] = 0 and 1.05 at free [Mg2+] = 10(-3) M. Taking the standard state of water to have activity equal to 1, the Kobs of glucose-6-P hydrolysis, sigma glucose-6-P + H2O equilibrium sigma glucose + sigma Pi, was found not to vary with free [Mg2+], being 110 M at both free [Mg2+] = 0 and free [Mg2+] = 10(-3) M. The Kobs of fructose-1,6-P2 hydrolysis, sigma fructose-1,6-P2 equilibrium sigma fructose-6-P + sigma Pi, was found to vary with free [Mg2+], being 272 M at free [Mg2+] = 0 and 174 M at free [Mg2+] = 0.89 x 10(-3) M.

Regulation of the interaction of purified human erythrocyte AMP deaminase and the human erythrocyte membrane.

The binding of purified human erythrocyte AMP deaminase to human erythrocyte membranes and the effect of binding on enzyme catalytic activity was investigated. AMP deaminase binds preferentially and specifically to the cytoplasmic surface of the erythrocyte membrane. The binding is saturable, reversible, and responsive to alterations of pH, of ionic strength, and of ATP and AMP concentrations. A limited number (approximately equal to 2.2 X 10(4) per erythrocyte) of apparently homogeneous high affinity (Ka approximately equal to 2.6 X 10(7) M-1) binding sites is present. The stability of purified and endogenously bound AMP deaminase is markedly improved by the interaction with the membrane, whereas the catalytic activity of AMP deaminase is sharply reduced. AMP deaminase displaces membrane bound glyceraldehyde 3-phosphate dehydrogenase in roughly a dose-response manner. No evidence for binding of AMP deaminase to spectrin or band 3 (the G3PD binding protein) was found in sucrose gradients, however. The interaction of AMP deaminase with the erythrocyte membrane may play an important role in the regulation of cellular adenine nucleotide metabolism.

Beta-adrenergic potentiation of the increased in vitro accumulation of cycloleucine by rat thymocytes induced by triiodothyronine.

We have previously demonstrated that 3,5,3'-triiodothyronine (T(3)), whether administered in vivo or added to suspending media in vitro, promptly stimulates the in vitro accumulation of the nonmetabolized amino acids, alpha-aminoisobutyric acid, and cycloleucine (CLE) by thymocytes isolated from weanling rats. In these studies, we have examined the in vitro interaction between catecholamines and T(3) with respect to this effect. The previously reported enhancement of CLE accumulation in thymocytes by T(3) in vitro (1 muM) was confirmed. When added alone in concentrations ranging between 10 nM and 0.1 mM, the adrenergic agonists, epinephrine and norepinephrine, had no effect on CLE accumulation. At a concentration of 1 muM, isoproterenol, terbutaline, and phenylephrine were also without effect. However, the effect of T(3) was clearly potentiated by the concomitant addition of epinephrine, norepinephrine, and possibly isoproterenol, whereas terbutaline and phenylephrine were without effect. Neither basal nor T(3)-enhanced CLE accumulation was affected by the addition alone of the adrenergic blocking agents, propranolol (0.1 mM), phentolamine (10 muM), or practolol (0.1 mM). Nevertheless, the beta(1)- and beta(2)-antagonist, propranol, and the beta(1)-antagonist, practolol, blocked the increment in CLE accumulation produced by epinephrine; the alpha-antagonist, phentolamine, was without effect. The enhancement of CLE accumulation that occurred in the presence of T(3), with or without epinephrine, was seen to be a result of an inhibition of CLE efflux, because T(3) alone inhibited CLE efflux, and this effect was increased when epinephrine was also present. On the other hand, neither T(3) alone nor T(3) plus epinephrine appreciably altered the rate of inward transport of CLE. As judged from studies of the ability of thymocytes to exclude trypan blue, neither T(3) alone nor T(3) plus epinephrine either enhanced or impaired viability of cells during 3-h periods of incubation. Cell water content, measured with [(3)H]urea, was unaffected by T(3), either alone or in the presence of epinephrine. In confirmation of previous results, the stimulatory effect of T(3) on CLE accumulation was unaffected by concentrations of puromycin sufficient to inhibit protein synthesis by at least 95%, and the potentiating action of epinephrine on the response to T(3) was similarly unaffected. From these findings, it is concluded that the effect of T(3) to increase CLE accumulation by thymocytes in vitro, though itself independent of adrenergic mediation, is potentiated by beta(1)-adrenergic stimulation. This interaction appears distinctly different from other thyroid hormone-catecholamine interactions, in which thyroid hormones enhance physiological responses to catecholamines. Its mechanism remains unclear, but the properties of the T(3) effect, and possibly the interaction itself, suggest that T(3) enhances CLE accumulation by an action at the level of the cell membrane.

Role of glucagon, catecholamines, and growth hormone in human glucose counterregulation. Effects of somatostatin and combined alpha- and beta-adrenergic blockade on plasma glucose recovery and glucose flux rates after insulin-induced hypoglycemia.

To further characterize mechanisms of glucose counterregulation in man, the effects of pharmacologically inducd deficiencies of glucagon, growth hormone, and catecholamines (alone and in combination) on recovery of plasma glucose from insulin-induced hypoglycemia and attendant changes in isotopically ([3-(3)H]glucose) determined glucose fluxes were studied in 13 normal subjects. In control studies, recovery of plasma glucose from hypoglycemia was primarily due to a compensatory increase in glucose production; the temporal relationship of glucagon, epinephrine, cortisol, and growth hormone responses with the compensatory increase in glucose appearance was compatible with potential participation of all these hormones in acute glucose counterregulation. Infusion of somatostatin (combined deficiency of glucagon and growth hormone) accentuated insulin-induced hypoglycemia (plasma glucose nadir: 36+/-2 ng/dl during infusion of somatostatin vs. 47+/-2 mg/dl in control studies, P < 0.01) and impaired restoration of normoglycemia (plasma glucose at min 90: 73+/-3 mg/dl at end of somatostatin infusion vs. 92+/-3 mg/dl in control studies, P<0.01). This impaired recovery of plasma glucose was due to blunting of the compensatory increase in glucose appearance since glucose disappearance was not augmented, and was attributable to suppression of glucagon secretion rather than growth hormone secretion since these effects of somatostatin were not observed during simultaneous infusion of somatostatin and glucagon whereas infusion of growth hormone along with somatostatin did not prevent the effect of somatostatin. The attenuated recovery of plasma glucose from hypoglycemia observed during somatostatin-induced glucagon deficiency was associated with plasma epinephrine levels twice those observed in control studies. Infusion of phentolamine plus propranolol (combined alpha-and beta-adrenergic blockade) had no effect on plasma glucose or glucose fluxes after insulin administration. However, infusion of somatostatin along with both phentolamine and propranolol further impaired recovery of plasma glucose from hypoglycemia compared to that observed with somatostatin alone (plasma glucose at end of infusions: 52+/-6 mg/dl for somatostatin-phentolamine-propranolol vs. 72+/-5 mg/dl for somatostatin alone, P < 0.01); this was due to further suppression of the compensatory increase in glucose appearance (maximal values: 1.93+/-0.41 mg/kg per min for somatostatin-phentolamine-propranolol vs. 2.86+/-0.32 mg/kg per min for somatostatin alone, P < 0.05). These results indicate that in man (a) restoration of normoglycemia after insulin-induced hypoglycemia is primarily due to a compensatory increase in glucose production; (b) intact glucagon secretion, but not growth hormone secretion, is necessary for normal glucose counterregulation, and (c) adrenergic mechanisms do not normally play an essential role in this process but become critical to recovery from hypoglycemia when glucagon secretion is impaired.

Four stimulants of the central nervous system: effects on short-term memory in young versus aged monkeys.

Aged Rhesus monkeys and young control monkeys were tested in a delayed-response procedure to assess the effects of central-nervous-system (CNS) stimulants on short-term memory (STM). Previous research had established that the aged monkeys showed specific impairments of STM in this procedure. Four different CNS stimulants (methylphenidate, magnesium pemoline, a pentylenetetrazole/niacin mixture, and caffeine) were chosen for evaluation on the basis of their relevancy to current geriatric-psychopharmacologic research. Four different doses of each of the four CNS stimulants were given to each monkey, counter-balanced for possible order effects. Methylphenidate and caffeine impaired the performance of both age groups in this non-human primate cognitive task, even at relatively low dose levels. Magnesium pemoline produced fewer adverse effects and some evidence of improving STM in the aged monkeys, although not within the levels of statistical significance. The pentylenetetrazole/niacin mixture produced a three-way interaction involving age, dose and retention interval. This reflected the fact that although no definite effects were noted under the zero-sec control condition, statistically significant age-related deficits did occur in the STM-dependent retention interval as the dose varied. The data demonstrate that, of these four CNS stimulants, none radily improves (and often may impair) performance of tasks requiring STM. Thus the results of this study offer little support for the hypothesis that general CNS stimulation may constitute significant therapy for cognitive impairments associated with advanced age.

Effect of reducing agents, catalase, and reuse of medium on toxicity of media for growth of Ureaplasma urealyticum.

The effects of catalase and reducing agents on the growth of Ureaplasma urealyticum type VIII in broth cultures were studied, and used medium was examined for the presence of toxic factors. Growth of U. urealyticum was slower in aged or pre-aerated medium than in fresh medium, although equivalent final yields were achieved. Generation times were 2.9 hr in pre-aerated broth and were 1.34 hr in pre-aerated broth supplemented with either 2mM dithioerythritol or catalase (300 units/ml). Growth and death of U. urealyticum in cultures rendered the broth medium deficient for growth when reinoculated. Filtration of spent medium, adjustment of pH, addition of urea, and reinoculation permitted growth at the same rate and to the same titer as that observed in unused medium. Omission of filtration allowed growth but the final yield was less than that in filtered medium. Addition of sonicated dead cells to filtered, supplemented, spent medium also resulted in only poor growth. Therefore, spent medium appears toxic because it contains organisms (either while or disrupted) that hydrolyze the urea added and again render the medium deficient for growth by depleting an essential growth factor.

Natural, genetically determined resistance toward influenza virus in hemopoietic mouse chimeras. Role of mononuclear phagocytes.

Radiation chimeras produced by crosswise transfers of bone-marrow cell among histocompatible mice susceptible, or genetically resistant, to lethal challenge by a number of myxoviruses were used to test whether macrophage resistance (as assessed in vitro) and resistance of the animal (as measured in vivo), both previously shown to be brought about by the gene Mx, were causally related. 49 chimeras were tested individually, both of resistance of their macrophages to in vitro challenge with M-TUR (a strain of avian influenza virus A/Turkey/England/63 adapted to grow in cultured mouse peritoneal macrophages), and for resistance of the animal in vivo upon challenge with pneumotropic, neurotropic, or hepatotropic influenza viruses. Cultivated Kupffer cells and peritoneal macrophages harvested from chimeric mice expressed the resistance phenotype of the bone-marrow donor irrespective of the host environment in which they had differentiated. However, susceptibility or resistance in vivo was according to the genotype of the host. Thus, inborn resistance of radiation chimeras was found to be independent of Mx-gene expression in cells of the hemopoietic system.

The effects of treatment and of withdrawal of treatment with guanfacine and clonidine on blood pressure and heart rate in normotensive and renal hypertensive rats.

Disagreement in the literature about the occurrence of rebound hypertension (hypertensive overshoot) in animal models initiated this investigation. Oral doses of clonidine (0.03 mg kg-1) or guanfacine (0.3 mg kg-1) were administered twice daily during three weeks to groups of normotensive and renal hypertensive rats. Blood pressure and heart rate were measured immediately before and 3 h after the first daily dose, and compared with values obtained from placebo-treated control rats. Treatment with either drug induced large daily fluctuations rather than sustained falls in blood pressure. In the normotensive, but not in the hypertensive groups, the morning blood pressures measured before the first daily dose were significantly elevated over those of the control groups after 9 and 5 days of treatment with clonidine or guanfacine. This elevation persisted for 3 days after drug withdrawal. It is concluded that in the rat the duration of action of both drugs was short, so that twice daily administration of both drugs similarly produced large daily fluctuations rather than sustained falls in blood pressure. Blood pressure rises developed during treatment rather than after withdrawal in normotensive rats only. Therefore, this type of study does not relate well to the human situation and different experimental approaches to this problem are needed.

Delayed-onset pulmonary insufficiency in primates resuscitated from hemorrhagic shock.

Forty-one unanesthetized cynomolgus monkeys were subjected to 2 hours of hemorrhagic hypotension at various mean arterial pressures (MAP) between 40 and 60 mm Hg. Resuscitation and maintenance of MAP were provided by lactated Ringer's solution and homologous blood. Thirty-eight per cent (57% of those surviving greater than 24 hours) developed a delayed-onset (18 to 24 hours) pulmonary insufficiency in spite of good urinary output, and which did not respond to furosemide. The group in which this caused death (24%) showed significantly decreased PaO2, PaCO2, dynamic compliance, and FECO2, and increased minute volume, Qs/Qt, pulmonary artery pressure, and VD/VT before death. Their lungs were heavier, with abnormal pressure/volume curves and increased minimum surface tension. During the entire post-resuscitation phase, this group remained in a high-output, low-resistance hemodynamic state in contract to survivors and those dying of shock. The implications regarding current practices of monitoring and resuscitating patients with traumatic shock are discussed.

Medical treatment of pancreatic insufficiency.

Treatment of exocrine pancreatic insufficiency with the use of eight tablets of pancreatin with meals consisting of 25 g of fat per meal will generally abolish azotorrhea. Although steatorrhea is not totally corrected, satisfactory nutritional status and relative relief of symptoms are usually achieved. For the occasional patient who continues to lose weight or remains symptomatic even after reduction of dietary fat, the addition of cimetidine to the standard pancreatin treatment will usually provide relief from the steatorrhea and alleviate troublesome diarrhea. In certain circumstances in which gastric pH is more than 4 for 1 hour after a meal, altering the dosage schedule to two tablets hourly may be effective in alleviating the steatorrhea. Conversely, in patients whose upper gastrointestinal tract is acidic for long periods postprandially (gastric pH less than 5, duodenal pH less than 4), Pancrease, an enteric-coated preparation, may be effective. In difficult cases in which symptoms and steatorrhea continue, special intraluminal studies need to be performed to ensure that intraluminal conditions are, in fact, present for certain dosage schedules to be effective or that intraluminal conditions have been altered by adjunctive therapy.

[Compact form of DNA in solution. XII. Double-stranded polyribonucleotide compacting in the presence of polyethylene glycol].

Double-stranded polyribonucleotides (a replicative form of phage f2 RNA--dsRNA and poly(A) poly(U), can adopt a compact from in solutions, containing NaCl and poly(ethylene glycol) (PEG). According to electron-microscopic observations dsRNA compact particles have the form of disks or doughnuts 200--400 A in diameter. X-ray diffraction patterns from dense slurries of dsRNA compact particles show a reflection at a spacing of 35 A, which is indicative of the existance of ordered regions in compact particles. The intense positive CD band, which is characteristic of dsRNA and poly(A) poly(U) compact particles, presumably results from the ordered regions in the particles. Heating of the solution leads to the disappearance of the intense positive CD band, probably as a result of the destruction of the ordered structure of compact particles. Heat or acid denatured dsRNA molecules as well as single-stranded molecules of ribosomal RNA also form large particles in PEG-containing solutions. However, X-ray diffraction patterns from these particles do not show the 35 A reflection and the specific positive band is not present in their CD spectra, which indicates that such particles lack ordered internal structure. It is suggested that similar mechanism of compactization of double-stranded polynucleotides (DNA and RNA) exist, and compact particles may be divided into two families (psi+ and psi-), differing by the secondary structure of double-stranded polynucleotides, which form the particles.

Effect of variations in blood hydrogen ion concentration on pulmonary gas exchange of artificially ventilated dogs.

The effect of variation of blood hydrogen ion concentration on arterial and mixed venous PO2, ideal alveolar-arterial O2 pressure difference (PAiO2--PaO2), venous admixture (Qs/Qt), arterio-alveolar CO2 pressure difference (a--A)DCO2, physiological dead space to tidal volume ratio (VD/VT), cardiac output (Qt) and mean pulmonary arterial pressure (PAP) has been studied. Arterial and mixed venous PO2 increased and (PAiO2--PaO2) decreased with increasing blood hydrogen ion concentration. No change in Qs/Qt, (a--A)DCO2, VD/VT, Qt and PAP was observed. The effect of hydrogen ion concentration on arterial and mixed venous PO2 and on (PAiO2--PaO2) is mainly due to a shift of the blood oxyhemoglobin dissociation curve (ODC), i.e. due to the Bohr effect. The upper part of the ODC is more flat in alkalosis (shift to the left) than in acidosis (shift to the right). Therefore the same end-capillary to arterial O2 content difference results in a greater (PAiO2--PaO2) in alkalosis than in acidosis. Any factor influencing the slope of the upper part of the ODC is expected to affect the arterial PO2 and the (PAiO2--PaO2) by this mechanism. Similarly any factor shifting the steep part of the ODC is expected to affect the PO2 of the mixed venous blood.

Translocation of glutathione from lymphoid cells that have markedly different gamma-glutamyl transpeptidase activities.

Translocation of intracellular glutathione to the medium was studied in lymphoid cells (grown in tissue culture) that have very high, very low, or intermediate levels of membrane-bound gamma-glutamyl transpeptidase, in the absence and presence of various inhibitors of this enzyme. The data show that glutathione is translocated to the medium by all of the cell lines studied, but that glutathione does not accumulate in the medium unless the cellular transpeptidase activity is either very low or substantially inhibited. Translocation of glutathione does not seem to be directly related to the activity of gamma-glutamyl transpeptidase. The present and previous [Griffith, O.W. & Meister, A. (1979) Proc. Natl. Acad. Sci. USA 76, 268--272] findings suggest that translocation of intracellular glutathione is a general property of many mammalian cells. Glutathione exported from cells that have membrane-bound transpeptidase may be recovered by the cell in the form of transpeptidation or degradation products. Translocation of glutathione may also reflect operation of a rather general mechanism that protects and maintains the integrity of cell membranes.

Genetic variation and relative catalytic efficiencies: lactate dehydrogenase B allozymes of Fundulus heteroclitus.

In order to evaluate whether functional differences exist between allelic variants of a B type lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) in the teleost fish Fundulus heteroclitus (Linnaeus), the kinetic properties of pyruvate reduction were examined. While the pH dependence and the temperature dependence for maximal catalysis were indistinguishable among the allozymes, reaction velocities at low pyruvate concentrations were significantly different. At pH values below 8.00, the LDH-BbBb allozyme showed a greater reaction rate at lower temperatures (e.g., 10 degrees C) than LDH-BaBa. The phenomenon was reversed at higher temperatures (e.g., greater than 25 degrees C) for pH values between 6.50 and 7.00. The rates for the heterozygous phenotype, LDH-BaBb, were not the arithmetic average of the two homotetrameric allozymes. When reaction rates were compared at constant relative alkalinity, that is, a constant [OH-]/[H+] ratio, the findings were similar. The differences in the temperature dependence and the pH dependence for pyruvate reduction found between the LDH-B allozymes may reflect a selective adaptation and help explain the geographical variation in the Ldh-B gene frequencies of F. heteroclitus.

The cell as an extreme environment.

Living cells and their intracellular parasites show many of the characteristics ascribed to extreme environments and their dominant species. The diversity of species colonizing intracellular habitats is low, and successful inhabitants exhibit special fitness traits that often render them obligately dependent on residence within a host cell. However, the diversity-limiting factor in the extreme environment of the host cell interior is not abiotic, as it is in conventional extreme environments. It is biotic: the living cell itself and its many activities. Host cells bar the entrance to most would-be parasites, they destroy most of those that do manage to get inside, and they deny parasites free access to many components of their soluble metabolite pools. Successful intracellular parasites have evolved fitness traits that give them the capacity to survive in the face of diversity-limiting factors or to modify the intracellular habitat so that those factors no longer operate. Looking on the cell as an extreme habitat emphasizes its simultaneous roles as environment, antagonist, and competitor.

Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host.

Agrobacterium tumefaciens is a gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing information determining the tumorous growth pattern and the synthesis of so-called 'opines', e.g. octopine (N-alpha-(D-1-carboxyethyl)-L-arginine) and nopaline (N-alpha-(1,3-dicarboxypropyl)-L-argine), is transferred and stably maintained and expressed in the transformed plant cells. This phenomenon can be understood as a 'genetic colonization' of the plant cells by bacterial plasmid DNA so that the transformed plant cells will produce and secrete into the medium amino acid derivatives (the opines) that Ti-plasmid carrying agrobacteria can selectively use as carbon and nitrogen sources.

Environmentally induced differences in susceptibility of rats to CNS stimulants and CNS depressants: evidence against a unitary explanation.

It has been suggested that socially isolated rats are more aroused then rats raised in social groups. This hypothesis was tested by examining amphetamine-induced activity and stereotypy in social and isolated rats of both sexes in both the active and inactive phases of their diurnal activity cycle. In socially raised rats it was possible to produce behavioural profiles similar to those of isolated rats by increasing the arousal level of the social rat. However, the complex interaction of housing conditions, diurnal variation and gender with drug dose suggests that one intervening variable such as arousal is too simplistic an explanation. In subsequent experiments, stereotypy was enhanced by a familiar environment, and there was a clear dissociation between the effects of CNS stimulants and CNS depressants. The increased susceptibility of isolates to CNS stimulants depends on social isolation for a short period before 45 days of age; the decreased susceptibility of isolates to CNS depressants may be produced by isolation at any age. We conclude that there is no evidence that isolated rats are hyperaroused.

Orchidopexy in cryptorchidism assessed by clinical, histologic and sperm examinations.

A study was undertaken of 187 patients with unilateral and 41 patients with bilateral cryptorchidism before and after orchidopexy. The mean tubular diameter and the mean tubular fertility index were used as quantitative criteria for assessment of the development of the testes. Before orchidopexy, no significant differences were found between scrotal and cryptorchid testes in patients up to six years of age. Thereafter, the scrotal testis showed marked development in distinction to the cryptorchid testis. After orchidopexy, follow-up examinations were carried out when the patients had reached the age, at least, 18 years. Based on the results of the mean tubular diameter and the mean tubular fertility index, a reasonable degree of fertility would be expected in both the unilateral and the bilateral cryptorchid testes. However, histologic assessment of spermatogenesis in the unilateral cryptorchid testes after orchidopexy showed spermatogenic arrest in 81 per cent and in all patients with bilateral cryptorchid testes after orchidopexy. In the unilateral orchidopexy patients, in distinction to the histologic assesment of spermatogenesis, sperm counts gave good results in 80 per cent; evidently, in these patients, the source of the spermatozoa was the contralateral scrotal testis. That spermatogenesis was defective in 20 per cent of the patients after unilateral orchidopexy suggests and underlying systemic factor affecting both testes.

[Investigations on the pathogenesis of distal renal tubular acidosis (author's transl)].

In distal (type 1) RTA, renal acid excretion is impaired by the inability to establish adequate pH gradients between plasma and distal tubular fluid at any level of acidosis. Main clinical signs in infancy are anorexia, vomiting and failure to thrive. Despite low serum bicarbonate levels the renal threshold of bicarbonate is normal, while urinary pH levels are high even with values below the threshold. Under conditions of bicarbonate-induced systemic alkalosis urinary the pCO2 exceeds blood pCO2 in normal subjects. by contrast, the urinary pCO2 tension is not significantly greater in distal RTA, indicating a failure of the cells of the distal nephron to secrete hydrogen ions even without a gradient. Red cell carbonic anhydrase is within the normal range, whilst the inhibition of carbonic anhydrase activity has no effect on distal tubular function. Until now no histological or enzymatic defect could be detected to explain the ineffective acidification. Bicarbonate loading is followed by a lowering of calcium excretion to within the normal range and a decrease in the uncharacteristic renal hyperaminoaciduria.

Acute central chest pain in the elderly. A review of 296 consecutive hospital admissions during 1976 with particular reference to the possible role of beta-adrenergic blocking agents in inducing substernal pain.

Two hundred and ninety-six patients were admitted to geriatric medical beds in Cardiff in 1976 with acute central chest pain. One hundred and eighty-six (63 per cent) had a confirmed acute myocardial infarction. Of the 37 per cent without evidence of cardiac infarction, 32 per cent were on beta-blocking drugs. The possible role of adrenergic blocking agents in producing acute central chest pain is discussed.

Comparison of hemoglobins Wood (alpha 2 beta 2 97 leu) and Malmö (alpha 2 beta 2 97 gln). Diagnostic value of citrate agar electrophoresis.

Diagnostic value of citrate agar electrophoresis. Am J Clin Pathol 71:668-671, 1979. Of approximately three dozen hemoglobin variants that have greater than usual oxygen affinity, nearly half are inseparable from hemoglobin A by electrophoresis at pH 8.6. A comparison of hemoglobins Wood (alpha2beta297leu) and Malmö (alpha2beta297gln) is of interest from several standpoints. They represent similar substitutions at the identical locus in the beta chain. They result in identical clinical and hematologic manifestations. Oxygen affinities of these variants are identical. Both are poorly resolved from hemoglobin A by electrophoresis at pH 8.6. The position of each is identical when studied by isoelectric focusing in polyacrylamide gel. Finally, they are easily distinguished by citrate agar electrophoresis at pH 6.2. The excellent resolution of hemoglobins Malmö and Wood from each other results neither from difference in charge, nor size, nor in quaternary structure. This technic provides a simple but effective means for identifying and differentiating these hemoglobin variants. Comparison with the results of citrate agar electrophoresis of other high oxygen-affinity hemoglobins indicates that the findings for hemoglobins Malmö and Wood are unique and unambiguous.

The ultrastructure of the human epidermis in chronic graft-versus-host disease.

The epidermal ultrastructure of 11 allogeneic bone marrow recipients with chronic graft-versus-host disease (GVHD) was compared with that of 4 recipients without chronic GVHD. This electron microscope study revealed three patterns of epidermal injury typical of chronic GVHD. The first type was a nonacantholytic (nondissecting) injury with a prominent cellular infiltrate consisting primarily of lymphocytes accompanied by a few macrophages. The second type was an acantholytic (dissecting) injury with a prominent infiltrate, while the third was a nondissecting injury with a sparse infiltrate. Broad-zone contact was observed between lymphocytes and all epidermal cell types as well as between other lymphocytes and macrophages. Point contact was only observed between lymphocytes and epidermal cells. Lymphocytes appeared to detach desmosomes from adjacent keratinocytes by isolating them with cytoplasmic projections, a phenomenon not previously described. Typical damage to the epidermal cells in the basal and spinous layers consisted of either swelling of the organelles or condensation of the cytoplasm and nucleus. In the keratinocyte, the condensation reaction resulted in the formation of colloid bodies, some of which were phagocytized by macrophages. Besides the cytolytic events, a concurrent stimulatory reaction occurred in the epidermal cells. The number of melanosomes in melanocytes and of Langerhans cell granules and dense bodies in the Langerhans cells all increased. Extensive areas of replication and disruption of the basal lamina were subjacent to areas of necrosis in the basal layer.

Effects of changes in maternal--fetal pH on the transplacental equilibrium of bupivacaine.

Increases in the maternal-fetal pH gradient that may occur during labor and delivery may increase the fetal concentration of local anesthetics. The authors evaluated effects of pH changes on the transplacental concentration equilibrium of bupivacaine. They increased the maternal-fetal pH gradient in each of six pregnant ewes from a control value of 0.15 to 0.54 by hyperventilating the lungs of the ewe and infusing lactic acid into her fetus. After infusion of bupivacaine, 0.15 mg/kg, intravenously into the mother, the drug rapidly appeared in fetal blood, with values significantly increased over control values at 1 and 5 min. The fetal/maternal (f/m) ratios were increased significantly at 5, 15, and 30 min. The f/m ratios had stabilized by 15 min in both control and experimental states, suggesting that equilibrium had been achieved. The consistently low f/m ratios are explained by the presumed similarity of the ovine maternal and fetal protein binding rates to those of man. It is concluded that the maternal and fetal pH values are major factors in the determination of the f/m ratios.

Concentration of poliovirus from tap water using positively charged microporous filters.

Microporous filters that are more electropositive than the negatively charged filters currently used for virus concentrations from water by filter adsorption-elution methods were evaluated for poliovirus recovery from tap water. Zeta Plus filters composed of diatomaceous earth-cellulose-"charge-modified" resin mixtures and having a net positive charge of up to pH 5 to 6 efficiently adsorbed poliovirus from tap water at ambient pH levels 7.0 to 7.5 without added multivalent cation salts. The adsorbed virus were eluted with glycine-NaOH, pH 9.5 to 11.5. Electropositive asbestos-cellulose filters efficiently adsorbed poliovirus from tap water without added multivalent cation salts between pH 3.5 and 9.0, and the absorbed viruses could be eluted with 3% beef extract, pH 9, but not with pH 9.5 to 11.5 glycine-NaOH. Under water quality conditions in which poliovirus recoveries from large volumes of water were less than 5% with conventional negatively charged filters and standard methods, recoveries with Zeta Plus filters averaged 64 and 22.5% for one- and two-stage concentration procedures, respectively. Electropositive filters appear to offer distinct advantages over conventional negatively charged filters for concentrating enteric viruses from water, and their behavior tends to confirm the importance of electrostatic forces in virus recovery from water by microporous filter adsorption-elution methods.

Effect of environmental parameters on the biodegradation of oil sludge.

A laboratory study was conducted with the aim of evaluating and optimizing the environmental parameters of "landfarming", i.e., the disposal by biodegradation in soil of oily sludges generated in the refining of crude oil and related operations. Oil sludge biodegradation was monitored by CO2 evolution and by periodic analysis of residual hydrocarbons. The parameters studied were soil moisture, pH, mineral nutrients, micronutrients, organic supplements, treatment rate, teratment frequency, and incubation temperature. Oil sludge biodegradation was optimal at a soil water-holding capacity of 30 to 90%, a pH of 7.5 to 7.8, C:N and C:P ratios of 60:1 and 800:1, respectively, and a temperature of 20 degrees C or above. Addition of micronutrients and organic supplements was not beneficial; sewage sludge interfered with hydrocarbon biodegradation. Breakdown of the saturated hydrocarbon (alkane and cycloalkane) fraction was the highest at low application rates, but higher application rates favored the biodegradation of the aromatic and asphaltic fractions. An application rate of 5% (wt/wt) oil sludge hydrocarbon to the soil (100,000 liters/hectare) achieved a good compromise between high biodegradation rates and efficient land use and resulted in the best overall biodegradation rate of all hydrocarbon classes. Frequent small applications resulted in higher biodegradation than single large applications. Two 100,000-liter/hectare (255 barrels per acre) or four 50,000-liter/hectare oil sludge hydrocarbon applications per growing season seem appropriate for most temperate zone disposal sites.

Lateral cerebral ventricular enlargement in chronic schizophrenia.

To investigate if cerebral ventricular enlargement is associated with chronic schizophrenia, computerized tomography scans from 73 psychiatric patients were compared with 56 asymptomatic volunteers all less than 50 years old. Ventricular size was significantly greater in the subgroup of 58 chronic schizophrenic patients than in the controls. Of the chronic schizophrenic patients, 40% were outside the control range; 53% exceeded 2 SDs of the control mean. Neither duration of illness nor length of hospitalization correlated with ventricular size. The 44 chronic schizophrenic patients who had never been treated with electroshock therapy (EST) had larger ventricles than controls. A group of seven nonchronic schizophrenic patients also had enlarged ventricles; the eight patients who were either schizoaffective or nonschizophrenic did not differ from controls. This study shows that lateral cerebral ventricular enlargement is associated with chronic schizophrenia; it suggests that this is not a result of treatment.

Inborn errors of cobalamin metabolism: effect of cobalamin supplementation in culture on methylmalonyl CoA mutase activity in normal and mutant human fibroblasts.

We have examined the effect of addition of hydroxocobalamin to growth medium on the activity of the adenosylcobalamin-requiring enzyme methylmalonyl CoA mutase in normal human fibroblasts and in mutant human fibroblasts derived from patients with inherited methylmalonicacidemia. The mutant cell lines were assigned to four distinct genetic complementation groups (cbl A, cbl B, cbl C, and cbl D), each deficient in some step in the synthesis of adenosylcobalamin from hydroxocobalamin. After control cells were grown in cobalamin-supplemented medium, mutase holoenzyme activitiy increased markedly in a time- and concentration-dependent fashion. Growth in cobalamin-supplemented medium had no effect on mutase activity in some mutant lines belonging to the cbl B group, while activity increased severalfold in other cbl B mutants and in all cbl A, cbl C, and cbl D mutants examined, although mutase activity was still less than 10% of control. Comparison of mutase holoenzyme activity and total propionate pathway activity suggests that enhancement of mutase activity in mutant cells after cobalamin supplementation to values 5--10% of control may be sufficient to overcome the inherited metabolic block and to restore total pathway activity to normal.

Quantitative estimation of the photosynthetic proton binding inside the thylakoids by correlating internal acidification to external alkalinisation and to oxygen evolution in chloroplasts.

The external alkalinisation delta pHe, or the rate of oxygen evolution vO2, of a suspension of envelope-free chlorplasts was correlated with their internal acidification, estimated from the transmembrane delta pHei. Knowing the external buffer value, the concentration of the total protons moved Hi was calculated from the delta pHe, measured with a glass electrode ([Hi] was also obtained from vO2), and the free proton concentration [Hi+] was determined from delta pHei, measured with 9-aminoacridine. This gives a ratio gamma i = theta [Hi]/theta [Hi+], which is independent of the thylakoids internal volume. Within a large pHi range, scanned by varying the light intensity, gamma i was kept reasonably constant; it was hardly sensitive to pHi. This apparent invariability implies a continuous change of the internal buffer value beta i with pHi, since beta i/gamma i = -2.3.....10pHi, a relationship which inlcudes neither the total concentration of protonizable groups [Ai] nor pKi. As gamma i approximately Ki[Ai]/(Ki + [Hi+i]2, to keep gamma i constant when pHi drops, pKi and [Ai] must increase. This may be achieved by a progressive unmasking of anionic functions, initially inaccessible in the membrane. The relative slowness of this process may explain why gamma i calculated from the initial kinetics was sometimes smaller in high than in low light, where it always equalled that measured from the steady-state amplitude at all intensities. A small deficit of [Hi+] deduced from what could have been expected from delta pHe may reflect a limited binding of protons in the membrane itself, about 1 H+ for 30--130 chlorophylls (gamma i could be between 70 and 240, more frequently around 100); these numbers varied depending on the samples, but were constant for a given preparation.

Active K+ transport in Mycoplasms mycoides var. Capri. Relationships between K+ distribution, electrical potential and ATPase activity.

The addition of 5 . 10(-5) M or less of dicyclohexylcarbodiimide to Mycoplasma mycoides var. Capri preferentially influences K+ influx rather than efflux and reduces by 30--40% the activity of the membrane-bound Mg2+- ATPase. Adding valinomycin to metabolizing cells does not markedly affect K+ distribution but induces a rapid and complete loss of intracellular K+ in non-metabolizing cells. Uncoupling agents such as dinitrophenol, carbonylcyanide p-trifluoromethoxyphenylhydrazone, dissipate the K+ concentration gradient only when combined with valinomycin. Variations in the merocyanine fluorescence intensity indicate that a transmembrane electrical potential (delta psi) is generated on cell energization. This delta psi, not affected by valinomycin or uncouplers when used alone, is collapsed by a mixture of both. No change in fluorescence intensity can be detected when glucose is added to dicyclohexylcarbodiimide treated organisms. These experiments suggest that the membrane-bound Mg-ATPase activity control K+ distribution in these organisms through the generation of a transmembrane electrical potential difference.

The destruction of serine and threonine thiohydantoins during the sequence determination of peptides by 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate.

1. A mechanism for the destruction of serine and threonine thiohydantoins during protein sequence analysis by the Edman-type degradation is proposed. The mechanism begins with the dehydration of serine and threonine side chains (at the cyclization stage) which occurs mainly in anhydrous acid solution. The dehydrated derivatives finally polymerize by way of the reactive methylene group (enamine) to form polymers with various molecular weights. In aqueous acid solution, the dehydrated thiohydantoins of serine and threonine undergo hydration (according to the Markovnikov rule) and ring fission, which leads to the irreversible breakdown of thiohydantoin ring. The serine derivative shows a much greater tendency to undergo these side-reactions than the threonine derivative. 2. In the presence of oxygen, the alkaline hydrolysis of amino acid thiohydantoins goes through an oxidation-deamination reaction at the C-N bond of the thiohydantoin ring and leads to the formation of thiourea derivative and keto acids. This reaction mechanism accounts for the low recoveries of amino acid obtained from the alkaline hydrolysis of amino acid thiohydantoins.

Energy transduction in the mitochondrionlike bacterium Paracoccus denitrificans during carbon- or sulphate-limited aerobic growth in continuous culture.

Paracoccus denitrificans was grown in carbon-limited aerobic continuous culture (critical dilution rate (Dc) = 0.48 h-1). The molar growth yield for carbon (succinate or malate) was constant at about 60 over a broad dilution range (growth rate) from 0.10 to 0.48 h-1. Measurements of the stoichiometry of proton translocation associated with the oxidation of endogenous substrates yielded a ratio of protons ejected from the cell per atom of oxygen consumed(leads to H+:O) of 8.55 which decreased to 5.85 in the presence of piericidin A (PA), a specific inhibitor of NADH dehydrogenase (EC 1.6.99.3). With starved cells, the observed leads to H+:O associated with the oxidation of added succinate in the presence of PA was 5.61. These observed leads to H:O's represent an underestimation since no correction was made for proton backflow during the short interval of respiratory activity. Aerobic growth of Pc. denitrificans in the chemostat becomes sulphate limited at entering concentrations of sulphate less than 300 is microM. Neither the maximum specific growth rate (measured at Dc) nor the observed molar growth yield for succinate decreased under sulphate limitation. The NADH oxidase in electron transport particles prepared from sulphate-limited cells was completely inhibited by PA. The stoichiometry of proton translocation associated with malate oxidation was similarly unaffected by sulphate limitation. It is concluded that (a) the respiratory chain of aerobic, heterotrophically grown Pc. denitrificans possesses three sites of energy conservation, including site III, (b) the number of protons ejected during the transfer of one pair of reducing equivalents along a region of the electron transport chain equivalent to a single energy-coupling site is 3, and (c) that sulphate limitation does not lead to a loss of proton translocation associated with the cytochrome-independent region of the respiratory chain.

DEAE-cellulose chromatography of creatine kinase isoenzymes--effect of pH and serum.

DEAE-cellulose chromatography (pH 7.0) of human heart extracts revealed the presence of three creatine kinase isoenzymes. The CK3 (skeletal muscle) isoenzyme was not retained on the column under these conditions. The CK2 (heart) and CK1 (brain) isoenzymes eluted at a conductivity of 5.5 +/- 0.6 m omega-1 and 11.4 +/- 1.2m omega-1, respectively. When DEAE-cellulose chromatography was performed at pH 8.0, CK2 eluted at a slightly higher conductivity, 6.5 m omega-1, whereas CK1 eluted as before 12.0 m omega-1. DEAE-cellulose chromatography of CK2 and CK1 isoenzymes in the presence of serum protein, and serum albumin had no significant effect on the elution of CK2 at pH 7.0 and 7.4, and on the elution of CK1 at pH 7.0 and 8.0 However, serum and serum albumin decreased the affinity of CK2 for DEAE-celluose at pH 8.0, and caused this isoenzyme to elute at a conductivity of 3.0-3.5 m omega-1. The decreased affinity of CK2 for DEAE-cellulose was not due to aggregation of CK2 with albumin or some other serum protein, but was related to the amount of albumin applied to the column.

Postischemic brain oxygenation with barbiturate therapy in rats.

We measured rat brain cortex PO2 (PtO2) with gold microelectrodes (tip diameter 5--10 micron) for up to 2 hours after 16 min of transient global brain ischemia with and without thiopental 90 mg/kg infused iv over 60 min beginning at 5 min postischemia. Seventeen rats were immobilized and mechanically ventilated on 1% halothane in oxygen with continuous monitoring of PtO2, ECG, end-expiratory CO2, rectal temperature, and arterial blood pressure. Global ischemia was induced by trimethaphan hypotension to an MAP of about 50 torr and a neck tourniquet inflated to 1500 torr. Postischemia, nine control rats (11 PtO2 measurements) were untreated and eight rats (8 PtO2 measurements) received thiopental 90 mg/kg. Preischemia, PtO2 values in both groups ranged from less than 5--70 torr with values of greatest frequency between 10 and 15 torr. Postischemia, PtO2 in control rats peaked at 45 +/- 8 (SEM) torr at 20 min. In thiopental treated rats, peak PtO2 was 24 +/- 6 torr at 10 min postischemia. Relative frequency histograms of PtO2 revealed that PtO2 in thiopental treated rats was lower (p less than 0.05) between 15 and 30 min postischemia. The magnitude of the decrease in PtO2 between 105 and 120 min postischemia appeared to correlate directly with the absolute preischemic value (i.e., the higher the preischemic PtO2, the greater the decrease in PtO2 postischemia). These results suggest that thiopental administered in large doses in early postischemia does not improve brain oxygenation secondary to a reduction in brain oxygen consumption. The relevance of the correlation between the magnitude of the fall in PtO2 postischemia and the magnitude of the preischemic value is discussed.

Effects of acid aspiration on pulmonary alveolar epithelial membrane permeability.

Employing a modification of the in vivo model of a liquid-filled canine lung, we measured the movement of substances of specific sizes (albumin, 69,000 daltons with a molecular radius of 35 A; and dextran with a molecular weight of 150,000 to 170,000 and an approximate molecular radius of 100 A) from the pulmonary capillary blood to the liquid-filled lung. A solution with a specific pH (1.5 to 4.5) was instilled into the left lung of the animals at a dosage of 3 to 5 ml/kg of body weight. For both albumin and dextran with a molecular weight of 150,000 to 170,000, the time for 50 percent equilibration between the specific substance in the blood and the same substance in the pulmonary liquid decreased significantly with instillation of pulmonary liquid with a pH of 1.5 and 2.5 but did not with a pH of 3.5 or above (P less than 0.05). In addition, since histamine has been implicated as a possible humoral mediator leading to increased permeability of alveolar membranes, the levels of histamine were measured in pulmonary liquids and blood in all groups. Levels of histamine in the pulmonary liquid (but not blood) were significantly higher in animals with instillation of liquids with a pH of 1.5 and 2.5 compared to all other groups.

Regulation of placental enzymes of the carbohydrate and lipid metabolic pathways.

The activity of enzymes with a regulatory function in the pathways of glycolysis, gluconeogenesis, NADPH generation and fatty acid synthesis was measured in the placenta and liver of rats. Compared with the liver, a high activity of pyruvate kinase was found in the placenta, indicating a high glycolytic potential; a small capacity for gluconeogenesis was also present and a moderate to low activity of enzymes associated with lipogenesis. The activity of all placental enzymes fell from day 15 to 20 of gestation irrespective of the pathway they represented. The pattern of decline continued when the gestation was prolonged up to day 26 by the administration of chorionic gonadotropin. The rates of activity disappearance over 11 days of gestation differed for each enzyme, with half-lives ranging from 2.7 days for NADP-malate dehydrogenase to 7 days for glucose-6-phosphate dehydrogenase. In contrast, the activity of hepatic enzymes either remained unchanged or showed individual adaptation to the advancing pregnancy. The regression in placental metabolic capacity after day 15 of gestation was also evident by the decrease in glucose uptake and its channelling to lactate, CO2, glycerol and fatty acids. In addition, placental ageing was associated with triglyceride accumulation, mainly due to the decrease in free fatty acid oxidation. Treatment of pregnant rats with several hormones, while markedly affecting the hepatic enzyme activities, failed to induce appreciable changes in the corresponding placental enzymes. This was illustrated in the case of triiodothyronine treatment. Similarly, insulin deficiency induced by streptozotocin failed to elicit adaptive changes in placental enzyme activities typical of diabetes like those occurring in the maternal liver; some converse responses in the placenta were attributed to hyperglycaemia. On the other hand, responses in some fetal liver enzymes were suggestive of fetal hyperinsulinaemia. These observations indicate that placental enzymes are not susceptible to endocrine regulation and imply that placental metabolism is largely independent of the physiopathological alterations affecting the maternal organism. The gradual activity decreases with gestation suggest that the enzyme complement of the placenta, once developed, is designed to last through its limited lifespan without continuous replenishment. Within this context, no mechanism seems to operate to ind1ce the adaptive synthesis of individual enzymes, and the age of the placenta appears to be the primary factor determining its enzyme activity and metabolic performance.

Light-induced proton transport by chloroplasts suspended in fluid media at sub-zero temperatures: kinetics and stoichiometry.

1. Chloroplasts suspended in a medium containing ethanediol and water (1 : 1, v/v) at -16 degrees C show light-induced proton uptake and subsequent dark efflux. Proton uptake in continuous light showed biphasic kinetics. 2. A 1 ms flash caused a single turnover of the photochemical centres at -16 degrees C. Under the same conditions 3H+ were taken up from the external medium in the presence of methyl viologen as electron acceptor. 3. The flash-induced proton uptake was exponential and monophasic with t1/2 = 3 s. The flash-induced proton release into the thylakoid interior was biphasic, with half-times of less than 0.1 s and 3 s. The fast phase represented approximately 30% of the total release and may be correlated with the oxidation of water. 4. The half-time of reduction of cytochrome f in the dark following illumination in the presence of 2 mM NH4Cl (2.5 s) is similar to the half-time of the slow phase of proton release, suggesting a correlation between the kinetics of cytochrome f reduction and plastoquinol oxidation.

Anaerobic respiration and energy conservation in Paracoccus denitrificans. Functioning of iron-sulfur centers and the uncoupling effect of nitrite.

1. Electron paramagnetic resonance spectra at 8-60 K of NADH-reduced membrane particles prepared from Paracoccus denitrificans grown anaerobically with nitrate as terminal electron acceptor show the presence of iron-sulfur centers 1-4 in the NADH-ubiquinone segment of the respiratory chain. In addition resonance lines at g = 2.058, g = 1.953 and g = 1.88 are detectable in the spectra of succinate-reduced membranes at 15 K, which are attributed to the iron-sulfur-containing nitrate reductase. 2. Sulphate-limited growth under anaerobic conditions does not affect the iron-sulfur pattern of NADH dehydrogenase or nitrate reductase. Furthermore respiratory chain-linked electron transport and its inhibition by rotenone are not influenced. These results contrast those observed for sulphate-limited growth of P. denitrificans under aerobic conditions [Eur. J. Biochem. (1977) 81, 267-275]. 3. Proton translocation studies of whole cells indicate that nitrite increases the proton conductance of the cytoplasmic membrane, resulting in a collapse of the proton gradient across the membrane. Nitrite accumulates under anaerobic growth conditions with nitrate as terminal electron acceptor; the extent of accumulation depends on the specific growth conditions. Thus the low efficiencies of respiratory chain-linked energy conservation observed during nitrate respiration [Arch. Microbiol. (1977) 112, 17-23] can be explained by the uncoupling action of nitrite.

Embryology of the diffuse neuroendocrine system and its relationship to the common peptides.

The diffuse neuroendocrine system is constituted by the cells, now more than 40 in number, of the central and peripheral divisions of the amine precursor uptake and decarboxylation (APUD) series. At one time presumed to be derived from a common "neural" ancestor, all are now deemed to be "neuroendocrine-programmed," arising either in the embryonic epiblast itself or in one of its principal descendants. The APUD cells produce more than 35 physiologically active peptides and a small number of equally active amines. Within the last 3 years, 17 of these peptides have been identified jointly in endocrine cells and in neuronal cell bodies or processes. Sharing in this way a neural and an endocrine location and site of production, they are called the "common peptides." The diffuse neuroendocrine system is to be regarded as a third division of the nervous system, whose products suppress, amplify, or modulate the activities of the other two divisions. The relationship of its products to the cells and processes of these two divisions is currently the object of intensive inquiry.

Electron microscopic radioautographic identification of the ECL cell as the histamine-synthesizing endocrine cell in the rat stomach.

Rat gastric oxyntic glands contain argyrophil "enterochromaffin-like" endocrine cells that synthesize and store histamine and also have APUD ability--they can take up exogenous L-5-hydroxytryptophan (5-HTP), can decarboxylate it to 5-hydroxytryptamine (5-HT, serotonin) by the enzyme DOPA-decarboxylase, and can store the amine. Previous cytochemical studies suggested that these cells correspond to both the ECL and A-like cells, the two predominant endocrine cells identified by electron microscopy (EM) in rat oxyntic glands. In a recent study, however, we demonstrated that the ECL but not the A-like cell exhibited APUD ability when rat gastric mucosa was incubated with H3-5-HTP and studied by EM radioautography. The purpose of the present study was to identify by EM radioautography the histamine-synthesizing endocrine cells in the rat stomach. Pieces of rat (male Sprague-Dawley) gastric mucosa were incubated in organ culture with L-H3-histidine (50 muCi, 1.8 x 10(-5) M) with and without inhibitors and were processed for LM and EM radioautography. H3-histidine labeled the ECL cells heavily but the A-like and other endocrine cells only lightly. The labeling of ECL cells was only modestly reduced by cycloheximide, an inhibitor of protein synthesis, whereas the labeling of A-like and other endocrine cells was almost abolished. In contrast, the labeling of ECL cells was markedly reduced by 4-bromo-3-hydroxybenzyloxyamine (NSD-1055), an inhibitor of histidine decarboxylase and DOPA decarboxylase, but was not appreciably affected by carbidopa, an inhibitor of only the DOPA decarboxylase. Incubations with H3-histamine (50 muCi, 0.9 x 10(-5) M) failed to label endocrine cells. Thus, this study demonstrates that the ECL but not the A-like cell is the histamine-synthesizing endocrine cell of the rat stomach.

Factors affecting release of heat-labile enterotoxin by enterotoxigenic Escherichia coli.

Various conditions affecting the release of heat-labile enterotoxin (LT) by enterotoxigenic Escherichia coli have been examined. The pH of a defined medium containing three amino acids, M-9 salts, and 0.5% glucose decreased to less than 7.0 in early log phase of growth, and no extracellular LT was detected. Adjustment of the pH at 8 h from 6.0 to 8.0 resulted in a concomitant increase in LT activity in culture supernatants. The release of cell-associated LT was significantly reduced by preincubation with protease inhibitors and increased by preincubation with trypsin. Cell-associated LT was not released by pH adjustment of cells grown at 21 degrees C; however, polymyxin B treatment released a toxin species active in only the pigeon erythrocyte lysate (PEL) assay system. As the growth temperature was increased, polymyxin B released toxin species which exhibited both PEL and Y-1 adrenal tumor cell activity. Polymyxin B extracts of enterotoxigenic E. coli in early log phase grown at 37 degrees C possessed only PEL activity, whereas extracts from cells in late-log and stationary phases had biological activity in both assay systems. Also, LT released by pH adjustment from mid-log to stationary phase was active in both PEL and Y-1 adrenal tumor cell assays. Gel electrophoresis of polymyxin B extracts revealed at least three molecular weight species active in either the PEL (22,000 daltons and 30,000 daltons) or both the PEL and the Y-1 adrenal tumor cell assay (72,000 daltons), depending on the growth temperature. These observations may help to explain the chemical and biological heterogeneity of most LT preparations and facilitate purification of LT by increasing the yield of enterotoxin.

Antibacterial activity of bladder surface mucin duplicated by exogenous glycosaminoglycan (heparin).

We have previously shown that the transitional cells lining the urinary bladder are capable of producing glycosaminoglycan (GAG). By use of a quantitative in vivo method of measuring bacterial adherence, we demonstrated that bacterial adherence to the mucosal cells is diminished in the presence of this GAG, rises when it is removed (by acid), and returns to normal when the GAG is resynthesized (in less than 24 h). We also found that this much layer could be removed (with a corresponding rise in bacterial adherence) and that addition of exogenous GAG (heparin) to the bladder prevented the expected rise in bacterial adherence. This study analyzed in depth the manner by which heparin prevents the rise in adherence seen when the mucin is removed. Pretreatment of bacteria with heparin had no effect on adherence, whereas pretreatment of the bladder with heparin inhibited adherence. To corroborate our impression that the heparin was coating the transitional cells, [3H]heparin was added to bladders after removal of mucin. Autoradiography revealed the heparin to be adherent to the surface of the transitional cells.

Chemical inducers of ovulation: comparative results.

Chemical inducers of ovulation are frequently used to reestablish a normal hypothalamic-ovarian function in the sterile woman. At present, several types are available and it is useful to evaluate comparatively their efficacy. In this paper we present our results in 396 cases treated with some of these drugs. Clomiphene citrate was administered to 307 patient. Ovulation was obtained in 85.99% and pregnancy in 35.50%. There were 16 abortions (14.68%) among the 109 pregnancies obtained. Cisclomiphene was used in 11 cases. The ovulation rate was 81.81%, with 54.5% of pregnancies. Thirty-eight patients were treated with Cyclophenil. Ovulation was obtained in 71.05% of the cases and pregnancy in 23.68%. In forty cases Tamoxifen was administered: the ovulation rate was 95% and the pregnancy rate was 35%, but the abortion rate was 35%. Tolerance was good with all medications. They had similar adverse side effects (mild and rare). None produced overstimulation. With Cyclophenil and specially with Tamoxifen the cycles were longer. The incidence of abortion in the 131 pregnancies obtained was 14.68% with Clomiphene; none with Cisclomiphene; 11.11% with Cyclophenil, and 35.10% with Tamoxifen. We conclude that Clomiphene is the drug which gives the best results at present.

Enhanced transformability with heterospecific deoxyribonucleic acid upon removal of nascent ribonucleic acid from the Streptococcus sanguis genome.

Treatment of Streptococcus sanguis recipient cells with rifampin (RIF) at the time of deoxyribonucleic acid (DNA) addition was an effective means of reducing discrimination, that is, of causing an increase in the number of transformants induced by irreversibly bound heterospecific DNA without significantly changing the number induced by bound homospecific DNA. RIF was unable to reduce discrimination when the recipient cells were RIF resistant due to an altered ribonucleic acid (RNA) polymerase. When recipient cells were treated at the time of DNA addition with concentrations of streptolydigin (STG) as inhibitory of RNA synthesis as RIF, discrimination was not reduced. The kinetics of RNA synthesis inhibition with these inhibitors indicated that, as reported for other bacterial species, RIF inhibited the initiation of transcription by RNA polymerase, whereas STG inhibited the progression of RNA polymerase at any point. Pulse-labeling of RNA immediately before STG addition showed that, if cells were incubated under STG inhibition for 10 to 15 min, their nascent RNA was degraded. Genome-bound RNA polymerase was not released under these conditions. When recipient cells were incubated with STG until nascent RNA was degraded and then exposed to transforming DNA, STG was as effective as RIF in reducing discrimination. The presence of nascent RNA was thereby implicated in the transforming inefficiency of incompletely homologous DNA.

Immunochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs.

Glutamine synthetase derived from two Neurospora crassa glutamine auxotrophs was characterized. Previous genetic studies indicated that the mutations responsible for the glutamine auxotrophy are allelic and map in chromosome V. When measured in crude extracts, both mutant strains had lower glutamine synthetase specific activity than that found in the wild-type strain. The enzyme from both auxotrophs and the wild-type strain was partially purified from cultures grown on glutamine as the sole nitrogen source, and immunochemical studies were performed in crude extracts and purified fractions. Quantitative rocket immunoelectrophoresis indicated that the activity per enzyme molecule is lower in the mutants than in the wild-type strain; immunoelectrophoresis and immunochemical titration of enzyme activity demonstrated structural differences between the enzymes from both auxotrophs. On the other hand, the monomer of glutamine synthetase of both mutants was found to be of a molecular weight similar to that of the wild-type strain. These data indicate that the mutations are located in the structural gene of N. crassa glutamine synthetase.

Transmembrane electrical and pH gradients across human erythrocytes and human peripheral lymphocytes.

Transmembrane electrical and pH gradients have been measured across human erythrocytes and peripheral blood lymphocytes using equilibrium distributions of radioactively labelled lipophilic ions, and of weak acids and weak bases, respectively. The distributions of methylamine, trimethylamine, acetic acid and trimethylacetic acid give calculated transmembrane pH gradients (pHe-pHi) for erythrocytes of between 0.14-0.21 for extracellular pH values of 7.28-7.16. The distributions of trimethylacetic acid. DMO and trimethylamine were determined for lymphocytes, establishing upper and lower limits of the calculated pH gradient over the external pH range of 6.7 to 7.7. Tritiated triphenylmethyl phosphonium ion (TPMP) and 14C-thiocyanate ion (SCN) equilibrium distributions were measured in order to calculate transmembrane electrical potentials, using tetraphenylboron as a catalyst to facilitate TPMP equilibrium. Transmembrane potentials of -7 to -10 mV were calculated from SCN and TPMP, respectively for red cells, and -35 to -52 mV respectively, in the case of lymphocytes. Distributions of TPMP and potassium ions were determined in the presence of valinomycin over a wide range of extracellular potassium concentrations for red cells and the calculated Nernst potentials for TPMP compared to the calculated potential using the Goldman equation for chloride and potassium ions. Distributions of TPMP, SCN and potassium ions were also determined for lymphocyte suspensions as a function of extracellular potassium and the calculated Nernst potentials for TPMP and SCN compared to the calculated potassium diffusion potential.

[The evolution of the thickness of rabbit corneas in hydratation, the influence of pH and composition of the solution (author's transl)].

The swelling of excised, de epitheliated, rabbit corneas has been measured every five minutes during their immersion in a hydrating medium. The following media have been used: distilled H2O, Krebs-bicarbonate Ringer solution, HCl 0,01 N and NaCl 1 M/l. The swelling is very moderate with Krebs and NaCl, much higher and practically proportional to the duration of immersion with HCl, still faster with H2O. On the other hand, the influence of pH has been studied using 13 different solutions buffered from pH 1 to pH 13. A pronounced minimum of the swelling was found for pH 4, along with 3 relative maxima: the first around pH 2, the second around pH 8 and the third around pH 12. The interpretation of those results on a biochemical basis is discussed. For all these media, the solvent was distilled H2O. At last, two statistical distributions, based on 131 corneas, have been determined: one concerning the initial thickness and the other concerning the difference between the initial thickness of the two corneas of the same rabbit.

Relative effects of different surfactants on intestinal absorption and the release of proteins and phospholipids from the tissue.

The actions of anionic, cationic and non-ionic surfactants on the absorptive capability of rat jejunal tissue in vivo were compared with their effects on the amounts of protein and phospholipid released from the mucosal surface under the same conditions. Release of a comparatively small amount of protein was accompanied by large increases in the absorption rates of both L-valine and salicylate, whereas much larger quantities of phospholipid were released before any increase in absorption were observed. Much of the released material appeared to be derived from mucus which was partly degraded after exposure to the higher concentrations of surfactants. The liberation of cholesterol by high concentrations of anionic surfactants suggested that some disruption of the mucosal membrane occurred under those conditions. The relative potency of the surfactants in stimulating both absorption of the solutes and the release of polypeptides and lipids followed the order: anionic greater than non-ionic greater than cationic. The possible pharmaceutical relevance of these findings is discussed.

Dissolution systems for chloramphenicol tablet bioavailability.

The relationship between chloramphenicol (I) tablet bioavailability and in vitro dissolution rates was examined. The effect of solid food on the I tablet and powder bioavailability was also studied. Five tablets of I were selected for bioavailability testing on the basis of the dissolution rates of 18 I tablets (250 mg) determined by several methods. Compound I, 500 mg, was administered orally to five subjects, following overnight fasting, according to a crossover design. The bioavailability parameters were obtained from urinary I excretion. Among the five formulations studied, only one tablet (F) showed significantly poorer bioavailability. The dissolution rates at pH 1.2 did not give the same rank order as the bioavailability. The dissolution rate of Tablet F showed remarkable pH dependency. The dissolution rates at pH 4 showed good correlation with in vivo bioavailability data. The bioavailability of I powder was not affected by solid food. Tablet F, which had poor bioavailability in the fasting state, showed good bioavailability when administered just after the standard breakfast.

Distortions in indexing methods and investing media for soldering and remount procedures.

A three-dimensional distortion analysis was made of seven indexing-investment systems and six indexing-remount systems. Measurements were made to within +/- 0.005 mm with a Nikon Profile Projector and a linear variable differential transformer (LVDT); distortions (linear and rotational) were calculated by means of a PDP 11/40 computer system. Mean vector displacements, square root deltaX2 + deltaY2 + deltaZ2, were calculated, and a statistical evaluation of the data was completed. The results of the study indicate: 1. The zinc oxide-eugenol (ZOE) indexing-investment system produced a narrower range and a significantly smaller mean distortion than the other six indexing-investment systems. 2. The ZOE-stone remount technique showed significantly less distortion than the ZOE-low-fusing metal or the ZOE-acrylic resin techniques. 3. The polyether-stone remount technique demonstrated significantly less distortion than either the polyether-low-fusing metal or the polyether-acrylic resin system. 4. The polyether-stone remount system was not significantly different from the ZOE-low-fusing metal system. 5. The ZOE-stone remount technique demonstrated a smaller range of distortions, but those distortions were not significantly different from those of the polyether-stone remount technique.

Microvascular and clinical effects of altered peritoneal dialysis solutions.

Blood flow in the peritoneum is one of the more important factors governing the efficiency of peritoneal dialysis. Yet there have been no previous studies which relate alterations and control of the peritoneal microcirculation to dialysis efficiency. Thus, we used closed-circuit television microscopy to quantitative the in vivo response (changes in diameter) to dialysis solutions of the small arteries on the mesothelial surface of the rat cecum and arterioles of the rat cremaster muscle. These responses were correlated wiht solute clearances from multiple peritoneal dialysis performed in humans. In the cremaster, a transient constriction was followed by a prolonged dilation. pH adjustments of the dialysis solution from 5.6 to 7.4 had no effect on the microvascular response and no effect on solute clearances during human peritoneal dialysis. In the cecum, dialysis solution caused a prolonged dilation which reached a maximum in about 10 min. Since dilation appears to be an important determinant of solute clearances during human peritoneal dialysis, the effects of a vasodilator, sodium nitroprusside, were determined. Sodium nitroprusside decreased the time to maximal dilation, which correlated clinically with an increased solute clearance during exchanges with this drug. Since nitroprusside increased clearances of the larger molecular weight solutes proportionally more than the smaller molecular weight solutes did, we hypothesize that nitroprusside increases solute clearances by both a vasodilatory effect and by an effect on vascular membrane permeability and area for solute exchange.

Reproducibility of clinical data and decisions in the management of upper respiratory illnesses: a comparison of physicians and non-physician providers.

The ability of non-physician providers to collect the data required by an algorithm for upper respiratory illness management, and the appropriateness of resulting key management decisions, were studied by comparing non-physician data and management decisions on 426 patients with those of internists. The internists, blinded to Amosists' findings and plans, evaluated the same patients and indicated management without using the algorithm (AM-MD) study). To control for variability of internists' data collecting and illness management, 171 additional patients were evaluated and managed consecutively by two internists, each also kept unaware of the other's findings and plans (MD-MD study). Overall AM-MD agreement on history and physical findings (90 per cent and 81 per cent) and on the need for tests (84 per cent) and treatment (87 per cent) was as high as MD-MD aggrement (91 per cent, 80 per cent, 88 per cent, and 75 per cent, respectively). In both studies, there was significantly more agreement on history data than on physical findings, evaluation, and therapy.

[DNA complexes with lysozyme].

Sedimentation method has been used to study hen egg-white lysozyme binding to glucosylated (from T2 phage) and non-glucosylated (from calf thymus) DNA under conditions similar to physiological ones (pH 7,3--7,4, ionic strength 0.07--0.24). The results indicate that lysozyme binds cooperatively to both DNA's. Binding parameters have been obtained by applying the theory of one-dimensional adsorption of small molecules on a linear homopolymer. X-ray patterns of complexes with different protein content have been obtained.

Action of androgenic steroids on brain neurotransmitters in rats.

The effects of androgenic steroids on the dopamine (DA), noradrenaline (NA) and 5-hydroxytryptamine (5-HT) contents of different brain regions have been studied in order to elucidate the possible involvement of neurotransmitters in the negative feedback action of androgens. Administration of testosterone propionate (TP); (100 micrograms/kg or 5 mg/kg, i.p.) increased plasma testosterones, which reached a maximum at about 30 min following injections. TP (100 micrograms/kg) decreased the DA level in the hypothalamus to a minimum after 30 min and returned to normal level after 120 min. There was no effect in the amygdala, striatum and mesencephalon. Subsequent to 5 mg/kg i.p. TP administration, the minimum in the DA level was observed between 90 and 120 min in the hypothalamus, and after 120 min in the amygdala, but the treatment was without effect in the striatum and mesencephalon. Both doses of TP were ineffective as regards for in altering NA and 5-HT levels in the brain areas studied. In a dose of 5 mg/kg, androgens of different activities, such as norandrostenolone, dihydrostestosterone and androstenedione, decreased the DA contents of the hypothalamus and amygdala regions, but pregnenolone was ineffective. None of the androgens affected the NA and 5-HT levels in the brain areas studied. The data suggest that some of the actions of androgens are mediated via a dopaminergic mechanism in which not only the hypothalamus but also the amygdala is involved.

Mitrochondrial NADH dehydrogenase in cystic fibrosis.

We have shown that skin fibroblast from patients with cystic fibrosis (CF) and from carriers for CF [heterozygotes (HZ)] consume more O2 than do their controls. When the mitochondrial electron transport inhibitor rotenone was added to the cells, the relative inhibition of O2 consumption was CF greater than HZ greater than controls (P less than 0.005 in both comparisons). Because rotenone specifically inhibits NADH dehydrogenase, [NADH: (acceptor) oxidoreductase, EC 1.6.99.3], which is the enzyme of energy-conserving site 1 of the mitochondrial electron transport system, activity and kinetics of this enzyme system were studied in fibroblast homogenates. NADH dehydrogenase activity was equal in cells from the three genotypes. At pH 8.0, affinity of the enzyme for its substrate was CF greater than HZ = controls; at pH 8.6, affinity was CF greater than HZ = controls (P less than 0.005 for the differences). pH optima for the genotypes were without exception 8.6 (CF), 8.3 (HZ), and 8.0 (control). HZ and control lines were distinguished unequivocally in a blind test on the basis of differences in pH optima. Purified mitochondrial preparations revealed pH optima identical to those found in whole cell homogenates. These data suggest that the mutant gene responsible for CF is expressed in the complex mitochondrial NADH dehydrogenase system.

The effect of destroying the whisker follicles in mice on the sensory nerve, the thalamocortical radiation and cortical barrel development.

Electrolytic destruction of whisker follicles in mice on the day of birth has been found to cause degeneration in the sensory nerve fibres supplying the follicles. The severity of the degeneration has been assessed in animals between 2 and 20 days old by counting the total number of myelinated fibres in the maxillary nerves on both normal and lesioned sides. The degeneration is apparent after 2 days and by 20 days the nerve on the lesioned side contains only 38% of the normal fibre content. This degeneration has also been shown to involve the trigeminal root, central to the ganglion. In addition, the lesioning procedure modifies the terminations of thalamocortical fibres in the barrel region of the sensory cortex. These terminations are normally in clusters, each corresponding to a barrel, but, after lesioning the follicles, the terminals appear to be evenly distributed in layer IV and cortical barrel structures no longer develop. In postnatal mice, electrolytic destruction of whisker follicles had less effect upon maxillary nerve fibres and cortical barrels. The number of myelinated axons surviving until day 20 increased progressively with later lesioning to reach nearly 80% of the control level when lesions were made on day 10. Cortical barrels became secure earlier than the maxillary nerve, for a normal number of cortical barrels was present at day 12 when follicles were destroyed on day 4. The implications of these results for the formation of cortical barrels is discussed.

Ultrastructure of graniferous tracheary elements in the haustorium of Exocarpus bidwillii, a root hemi-parasite of the Santalaceae.

The xylem in the body of the haustorium of E. bidwillii has the shape of an inverted conical flask with the expanded portion being known as the vascular core. The tracheary elements of the vascular core are notable for the occurrence of numerous granules within their lumina and the presence of mostly imperforate walls. Elsewhere in the haustorium graniferous tracheary elements are absent and the cells are usually ordinary vessel elements. Thin sections for transmission electron microscopy, post-stained in potassium permanganate, show that the secondary wall thickenings of the graniferous tracheary elements consist of eccentric layers in which the microfibrils of each successive layer run alternately longitudinally and transversely. The granules of the tracheary elements average 2 micrometer in diameter and consist of a homogeneous matrix which shows a fine fibrillar structure on high resolution. The granules are naked and mostly remain as separate structures within the lumen of the cell, but occasionally they fuse into small groups or irregular masses. In some cells the granules become transformed into fibrillar material that disperses throughout the lumen. This dispersed material may accumulate in vessels of the interrupted zone proximal to the vascular core. Occasionally, the granules also change into compacted amorphous masses that adhere to the walls of the cell. Ultrastructural cytochemistry confirms that the granules are protein and not starch as was originally believed for the Santalaceae. The function of the vascular core and its graniferous tracheary elements is discussed and we suggest that it might help regulate the pressure and flow of xylem sap entering the parasite from the host. Graniferous tracheary elements in the Santalaceae and in root parasites of the Serophulariaceae are compared and it is concluded that they represent examples of convergent evolution.

[Sociopsychiatric treatment combination in patients who are partly inpatients and genuine outpatients: definition and comparison (author's transl)].

The article gives a definition and brief description of those possibilities of treatment which are important in the sociopsychiatric therapy of psychoses: sociotherapy, therapy with neuroleptics, and psychotherapy as such, as well as their various combinations. Basing on a representative random trial from the fields of outpatient and nightpatient wards, the distribution of therapeutic potential among these combinations is described and outlined in a manner customary for the work bone by the Zürich Sociopsychiatric Service. It is evident, despite the fact that the work performed at our hospital is basically of the psychotherapeutic nature, patients can be rehabilitated with a lower level of neuroleptics provided sufficient sociotherapeutic measures are offered, including satisfactory psychotherapeutic efforts--the chances of meeting these demands being greater in our night ward. Patients who cannot be fully rehabilitated but more or less integrated, did not show such obvious differences, whereas the more intense sociotherapeutic programme of the nigth ward enabled the reintegration even of the nigth ward enabled the reintegration even of more seriously disturbed patients than could be achieved by mere outpatient follow-up treatment.

[Comparative study of chicken liver xanthine dehydrogenase and bovine liver xanthine oxidase. dehydrogenase activity of xanthine oxidase (author's transl)].

A method to purify bovine liver xanthine oxidase in described, with which samples of 256-fold specific activity with respect to the initial homogenate are obtained. Bovine liver xanthine oxidase and chicken liver xanthine dehydrogenase with oxygen as electron acceptor exhibit similar profile in pKM and log V versus pH plots. With NAD+ as electron acceptor a different profile in the pKM xanthine plot is obtained for chicken liver xanthine dehydrogenase. However three inflection points at the same pH values appear in all plots. Both enzymes are irreversibly inhibited by pCMB and reversibly by N-ethylmaleimide and by iodoacetamide, with competitive and uncompetitive type inhibitions respectively. These results suggest that NAD+ alters the enzymatic action since its binding to the enzyme antecedes the binding of xanthine to the xanthine oxidase molecule, without undergoing itself any modification. 0.15 M DDT of DTE treatment of bovine liver xanthine oxidase gives to the enzyme a permanent activity with NAD+ without modifying its activity with oxygen. The enzyme thus treated produces parallel straight lines in Lineweaver-Burk plots.

Endorphins, dopamine, and schizophrenia.

The theory that alterations of dopaminergic synaptic transmission may play a role in the pathogenesis of schizophrenia is widely accepted. A more recent theory links the endorphin system to the etiology of schizophrenia. We propose that these two theories may be combined into a single model. Recent neurochemical and pharmacological findings have indicated close functional relationships between the endorphin and dopamine systems. Endorphins modulate dopaminergic synaptic transmission by exerting both presynaptic and postsynaptic effects. On the molecular level, this modulation may involve the activity of nucleotide cyclases and protein phosphorylation systems. Thus, the dopaminergic neuronal hyperactivity, currently believed to be related to schizophrenia, may be caused by a primary alteration in the endorphin system. Several hypotheses about the nature of that alteration have been advanced and tested in therapeutic experiments with schizophrenic patients. These experiments have not yet yielded definitive results.

Cerebral anoxia: effect of deep hypothermia and pH.

Deep hypothermic circulatory arrest facilitates repair of congenital cardiac anomalies in infants. It is known empirically that hypothermia protects against central nervous system (CNS) ischemic damage. The Q10O2 is only 2.2 for brain and thus a decrease in metabolic rate does not fully account for protective effects of hypothermia. Since enthalpy of dissociation of H2O is high (approximately 7 kcal/mole), its pH is temperature dependent (7.0 at 25 degrees C, 7.4 at 20 degrees C) and hypothermia may in part protect by its influence on hydrogen ion concentration. A manifestation of CNS susceptibility to ischemia is an obstruction of the microcirculation [no-reflow lesion (NRL)] demonstrated by infusion of carbon black into the cerebral circulation after a period of circulatory arrest. White lesions (NRL) against a gray background on cut section of brain increase in size with increasing time of arrest. The effect of anoxia versus circulatory arrest, brain temperature, and extracellular brain pH on NRL was studied in 45 mongrel dogs, subjected to varying periods of N2-induced anoxia on cardiopulmonary bypass (CPB) at 37 degrees C or 20 degrees C. In some studies jugular venous pH was adjusted by infusion of NaHCO3 or HCl. Control groups included normothermic CPB without anoxic and normothermic CPB, anoxia, and equimolar NaCl infusion. NRL was quantified by planimetry of photographs of cut sections of brain. These results confirm that NRL is abated by hypothermia and suggest that (1) NRL is a function of anoxia and not arrested circulation since perfusion with N2 at 37 degrees C does not protect the brain (i.e., NRL is not solely related to "critical reopening pressure") and (2) NRL is in part a function of extracellular pH.

Characterization of two new enzymatic activities of the rat ventral prostate: 5 alpha-androstane-3 beta, 17 beta-diol 6 alpha-hydroxylase and 5 alpha-androstane-3 beta, 17 beta-diol 7 alpha-hydroxylase.

This study has characterized two new enzymatic hydroxylase activities specific for 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) in the rat ventral prostate: 5 alpha-androstane-3 beta, 17 beta-diol 6 alpha-hydroxylase (6 alpha-hydroxylase) and 5 alpha-androstane-3 beta, 17 beta-diol 7 alpha-hydroxylase (7 alpha-hydroxylase). Both of these irreversible hydroxylase activities require NADPH and are localized in the microsomal fraction of the prostate. The apparent Km for 3 beta-diol is 2.5 microM for both the 6 alpha- and 7 alpha-hydroxylase activities. The apparent Km for NADPH is 7.6 microM for the 6 alpha-hydroxylase and 7.0 microM for the 7 alpha-hydroxylase. The pH optimum for both activities is 7.4. Several steroid inhibitors of these hydroxylase activities in vitro were identified including cholesterol, progesterone, and estradiol. Estradiol was found in vitro to be a noncompetitive inhibitor (Ki = 5 microM). Injection of estradiol into intact male rats, simultaneously receiving exogenous testosterone, also produced a significant lowering of the 6 alpha-plus 7 alpha-hydroxylase activities. Both the 6 alpha- and 7 alpha-hydroxylase were found to be androgen sensitive. Following castration there is a rapid decrease in both activities.

Lymphocyte traffic within the bone marrow and selective retention of alloreactive cells.

Earlier studies showed that large numbers of isotopically labelled thoracic duct lymphocytes (TDLs) enter the bone marrow (BM) within hours of injection but depart equally as rapidly by 12 to 24 hr. The significance of this rapid flux was investigated further. Early (1/2 to 2 hr) after the i.v. injection of TDLs, BM was shown to contain T cells capable of initiating a graft-versus-host (GVH) reaction in F1 hybrids and in other experiments memory cells against human serum albumin (HSA). Both GVH and memory cell activity had markedly declined in the BM by 12 hr. In contrast to the rapid departure of TDLs from syngeneic BM, F1 hybrid BM retained parental lymphocytes with GVH activity for alloantigens of the opposite parent. F1 hybrid BM under these circumstances supported the transformation and proliferation of lymphocytes activated in situ by alloantigens. These selectively retained T cells also reacted to third-party alloantigens. In addition, TDLs with memory for HSA were retained in the BM of F1 hybrids. The BM is a site in which alloreactive immune responses may be initiated or sustained.

Methods of study of the invasion of malignant C3H-mouse fibroblasts into embryonic chick heart in vitro.

Methods of study of tumour invasiveness in vitro were investigated using the interactions between malignant virally transformed C3H mouse fibroblasts (MO4) with fragments from embryonic chick heart. Invasion of MO4 cells into the heart tissue could be demonstrated in all three-dimensional cultures. On the contrary, seeding of MO4 cells on top of a monolayer of heart cells did not mimic invasion. Precultured heart fragments were more suitable for the study of the early phase of invasion than freshly cut ones because they presented healthy well-delineated borders. Aggregates of MO4 cells proved more suitable than suspensions or monolayer fragments because they were amenable to quantitation, were not traumatized or treated with enzymes during harvest. They allowed precise control of the initial site of contact with the heart tissue. The nature of the culture medium predominantly affected the growth of the MO4 population. This factor influenced the pattern of invasion quantitatively, but did not alter the invasive capacity of the MO4 cells itself. In cultures on adhesive substrates the interaction between the MO4 cells and the heart tissue was complicated by the fact that both also interacted with the aritficial substrate. Shaker cultures appeared better than comparable static cultures because they allowed better aeration and so delayed central necrosis.

The effect of duodenal acidification on plasma secretin and gastrin and pancreatic bicarbonate secretion in man.

Plasma secretin, plasma gastrin and pancreatic bicarbonate output were measured in three healthy youths before and after a 10 min period of duodenal infusion of 50, 75 and 100 ml 100 mmol/1 HCl. Plasma secretin rose to a shortlived peak within 10 min, whereas plasma gastrin fell gradually to values significantly below the basal level 60 min after the start of duodenal acidification. Pancreatic bicarbonate output showed a more sustained increase following duodenal acidification. Significant positive correlations were obtained between plasma secretin and infused dose of HCl, between pancreatic bicarbonate output and infused dose of HCl and between plasma secretin and pancreatic bicarbonate output. The calculated maximal pancreatic bicarbonate output (Vmax) of 30.6 mEq/h and the calculated dose of secretin to elicit half maximal pancreatic bicarbonate output (S50) of 0.2 CU/kg-h following duodenal acidification were comparable to that seen after intravenous infusion of secretin. No significant correlation was found between plasma secretin and plasma gastrin. It is suggested that the pancreatic stimulation subsequent to duodenal acidification is mainly effected by release of secretin, and that the fall in plasma gastrin may be caused by a HCl-induced inhibition of gastrin release from the duodenum.

Histopathologic sequence of events in adult mice undergoing lethal graft-versus-host reaction developed across H-2 and/or non-H-2 histocompatibility barriers.

The sequence of histologic events in graft-versus-host reaction (GVHR) caused by major and/or minor histoincompatibilities was studied. It was discovered that GVHR may manifest itself in the form of two distinct multiphasic disease entities, depending on whether the donor cells are incompatible with the host for both major and minor histocompatibility antigens ("major GVHR") or for minor histocompatibility antigens alone ("minor GVHR"). The acute or major GVHR has four phases: 1) a transient phase of aplasia, 2) a repopulation phase, 3) a proliferative phase involving lymphoid, presumably immunocompetent, cells, and 4) a phase of acute organ rejection (terminal). The chronic or minor GVHR is characterized by six phases, namely: 1) a transient phase of aplasia, 2) a repopulation phase, 3) a phase of proliferation and tissue infiltration by lymphoid, presumably immunocompetent cells, 4) a phase of major immunologic injuries, 5) a phase of repair, and 6)a terminal phase with advanced sclerosis and proliferative glomerulonephritis. In acute or major GVHR the disease was manifested by the tissue reactions characteristic of acute organ rejection. Lesions were seen in the kidney, liver, bone marrow, lymph nodes, spleen, thymus, intestine, and skin. In the chronic or minor GVHR, tissue injuries were more widespread, affecting the collagen, vessel walls, adipose tissue, renal glomeruli, heart muscle, fascias of skeletal muscles, lymph nodes, spleen, thymus, bone marrow, intestine, skin, esophageal mucosa, and urinary tract. A pronounced plasma cell proliferation was a striking feature in the minor GVHR. Its evolution coincided with advanced thymic epithelial atrophy. It is suggested that the destruction of thymic epithelium resulted in depletion of suppressor T cells and, consequently, in an unopposed proliferation of plasma cells.

Influence of volume dilution, lactate, phosphate, and calcium on mitochondrial functions.

Oxidative phosphorylation of isolated canine myocardial mitochondria has been evaluated after exposure to different concentrations of phosphate (5--50 mM), lactate ion in excess (5--40 mM, pH 7.4), calcium (50--270 nmol/mg protein), to lactic acidosis (pH 6.3), and to mitochondrial protein dilution (in vitro volume expansion) for 10 min to 8 h. The influence of phosphate and lactate ion addition, lactic acidosis, and in vitro volume expansion on mitochondrial function were studied in the isolation medium (0.18 M KCl, 0.5% BSA (bovine serum albumin), with or without Tris-EDTA, pH 7.4) prior to evaluation of mitochondrial function in the assay medium (0.25 M sucrose, 10 mM Tris-HCl, and 10 mM inorganic phosphate, pH 7.4). The effect of calcium addition was assessed in the assay medium. The results of these studies demonstrate that each of these interventions detrimentally alters mitochondrial oxidative phosphorylative ability. The most severe mitochondrial functional impairment resulted from phosphate or calcium addition. The detrimental effect of phosphate and in vitro volume expansion was partially corrected by the addition of cytochrome c.