[Effect of estrogens on interaction of calcium ions with mitochondrial membranes]. It has been shown that steroid hormones do not affect the fluorescence parameters in solutions of the complex NADH-Ca. Addition of discoupling concentrations of Ca2+ to the liver mitochondria induced the elimination of pyridine nucleotides. The hormones studied inhibit the oxidation of NAD-dependent substrates, thus suppressing an active transport of Ca2+. An analysis of the effect of steroid hormones on Ca2+ ions transport under succinate oxidation suggests that these hormones affect the polarity of hydrophobic regions of the mitochondria membranes. It is suggested that steroid hormones can perform their regulatory functions by influencing the transport of Ca2+ ions in the living cell.


[Quaternary structure of a cytoplasmic protein with ribonuclease activity from wheate seedling leaves. Kinetic manifestation of its "native", dissociated and reassociated forms]. Previous  data on oligomeric nature and values of molecular masses of "native" and dissociated forms of cytoplasmic protein with ribonulcease activity from wheat seedling leaves (Bezenchukskaya 98) are confirmed by means of polyacrylamide gel disc electrophoresis. Possible relation of the "native" enzyme form with 3':5'-cAMP is demonstrated by paper chromatography and the enzyme reassociation by the nucleotide. Study of kinetic manifestation of "native", dissociated and reassociated enzyme forms has revealed that the enzyme in the "normal" state has probably at least two active sites, "negative" homotropic cooperative interaction being establish-d between them at the definite degree of substrate saturation. The cooperativity disappears under dissociation, and it is discovered again under reassociation.


[Effect of liver chromatin DNAse on closed circular DNA of PM-2 phage and simian virus 40]. Comparative effect of the DNAse from rat liver chromatin and Neurospora crassa endonuclease S1 on closed circular superhelical DNA of PM-2 phage and Simian Virus 40 is studied. It is shown that both of them--the DNAse from chromatin proteins and endonuclease S1--are specific to single-stranded regions in DNA molecular. It is suggested that chromatin protein DNAse participates in reparation processes.


[Investigation of lipid-exchange lipoproteins from rat liver and hepatoma 27]. A phospholipid exchange lipoprotein from the postmicrosomal supernatant of rat hepatoma 27, which stimulated in vitro the exchange of sphingomyelin between mitochondria and microsomes, was found. Sphingomyelin is incorporated into the mitochondria under incubation of this complex with rat liver mitochondria (in which sphingomyelin is absent) an microsomes. Under the same conditions the phospholipid exchange lipoproteins of rat liver do not transfer sphingomyelin form microsomes to mitochrondria.


[A mathematical model of the pyruvate oxidation in liver mitochondria. 1. Regulation of the Krebs cycle by adenine and pyridine nucleotides]. A mathematical model is proposed to describe the behavior of the pyruvate metabolic reactions, Krebs cycle and oxidative phosphorylation over a wide range of changes in the pyruvate influx rate and the activities of ATPase and NADH-reoxidating dehydrogenase. The role of adenine and pyridine nucleotides in various allosteric regulations of the Krebs cycle enzymes is discussed. The accumulation of ATP and NADH has been shown to proceed in definite succession, which makes the allosteric regulation of the Krebs cycle enzymes successive too. First "works" the inhibition by ATP, then by NADH. It has been shown that the properties of the model are in qualitative agreement with the experimental data (Garber A., Hanson R. [1]) on pyruvate oxidation by mitochondria from guinea pig liver, when allosteric regulation of isocitrate dehydrogenase by adenine nucleotides is taken into account.


[Isolation and some properties of protein kinase from pigeon breast muscle]. Protein kinase was isolated from pigeon breast muscle. The preparation obtained was chromatographically homogeneous. The apparent Km varlue for histone H1 and ATP were 3,5-10(-5) M and 1,6-10(-5) M respectively. The purified enzyme displays high specificity for the lysine-rich histones (H1, H2b, H2a). The protein kinase activity is stimulated, 1,6-fold by cyclic AMP.


[The actions of low-molecular fragments of substrate and their analogs on the activity of isoenzymes of phospholipase C from Clostridium perfringens]. The interaction of the lecithin molecule fragments and their analogues with phospholipase C Cl. perfringens was studied by gel-diffusion in agarose-lecithin gels. It was found intense inhibition of phospholipase C activity in the presence of cathionic compounds; this phenomenon shows the existence of anionic centre in the active site of enzyme. The esteric centre is probably hydrophobic nature and is not capable to bind the negatively charged groups. However, phosphoserine, phosphothreonine, gamma-aminobutyric, aspartic and glutamic acids can interact with an additional cathionic centre, whose location in phospholipase C differs from that in pancreatic phospholipase A2.


[The role of 3',5'-AMP in regulation of activity of histidase from rat liver and skin]. A dependence of activity of histidase from rat liver and skin on the agents affecting the activity of the adenylate cyclase systeme was studied in vitro. Under conditions optimal for the activity of liver phosphorylase protein kinase the skin extract histidase was activated 2-3-fold. This is indicative of a possibility of regulation of the skin histidase activity via the adenylate cyclase system by modification of enzyme by phosphorylation-dephosphorylation, which is performed by 3':5'-AMP-dependent protein kinase. Theophylline at concentrations of 10(-4) M and 10(-3) M activates partially purified histidase (both liver and skin forms), probably in the course of direct interaction with the enzyme.


[Effect of some organosilicon compounds (silatranes) on the biosynthesis of collagen in cartilagenous tissue of chick embryo]. Effect of some organosilicon compounds from the class of silatranes on the biosynthesis of collagen in cartilagenous tissue of chick embryos in vitro was studied. The criteria for assessing the intensity of collagen biosynthesis was the formation of peptide bond 14C-hydroxyproline from 14C-proline of incubation medium. All the compounds studied (methylsilatrane, ethoxysilatrane and chloromethylsilatrane) stimulated total protein biosynthesis and collagen biosynthesis in cartilagenous tissue of chick embryos. The most pronounced stimulation of the biosynthesis of collagen was observed at 3-10(-3) M concentration of silatranes within 180 min. of incubation (14C-proline was added to the incubation medium 60 min after the beginning of the incubation). The activity of partly purified collagen prolyl-hydroxylase in the presence of silatranes was also studied.


Motoneuron excitability in psychiatric patients. The excitability at the alpha-motoneuron pool was studied by measuring the spinal monosynaptic reflex responses of soleus muscle to paired stimuli in psychiatric patients and controls. Patients showed significant alterations in the recovery of excitability when compared with normal volunteers. Specifically, the value for the peak of "secondary facilitation" was reduced in 5 of 8 chronic schizophrenic patients, while the following trough in the recovery cycle was reduced in 9 of 18 acutely schizophrenic patients. Drug effects were studied in 10 patients. Phenothiazine antipsychotic medication tended to enhance recovery of excitability. Patients of all types showed alterations of excitability. The altered excitability may relfect one aspect of a widespread abnormality of CNS function or may be a specific response to a supraspinal defect in neurophysiological activity in psychotic patients.


Thrombin decreases platelet cyclic AMP in the absence of prostaglandin synthesis. Cyclic AMP first decreased in wahed platelets after the addition of 0.1 IU thrombin/ml and then increased progressively. Platelets pre-incubated with acetylsalicylic acid showed only a decrease of cyclic AMP upon addition of thrombin. Furthermore, two patients  with defective prostaglandin synthesis had always reduced cyclic AMP whether their platelets were pre-incubated with acetylsalicylic acid or not. Thus, if prostaglandin synthesis is inhibited the level of cyclic AMP decreased and did not increase with time.


Phosphorylation of rhodopsin as a possible mechanism of adaptation. Light-induced phosphorylation of rhodopsin has been extensively studied by a number of investigators from a biochemical point of view. However, little is known about the physiological function of this reaction. The slow rates measured for phosphorylation and dephosphorylation suggest that it may be involved in visual adaptation rather than in excitation. This paper presents biochemical data obtained from phosphorylation experiments in isolated photoreceptor membranes as well as in the physiological system of whole retinas and living animals. An attempt is made to compare the phosphorylation reaction with visual adaptation hypotheses taken from the electrophysiological literature. Finally, effects of cyclic nucleotide metabolism on the sensitivity of photoreceptors are presented and discussed.


The natural history of aminopterin-induced embryopathy. A boy whose mother ingested aminopterin to induce abortion and who has the characteristic anomalies caused by fetal exposure to this drug, has been followed for 12.75 yr. Despite short stature, his growth velocity is normal, and both normal endocrine studies and a radiologic bone age, retarded consistently between 2.25 and 3.0 yr, allow a predicted adult height of over 150 cm. Intelligence testing has shown a rise in full scale IQ on the Wechsler Intelligence Scale from 64 to 80 with performance increasing from 70 to 92. Psychologic assessment suggests potential for self-support in adult years.


[Changes in the electric activity of the cerebral cortex in poisoning caused by Cl. perfringens toxin type A]. The electrical activity of the cerebral cortex was recorded in cats under mild nembutal anesthesia (15-20 mg/kg of body weight) during the development of Cl. perfringens, type A, toxin poisoning (the toxin was injected intramuscularly in a dose of 100 MLD per kg of body weight). Two phases of the changes in the electrical activity of the cerebral cortex were noted. The first phase was attended by the desynchronization of the electrical activity, persistence of the induced potentials and of the reaction of the rhythm reconstruction to the rhythmic light stimulus. No desynchronization occurred under conditions of preliminary section of the midbrain (on the mesencephalic preparation), this indicating the involvement of the reticular formation into the pathological process and pointing to its role in the desynchronization effect. A profound depression of the electrical activity of the brain, depression of induced potentials and disturbance of the reaction of the rhythm reconstruction occurred during the second phase.


[Study of the relationship between the glucocorticoid function of the adrenal cortex and blood lipoproteins]. Single administration of hydrocortisone to rats led to elevation in the blood serum of lipoproteins of very low density; and repeated administration - of lipoproteins of very low density, low density and of high density. Lipoproteins with d less than 1.063 g/ml produced an inhibitory action on the production of steroid hormones by the adrenal glands of rats in vitro.


[Effect of ethyl ether of p-chlorphenoxyisobutyric acid on the glucocorticoid function of the adrenal cortex of guinea pigs]. In administration to quinea pigs of ethyl-p-chlorophenoxyisobutyrate daily in a dose of 0.2 g/kg per os for 15-26 days decreased the 17-OHCS concentration in the peripheral blood plasma. A fall of the urinary excretion of individual 17-OHCS occurred chiefly at the expense of free unchanged cortisol, tetrahydrocortisol and tetrahydro-11-desoxycortisol. The zona fasciculate displays the abundance of the sudanophilic material. A test with ACTH demonstrated that the present functional reserves of the adrenal cortex failed to change under the effect of the preparation. The content of NEFA decreased in the blood serum; as to cholesterol level - it reamined unchanged. Possible mechanisms of the action of the preparation and prospects of its use for the treatment of hypercorticism are discussed.


[Cytochrome content in the liver of rabbits in chronic carbon tetrachloride poisoning after perenteral administration of exogenous cytochrome C]. Administration of exogenous cytochrome C to rats poisoned with CCl4 prevented reduction of the cytochrome C content in the homogenates, and of cytochromes a+a3, b and c+c1 in the hepatic mitochondria; it also promoted the normalization of the histostructure of the organ.


[Diurnal rhythm of cell proliferation at the late stages of precancerous changes in the liver induced by ortho-aminoazo-toluene]. Orthoaminoazotoluol was administered to mice for a period of nine months. Circadian rhythms of mitotic activity and the number of the DNA-synthesizing cells were determined by autoradiography (with the use of 3H-thymidine) at the second (adenomatous nodes) and the third (primary hepatoma) stages of carcinogenesis in the developing tumours and the surrounding liver parenchyma. The listed structures proved to have a single-peak rhythm of mitotic activity with the maximum of mitoses at 4-7 a. m.; the labeled nuclei circadian rhythm was double-peaked with the maximum at 7 p. m. and 4 a. m. The mean 24-hour values of both indices at the second and third stages of hepatocarcinogenesis were much greater than in the surrounding non-tumour tissue of the liver.


[Effect of heat and cold on the amylolytic and invertase activity of the small intestine of growing rats]. Acute experiments were conducted on ratlings; it was shown that the action of heat (40-41 degrees C), cold (5-6 degrees C), and ACTH injections (4 units per 100 g of body weight) during the first week after birth led to a sharp reduction of the total (alpha-gamma-conditioned) amylolytic and invertase activity of the homogenates and of the portions of the small intestine (turned inside out) of the growing rats. This depressed condition of the intestinal function was revealed directly after the arrest of the action of the unfavourable factors (7-day rats) and persisted for the subsequent two weeks of the animal life.


Effects of dibutyryl cyclic adenosine 3',5'-monophosphate and theophylline on the bullfrog sympathetic ganglion cells. 1 Effects of dibutyryl cyclic adenosine 3',5'-monophosphate (dibutyryl cyclic AMP) and theophylline on bullfrog sympathetic ganglion cells were examined in order to test the hypothesis that cyclic AMP is essential for the generation of slow inhibitory postsynaptic potentials (i.p.s.ps) in these cells. 2 In the absence or presence of theophylline, dibutyryl cyclic AMP did not hyperpolarize but rather tended to depolarize ganglia that were hyperpolarized by adrenaline. 3 Theophylline augmented neither the P-potential (slow i.p.s.p.) nor adrenaline-induced hyperpolarization. 4 Thus, cyclic AMP does not seem to be essential for the generation of the slow i.p.s.p., at least in amphibian sympathetic ganglion cells.


Gas gangrene and related infection: classification, clinical features and aetiology, management and mortality. A report of 88 cases. The clinical features of gas gangrene and related infection seen in 88 patients over a 10-year period are described. It is suggested that clostridial infection could be simply classified as either 'gas-forming' or 'non-gas-forming'. The gas-forming group represents the more severe form of infection. Non-clostridial gas gangrene may present in a variety of forms. The anaerobic streptococcus was the organism most frequently responsible, but these cases were indistinguishable from clostridial infection on clinical grounds. The treatment of gas gangrene in this series of patients is reported. Emphasis is laid on the importance of adequate prophylaxis with penicillin in patients at risk. The value of antibiotics in established infection remains equivocal. The evidence supporting the value of hyperbaric oxygen therapy is assessed and an attempt made to quantify the response to this treatment. Benefit was apparent in only a proportion of patients. A favourable response indicated clostridial infection and guaranteed immediate survival. Extensive debridement or amputation is unnecessary in this group. No response following hyperbaric oxygen therapy indicated widespread mixed clostridial and non-clostridial infection, or infection due to organisms other than clostridia. Urgent and extensive debridement and amputation remain the predominant measures in this group.


Successful treatment of early cancer of the liver and portal hypertension in patients presenting with bleeding oesophageal varices. A small hepatoma was diagnosed by selective arteriography in 2 patients admitted for treatment of bleeding oesophageal varices. In both cases, hepatoma and variceal lesions were successfully treated. In the first case, left gastric vena caval shunt and local excision of the hepatoma were performed. In the second case, distal splenorenal shunt and local excision of the tumour were carried out. Although a combination of hepatoma and oesophageal varices may be an embarrassing problem, both lesions can be remedied provided that hepatoma is diagnosed at an early stage. It is stressed that arteriography is of value in assessment.


Dopamine-sensitive adenylate cyclase within laminae of the olfactory tubercle. There are three histological laminae within the rat olfactory tubercle: plexiform, pyramidal and polymorphic. We have assayed dopamine-sensitive adenylate cyclase in homogenates of frozen sections cut parallel to these laminae. Consecutive sections were cut of alternating thickness, 16 micron and 100 micron. The 16 micron sections were stained with toluidine blue to ascertain the depth and orientation of each section. The 100 micron sections were homogenized and assayed for dopamine-sensitive adenylate cyclase. Substantial levels of dopamine-sensitive adenylate cyclase were found within all three laminae. The results suggest that the enzyme occurs in cell processes, including dendrites, of the plexiform layer, and they are consistent with the localization of dopamine-sensitive adenylate cyclase in the pyramidal cells.


[3H]dihydroergocryptine binding in rat brain. [3H]dihydroergocryptine (DHE) appears to bind to alpha-adrenergic receptor sites in rabbit uterine membranes. We have characterized the binding of [3H]DHE to membranes prepared from rat cerebral cortex. alpha-Adrenergic agents were less potent and dopamine and serotonin, more potent, in displacing brain DHE binding than in uterus. Furthermore brain DHE binding sites demonstrated less stereospecificity for catecholamines than sites in uterus. Dopamine displaced DHE binding with about the same potency in cerebellar and cerebral cortical membranes, but was 10 times as potent in displacing DHE binding in the striatum. The binding of [3H]DHE in brain is complex and differs significantly from the rabbit uterus. There are two possible explanations for this discrepancy. [3H]DHE may bind a single site in brain with properties differing from known peripheral adrenergic receptors or DHE may bind to multiple sites in brain, sites which may or may not represent other neurotransmitter receptors.


Suppression of radioiodide accumulation by the thyroids and oocytes of Japanese quail with perrhenate. Japanese quail, 50% inhibition of the 3-h accumulation of 131I occurred with 2.0 micromol KReO4, 2.2 micromol KClO4 and 13 micromol KSCN for the growing oocytes, 1.7, 1.9 and 4.2 micromol for the thyroids of females and 0.20, 0.47 and 5.6 micromol for the thyroids of males. 2. KReO4 inhibition failed after 24 h in males and 36 h in females. 3. Maxima at 6 h in the 131I curves for the plasma, the livers and the testes of KReO4-treated birds were attributed to retention of 131I in the plasma.


[Sodium diethyldithiocarbamate is a stimulating agent of immunity]. A single dose of DTC was administered, in a dose-range from 0.6 mg/kg to 25 mg/kg, to mice immunized with 10(8) sheep red cells (SRC). All doses strongly enhanced plaque-forming spleen cell (PFC) responses, when given either 18 h before, simultaneously to, 6 h or 24 h after SRC immunization. However, the higher levels of immunostimulation were attained by DTC doses above 5 mg/kg. DTC-induced immunopotentiation was not accompanied by untoward effects, such as acute toxicity, splenomegalia or modifcations in counts of viable spleen lymphocytes.


[Demonstration by immunofluorescence of adenohyphysis corticotropin and melanotropin cells in Erythrocebus patas, Cercopithecus aethiops and Papio hamadryas]. Using antibodies against synthetic corticotropic hormones (1-24 ACTH and 17-39 ACTH), and melanotropic hormones (alpha-MSH and beta-MSH), it is possible to identify corticotropic and melanotropic cells in the adenohypophysis of three species of monkeys : Erythrocebus patas, Cercopithecus aethiops and Papio hamadryas. The corticotropic cells are numerous in the anterior lobe in both the adult and infant male and female monkeys of these three species. The intermediate lobe reacts with antibodies against ACTH and also with antibodies against the two MSH. In the anterior lobe, the corticotropic cells react also with anti-beta MSH antibody but not with the anti-alpha MSH antibody.


Diencephalic sites responsive to prostaglandin E2 facilitation of sexual receptivity in estrogen-primed ovariectomized rats. Crystalline prostaglandin E2 (PGE2) induced high levels of sexual receptivity when stereotaxically implanted into the antirior-bala hypothalamic and preoptic regions of estrogen-primed, ovariectomized rats. Anterior- as well as medial basal hypothalamic implants of PGE2 induced a significant elevation of rectal body temperature, but had no effect on open-field activity scores. The possibility that PGE2 exerts its facilitatory effects upon sexual receptivity via an LHRH mechanism is discussed.


Initial studies on the crystallinity of the mineral fraction and ash content of isolated human and bovine osteons differing in their degree of calcification. Several groups containing 10--15 isolated osteons differing in their degree of maturity were analysed. Samples were isolated from undecalcified human and bovine bone sections. The crystallinity coefficient, defined as the ratio of the number of radiation-induced paramagnetic defects in the crystalline lattice of hydroxyapatite to the total ash content, was calculated. The results were compared with measurements performed on fragments of total cortical bone, primary periosteal bone, and inner circumferential lamellar bone. The results show a higher crystallinity of fully calcified osteons as compared with that found at the initial stage of calcification. No differences in the ash content were observed between human osteons, from different stages of calcification evaluated morphologically and by X-ray absorption. These differences were evident when bovine osteons differing in their stage of calcification were compared. Human fully calcified osteons contain 60% ash and their crystallinity coefficient is 52.1. Human osteons at the initial stage of calcification contain 57% ash and their crystallinity coefficient is 40.6. The same parameters for fully calcified bovine osteons and for bovine osteons at the initial stage of calcification are 59% ash, 62.6 crystallinity coefficient and 46% ash, 43.0 crystallinity coefficient, respectively.


The effect of induced metabolic acidosis on vitamin D3 metabolism in rachitic chicks. The metabolism of vitamin D3 was studied in 3-week-old, vitamin D deficient chicks, fed since hatching with a diet containing 3% ammonium chloride, 1% calcium, and 0.7% phosphorus. When kidney homogenates were incubated in vitro with [3H]25-(OH)D3, the production of 1,25-(OH)2D3 was reduced by 40% in acidotic birds. During in vivo experiments, after injection of [3H]D3 (1220 pM/bird), the level of 1,25-(OH)2D3 was also reduced in blood plasma, intestine, and tibiae in acidotic chicks as compared with the controls. As a large increase in plasma phosphate was found during acidosis, these results are discussed in relation to the possible role of phosphorus in the control of 1,25-(OH)2D3 synthesis.


Neuromuscular block by circulating D-tubocurarine residue following uptake and distribution. Serum concentration of d-tubocurarine decreases rapidly after intravenous injection because of uptake and distribution. The circulating residue of an ED 50 dose of d-tubocurarine five minutes after injection will produce no block in a previously unexposed neuromuscular junction. To produce a 50 per cent block with the circulating d-tubocurarine residue in a previously unexposed neuromuscular junction requires an initial injection of 2.5 x ED 50 dose. Five minutes after a dose 5 to 6 times the ED 50, the plasma d-tubocurarine residue is sufficient to produce a total block.


Regulation of selectivity of CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase in rat liver microsomes towards different molecular species of 1,2-diacyl-sn-glycerols. The suitability of monoenoic, dienoic, tetraenoic, and hexaenoic molecular species of 1,2-diacyl-sn-glycerols as substrates for the CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied in rat liver microsomes. No statistically significant difference in the rates of phosphatidylcholine synthesis with the various diacylglycerols was found at 0.40 mM, although a moderate discrimination against hexaenoic species relative to monoenoic and dienoic species was observed at 0.25 mM. The addition of palmitoyl-CoA (7.5 micron) significantly enhanced cholinephosphotransferase activity when tetraenoic diacylglycerols were added at 0.25 or 0.40 mM. CDPethanolamine at 24.4 micron was found to inhibit the rates of phophatidylcholine biosynthesis by 54 and 39% with hexaenoic and monoenoic 1,2-diacyl-sn-glycerols, respectively, whereas no significant effects were observed in the case of dienoic and tetraenoic species. These latter findings may partially explain why 1-saturated 2-docosahexaenoyl diacylglycerols are used to a greater extent for phosphatidylethanolamine than for phosphatidylcholine synthesis in rat liver in vivo. The present results also suggest that the selectivity of the cholinephosphotransferase for certain molecular species of 1,2-diacyl-sn-glycerols is a function of diacylglycerol concentration and may be mediated under physiological conditions by substrates for enzymes which compete for common diacylglycerol precursors.


A new method for the fractionation of human plasma high density lipoprotein. We have devised a new method for the fractionation of human plasma high density lipoprotein (HDL). The HDL was chromatographed on DEAE-agarose columns using a continuous gradient of 0.06--0.15 M NaCl. The elution pattern obtained showed three phases, each with differing peptide composition. Examination of the three subfraction showed that each contained both apoA-I and apo A-II, but in different proportions. Subfraction 1 contained no apo C-II or C-III-1 and only a trace of apo C-III-2, subfraction 2 contained apo C-II and C-III-1 but no C-III-2, while subfraction 3 contained considerable apo C-III-2 with only traces of apo C-II or C-III-1.


The oxidation of ferrocytochrome c in nonbinding buffer. The apparent equilibrium constant and rate of oxidation was investigated for the reaction of cytochrome c with iron hexacyanide. It was found that if horse heart ferricytochrome c was exposed to ferricyanide (to oxidize traces of reduced protein) the cytochrome subsequently, even after extensive dialysis, had an apparent equilibrium constant different from that of electrodialyzed protein. The effect of ferricyanide ion apparently cannot be removed by ordinary dialysis. The ionic strength dependence of the apparent equilibrium constant and bimolecular oxidation rate constant was measured in the range 1--200 mM using Tris--cacodylate or potassium phosphate buffers at pH 7.0, and electrodialyzed horse heart cytochrome c. The oxidation reaction proceeded very rapidly. Extrapolated to zero ionic strength, kox (approximately 9 X 10(9) M-1 S-1) was about 7% of that calculated for a diffusion-limited reaction. Since the exposed heme edge occupies only the order of 3% of the surface area, electron transfer apparently results at nearly every collision with the active-site region. An effective charge of + 7.8 units was estimated for the oxidation reaction. The rate of oxidation of Pseudomonas aeruginosa c551 was much slower (kox at mu = 0 was the order of 6 X 10(3)), and was not consistent with diffusion-limited kinetics.


Studies on phosphoglyceromutase from chicken breast muscle: chemical modification of lysyl residues. To examine the role of lysyl residues in the activity of the enzyme, phosphoglyceromutase (PGM) from chicken breast muscle was chemically modified with trinitrobenzenesulfonate (TNBS) and pyridoxal 5'-phosphate. Trinitrophenylation resulted in modification of about nine lysines per mole of PGM with almost complete activity loss. Substrate (3-PGA) offered some protection to TNBS inactivation but cofactor (2,3-DPGA) did not. Reduction of the Schiff's base complex between pyridoxal 5'-phosphate and PGM gave irreversible inactivation of the enzyme. Inactivation was due to incorporation of 1 mol of pyridoxal 5'-phosphate per mole of PGM dimer through the epsilon-amino group of a lysyl residue. The effect of pyridoxal 5'-phosphate was specific for intact native enzyme and reaction with only one lysine per dimer was not due to induced conformational changes nor to dissociation of the reacted enzyme. 3-PGA prevented much of the reaction with pyridoxal 5'-phosphate with preservation of 70% of the activity and was a competitive inhibitor of the active site directed reagent. Cofactor (2,3-DPGA) acting noncompetitively, reduced the rate at which inactivation occurred with pyridoxal 5'-phosphate. Incorporation of 2,3-[32P]DPGA into PGM irreversibly inactivated with pyridoxal 5'-phosphate and NaBH4 was incomplete indicating hindrance to phosphorylation in the modified enzyme. The results indicate that a lysyl residue is located at or near the active site of PGM and that it is probably involved in the binding of 3-PGA.


Some biochemical effects of 4-deoxy-4-fluoro-D-glucose on Escherichia coli. The uptake of 4-deoxy-4-fluoro-D-glucose (4FG), without subsequent catabolism, by resting cells of Escherichia coli (ATCC 11775) is 0.06 mg/mg dry weight. In frozen-thawed cells of this organism, 4FG is a substrate for the phosphoenolpyruvate phosphotransferase system with a rate of phosphorylation twice that found for the isomeric 3-deoxy-3-fluoro-D-glucose. 4FG is not a carbon source for growth of this organism and it inhibits the extent of growth of cells in the presence of glucose. The inhibition of growth of E. coli K12 on lactose by 4FG is also observed and this is considered to be consistent with the fact that 4FG is an uncompetitive inhibitor of beta-galactosidase (EC 3.2.1.23) activity and that 4FG or 4-deoxy-4-fluoro-D-glucose-6 phosphate repress beta-galactosidase synthesis. These results support the view that catabolite repression may be produced by compounds which are not necessarily metabolised further than hexose-6-phosphates.


Estimation of time of divergence from phylogenetic studies. Recent studies with comparative data on base sequences of homologous DNA's or amino acid sequences of homologous proteins indicate that simultaneous estimation of phylogenetic structure and time of divergence is often cumbersome and time consuming. On the other hand, when the topology of an evolutionary tree is known, it is shown in this paper that the least squares theory may be applied to obtain simple estimates of the relative time lengths for each segment of the tree under the assumption of uniform random substitutions in each segment. The method is illustrated with amino acid sequence data on various globin molecules and cytochrome c. The evolutionary significance of some of the estimates is also discussed.


A comparison of six methods for the concentration of a porcine enterovirus. Single-step concentration of porcine enterovirus strain T80 by adsorption to the polyelectrolyte PE60 gave virus concentration factors of 35- to 88-fold in terms of plaque-forming units, with recovery rates of 22 to 75% of total virus present in the original virus suspension. Concentration by separation in an aqueous polymer two-phase system gave virus concentration factors of 56- to 105-fold and recovery rates of 37 to 107%. In the latter procedure, sodium dextran sulfate appeared to have no effect on plaque numbers, although plaques were sharper and clearer when this substance was incorporated in the overlay. The failure of sonication of virus concentrated by either procedure to increase plaque numbers indicated the absence of virus aggregates in the concentrates. T80 virus was not effectively concentrated by cobalt chloride or polyethylene glycol precipitation or by adsorption to either aluminium hydroxide or calcium hydrogen phosphate.


The effect of the antiherpesvirus drug 5-methoxymethyl-2'-deoxyuridine on the humoral immune response in vivo. 5-Methoxymethyl-2'-deoxyuridine (MMUdR), a drug with potent antiviral activity in vitro against Herpes simplex virus, was investigated for its immunosuppressive effects. Doses as high as 2000mg/kg given daily for 9 days were not immunosupporessive as judged by the fact that treated animals produced normal immune responses to sheep erythrocytes, Brucella bacteria, and Herpes simplex virus.


Elastosis and other stromal reactions in benign and malignant breast tissue: an ultrastructural study. The stroma and stromal reaction in normal breast, benign, and malignant breast tissue was studied by electron microscopy. Elastosis is the main stromal response in infiltrating duct and lobular carcinomas. Medullary carcinoma elicits no significant elastosis but intraductal carcinoma has significant local elastosis. Benign conditions such as fibroadenoma and sclerosing adenosis produced no significant elastosis. The high number of elastic fibers and the high microfibril:elastin ratio indicate that most of the elastic fibers are recently secreted, probably by fibroblasts and myofibroblasts. The factors stimulating the increased production of elastic fibers and the prognostic significance of elastosis remain unclear.


Chemotherapy of malignant major salivary gland neoplasms: a 25-year review of M. D. Anderson Hospital experience. From 1950 through 1975, 671 patients with malignant major salivary gland neoplasms were referred to M. D. Anderson Hospital and Tumor Institute. Thirty-six patients with advanced local or metastatic disease subsequently underwent 62 evaluable trials with a variety of chemotherapeutic agents, either alone or in combination. Six patients achieved a partial response, with a median duration of 3 months. Ten additional patients had stable disease for 2 or more months. Anthracyclines appeared to be the most effective agents in this study, with three partial responses of six evaluable trials. The longest partial response (10 months) occurred in a patient receiving combination chemotherapy plus BCG immunotherapy. Pulmonary metastases were most commonly responsive to chemotherapy. The median intervals from diagnosis to death or to last follow-up and from initiation of chemotherapy to death or to last follow-up were 30 months and 6 months, respectively. Further therapeutic trials are necessary before response rates to single chemotherapeutic agents or combinations can be accurately assessed. In view of the poor prognosis of patients with recurrent disease, postoperative adjuvant studies with chemoimmunotherapy in patients with a high risk of recurrence are planned.


Treatment of small cell anaplastic carcinoma of the lung with the oral solution of VP-16-213 (NSC 141540, 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-beta-D-glucopyranoside). The semisynthetic podophyllotoxin derivative VP-16-213 (NSC 141540) has been evaluated in a phase II study in patients with small cell anaplastic carcinoma of the lung. The drug was administered as an oral solution, the drinking ampoule, in doses of 100 mg twice a day for 4 days in 30 patients previously treated with intensive combination chemotherapy and for 5 days in 10 untreated patients. The courses were repeated every third week with dose modifications according to individual tolerance. All patients had measurable disease and objective responses were obtained in 20 patients (50%), 15 previously treated (50%) and 5 untreated patients (50%). The median time for response after the start of treatment was 15 days (range 6-42) and the median duration of response was 56 days (range 16-147). Dose-limiting toxicity was principally hematologic, consisting of leukopenia, but gastrointestinal toxicity and alopecia were also observed. The study demonstrated that VP-16-213 administered as an oral solution is highly effective against small cell anaplastic carcinoma of the lung without clinical cross-resistance to CCNU, cyclophosphamide, methotrexate, or vincristine.


Soluble membrane antigens of brain tumors. I. Controlled testing for cell-mediated immune responses in a long surviving glioblastoma multiforme patient. A patient with glioblastoma multiforme survived 18 years after diagnosis and underwent 20 operations for extracranial metastasis. An immunologic survey of the patient was made over a 1-year-period using in vitro tests of lymphocyte responsiveness and skin tests with control and tumor antigens isolated from autologous and allogenic brain cell membranes. Two tissue-associated soluble cell membrane antigens also present in normal white matter, and two tumor-associated antigens (TAA) produce cell-mediated immune responses in patients with brain tumors. One of these tumor-associated antigens predominates in meningioma cells. In addition some low molecular weight components appeared, which seemed to be unique for the glioblastoma cells from the long-surviving patient.


Viral oncolysate in the management of malignant melanoma. I. Preparation of the oncolysate and measurement of immunologic responses. Primary explants of human malignant melanoma were utilized in the preparation of oncolysates by Newcastle disease virus. The concentrated lysate, administered parenterally, was employed in an effort to augment antitumor immunologic responses in patients with metastatic melanoma. Observed cellular changes suggested a benefit, but humoral antibody measurements were not impressive.


Viral oncolysate in the management of malignant melanoma. II. Clinical studies. Melanoma cell oncolysate, prepared with Newcastle disease virus, was administered as an immunostimulant to 13 patients with metastatic melanoma. The oncolysate was well tolerated. Six treated patients evidenced a decrease in the size of skin nodules or diseased lymph nodes. Visceral lesions were not favorably influenced to any marked degree. One case of fulminating disease showed a change to slow progression and survived a year longer than was otherwise expected. Another patient, whose melanoma could not be controlled by surgery or chemotherapy, has been in complete remission for 2 years. It appears that viral oncolysate might be particularly helpful to patients with early disease.


A scanning electron microscopic study of diffuse mesothelioma and some lung carcinomas. Seventeen cancers from lung and pleura were studied with scanning, transmission electron, and light microscopy (SEM, TEM and LM). Diffuse mesothelioma mimics bronchioloalveolar carcinoma at LM but shaggy microvilli were found on the cellular surface of the former, and short sprouts densely packed or loosely scattered, on that of the latter. Neolumen formation was found in both. Oat cell carcinoma had a smooth surface with occasional tiny projections and minute surface depressions. The cellular projections of squamous cell carcinoma were quite irregular. Differentiation between diffuse mesothelioma and bronchioloalveolar carcinoma appears feasible with SEM in tissue appropriately fixed either with formaldehyde or glutaraldehyde. The role SEM can play in diagnostic pathology is yet to be explored.


Ultrastructure of atypical fibroxanthoma. Electron microscopic examination of an atypical fibroxanthoma (AFX) confirmed the fibrohistiocytic nature of this lesion. Ultrastructural evidence suggested a transition from fibroblasts to large giant cells with intermediate forms exhibiting features of both. A comparison of AFX ultrastructural features to those of other similar neoplasms suggested a possible relationship to malignant fibrous histiocytoma. As diagnosed by light microscopy, AFX probably represents a spectrum of neoplasms; this concept is discussed. It is urged, when possible, that electron microscopy be performed on lesions diagnosed as atypical fibroxanthoma since ultrastructure studies represent the most valid basis on which a correct diagnosis can be made.


Ectopic ACTH, prostatic oat cell carcinoma, and marked hypernatremia. Locally recurrent, poorly differentiated carcinoma of the prostate was associated with hypokalemic alkalosis, marked hypernatremia, diabetes mellitus of recent onset, and hyperosmolar syndrome. These findings, with mild hypertension, in the absence of clinical features of Cushing's syndrome, suggested an ectopic ACTH syndrome. Plasma ACTH and cortisol levels were markedly elevated, and failed to suppress in response to either low or high-dose dexamethazone administration. The patient's condition deteriorated rapidly. Autopsy findings included carcinoma extensively infiltrating the prostate with extension to the urinary bladder, and metastases confined to the pelvic nodes and soft tissues. The adrenal glands weighed 23 g and showed diffuse hyperplasia. Extract of the prostatic tumor was analyzed for ACTH and showed approximately 40 times normal plasma levels (or about 4,010 pg/g of tissue); ultrastructural features showed secretory granules consistent with ACTH content of the tumor cells. Such cells were positive when stained for ACTH by peroxidase-tagged immunochemical methods. The case fulfills all established criteria for relating excess corticosteroid production and nonpituitary tumors.


Cystic partially differentiated nephroblastoma: a clinicopathologic entity in the spectrum of infantile renal neoplasia. Three cases of cystic partially differentiated nephroblastoma (CPDN) are presented and ten cases from literature are reviewed. CPDN has been designated by various terms; it is a cystic encapsulated tumor occurring before 2 years of age. Cysts are lined by epithelium; septa of the cysts show a mixture of partially differentiated and undifferentiated metanephrogenic blastema. This histologic feature distinguishes CPDN from multilocular cyst of kidney. In seven cases simple nephrectomy, and in remaining cases nephrectomy with radiation and/or chemotherapy, had been the treatment. The disease-free interval ranged from 5 to 72 months, without reports of recurrence or metastasis. CPDN appears to take a benign course and simple nephrectomy seems to be the treatemtn of choice. However, in view of the possibility of recurrence as shown in rare instances by congenital mesoblastic nephroma, another less aggressive lesion in the spectrum of infantile renal neoplasia, regular follow up is recommended.


Histologic types of bronchogenic cancer in relation to different conditions of radiation exposure. The rish of lung cancer among the uranium miner study group was found to be not only connected with the increase in frequency of different histologic types of lung cancer, but mainly with the increase of small cell undifferentiated and epidermoid type frequencies. It appears that the frequency of these two major histologic types may be influenced by the level of cumulated radiation exposure and by the time course of cumulation of exposure in a different way. These findings, in agreement wit recent data of Archer et al., eliminate the former assumption that radiation can induce an elevated frequency of only one histologic type of lung cancer.


Hepatocellular carcinoma in a woman on long-term oral contraceptives. A 32-year-old woman on oral contraceptives for 12 years developed a hepatocellular carcinoma, which resulted in death 15 months after initial diagnosis. The possible association between this malignant hepatic tumor and the long-term use of oral contraceptives is reviewed.


Mixed germ cell-sex cord stroma tumor of the ovary associated with isosexual precocious puberty in a normal girl. A case of mixed germ cell-sex cord stroma tumor occurring in a normal 8-year-old girl with 46XX karyotype is reported. The child presented with evidence of isosexual precocious puberty 3 years prior to the excision of the tumor. Although the tumor was known to be present at least 2 years prior to its excision and weighed more than 1 kg, there was no evidence of involvement of the adjacent organs or metastases at the time of excision. The contralateral ovary was normal. The endocrine activity abated following the excision of the tumor and the patient is well and free of disease 2 years after the operation. The literature concerning this entity is discussed and reviewed.


A lack of correlation between 'T' antigen and any particular human chromosome in hybrids made between SV40 transformed human fibroblasts and mouse LMTK cells. Lines of hybrid cells were established by fusion of mouse LMTK- cells with SV40 transformed human fibroblasts. The lines were examined for the presence of SV40 'T' antigen and the human chromosomes they carried identified by Giemsa banding. No significant correlation could be found between any particular human chromosome and the presence of SV40 virus.


Correlation of bacterial mutagenicity and hamster cell transformation with tumorigenicity induced by 2,4-toluenediamine. In the presence of rat liver microsome enzymes, 2,4-toluenediamine (TDA) was mutagenic for several tester strains of Salmonella typhimurium. TDA induced morphological transformation in an in vitro carcinogenesis system using secondary culture target cells prepared from cryopreserved primary Syrian hamster embryo cells. These results now correlate bacterial mutagenicity and in vitro morphological transformation with the reported tumorigenicity of this compound.


C-type virus particles in the transplantable mouse bladder tumour FCB grown in vitro. Electron microscopic studies were made on an in vitro cultured cell line obtained from the FCB transplantable murine bladder tumour. The cells exhibited a loss of differentiation compared with solid tumour and the presence of C-type virus particles, many in the process of budding. The possible relation of this virus to murine bladder carcinogenesis is discussed.


Spontaneous and induced patterns of the Epstein-Barr virus (EBV) cycle in a new set of somatic cell hybrids. A somatic cell hybrid line and its subclones obtained by fusing two Burkitt lymphoma lines (Raji and Namalwa) were examined for the expression of EBV-specific antigens both spontaneously and after induction. The hybrids retained spontaneous early antigen (EA) production at the level characteristic of Raji. Similarly the more permissive Raji pattern dominated the induction of EA by IUDR treatment or P3HR-1 virus superinfection. These findings accord with our previous results on independently derived Raji/Namalwa hybrids. Virus capsid antigen was induced in the hybrids by P3HR-1 virus superinfection though at a lower level than in the Raji parental cell.


Effect of the radioprotective drugs MEA, DMSO, and WR-2721 on tumor control and skin tolerance in the rat. Mercaptoethylamine (MEA), dimethylsulfoxide (DMSO), and S-2-(3-aminopropylamino)ethylphosphorothioate (WR-2721) were tested for their protective effects against single and fractionated doses of 250 kv X-rays. Acute and late skin reactions and control of the BA-1112 rhabdomyosarcoma were examined in protected and unprotected WAG/Rij rats. All drugs protected skin in both single and fractionated treatment regimens, with MEA giving the most protection and DMSO the least. DMSO and WR-2721 protected tumors against single doses of radiation, and all three drugs tested protected tumors against fractionated irradiation. As a result, only single-fraction treatments of MEA-protected animals showed therapeutically favorable differential protection.


Collagen metabolism in experimental cardiac hypertrophy in the rat and the effect of digitoxin treatment. It is generally agreed that cardiac hypertrophy is accompanied by the hyperplasia of connective tissue cells. In the present work, collagen metabolism was studied in the heart of nondigitalised and digitalised rats after the constriction of the aorta. The activity of prolyl hydroxylase was maximally increased 2 days after the operation. The incorporation of proline into collagen hydroxyproline increased without any increase in the specific radioactivity of free intracellular proline, the peak labelling of collagen occurring at 4 days. Although the treatment of the rats with digitoxin prevented the development of cardiac hypertrophy and an increase in collagen labelling, an increase in the activity of prolyl hydroxylase was observed. The intracellular free proline pool and its specific radioactivity were significantly lower in digitalised rats as compared with non-digitalised rats. The results indicate that constriction of the aorta is accompanied by an activation of connective tissue cells leading to increased synthesis of collagen. However, digitoxin treatment can prevent the increase in collagen labelling, possibly by inhibiting the amino acid transport, but it is unable to remove the stimulus for hypertrophy.


Analysis of VSV mutant with attenuated cytopathogenicity: mutation in viral function, P, for inhibition of protein synthesis. T1026, a ts mutant of VSV which is much less cytopathogenic than its parent, HR, and which can establish persistent infection under certain conditions, is a double mutant. In addition to its ts mutation in the virion RNA polymerase, T1026 has a second non-ts mutation in a viral function termed "P". This function is responsible for the inhibition of total protein synthesis in infected cells and acts chiefly at the level of translational initiation. In some cell systems, the inhibition of protein synthesis produced by P appears to be selective for cellular protein synthesis, whereas in other cell systems, both cellular and viral protein synthesis are inhibited. T1026 and its ts revertants are phenotypically P- -that is, cells infected with them show total protein synthesis rates equal to or greater than uninfected cells, while synthesizing viral proteins at the same or even greater rates than HR-infected cells. The P- mutation is correlated with failure to increase plaque size after 2-3 days of incubation. Since viral mutants obtained from persistently infected cultures in a variety of systems appear to be double mutants with a ts mutation in the virion RNA polymerase and a small plaque marker, we suggest that T1026 could represent a model for such mutants.


The early region of SV40 DNA may have more than one gene. The nucleotide sequence of 70 base pairs (bp) around 0.545 map units (Alu I C and B junction) of the genome from the single Eco RI cleavage site within SV40 DNA is presented. The mRNA transcribed from the early strand template from this stretch contains two copies of the nonsense triplet UAA in each of the three reading frames. Thus at least 25% of the early region of SV40 DNA does not code for the SV40 "A" protein, and the viral contribution to events in the lytic cycle and transformation may be more complex than is generally appreciated.


Initiation points for DNA replication in nontransformed and simian virus 40-transformed Chinese hamster lung cells. Randomly growing Chinese hamster lung cells were pulse-labeled with 3H-thymidine, and the replicating forks of individual DNA fibers were visualized by autoradiography. When grown in complete medium, wild-type SV40-transformed cells had more forks per unit length of DNA than nontransformed cells. In isoleucine-depleted medium, wild-type SV40-transformed cells had fewer forks per unit length than those few nontransformed cells (1-3% of the population) which continued DNA replication. Cells transformed by a tsA mutant of SV40 when grown at the permissive temperature had more forks per unit length in complete medium and fewer forks per unit length in depleted medium than nontransformed cells, but when grown at the restrictive temperature, the tsA-transformed cells behaved like nontransformed cells.


Transformation of chicken embryo retinal melanoblasts by a temperature-sensitive mutant of Rous sarcoma virus. Retinal melanoblasts were transformed by a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV). At the permissive temperature for transformation, the cells cease melanin synthesis, degrade their melanosomes and release much of their accumulated melanin into the medium. At the nonpermissive temperature, the cells assume an epithelioid morphology, actively synthesize melanin and become difficult to distinguish from normal uninfected control cultures. Both the transformed phenotype and the differentiated cell phenotype are temperature-dependent. Infected retinal melanoblasts which are incubated at the nonpermissive temperature and which accumulate a large amount of melanin are unable to transform in response to a temperature shift; instead, the cells degenerate and die. Retinal melanoblasts can be infected by subgroups A, B, C and D of RSV; however, their level of susceptibility to infection is about 1/40 compared to fibroblasts. Cultures infected by ts-RSV produce virus at both temperatures, suggesting that cell phenotype does not regulate virus synthesis.


Transformation of chondroblasts by Rous sarcoma virus and synthesis of the sulfated proteoglycan matrix. The presence of the extracellular matrix synthesized by chondroblasts provides a barrier to virus penetration. Chondroblasts can be infected and transformed following treatment with proteolytic enzymes. Using a temperature-sensitive transformation mutant of Rous sarcoma virus and rearing the cells at permissive temperature, we demonstrate that transformed chondroblasts stop synthesizing their cell-unique sulfated proteoglycan. If such transformed chondroblasts are shifted to nonpermissive temperature, the cells reinitiate the synthesis of their cell-unique sulfated proteoglycan.


The extensive homology between mRNA sequences of normal and SV40-transformed human fibroblasts. The poly(A)-containing messenger RNA of normal diploid fibroblast and SV40-transformed progeny cells are compared by cross-hybridizing cDNA. We find a high degree of homology between the mRNA from normal and transformed cells. Despite imperfections in the procedure, the technique permits the conclusion that, at most, 3% of the mRNA in the transformed cell has sequences not present in the normal parental cell. Furthermore, much of the difference appears to occur in low and intermediate complexity classes of mRNA molecules. Extension homology in the mRNA sequences of disparate cell lines may be a general phenomenon, and even HeLa cell mRNA is nearly identical to that of diploid human fibroblasts.


Dibutyryl cAMP-induced protein changes in differentiating mouse neuroblastoma cells. Proteins from mouse neuroblastoma cells treated with dibutyryl adenosine-3',5'-monophosphate (B2cAMP) were analyzed by high resolution, two-dimensional gel electrophoresis. Quantitative changes in proteins and charge modifications of proteins apparently induced B2cAMP were detected by isoelectric focusing. Some proteins appeared to be modified and one protein was increased 7- to 8-fold in cells treated with B2cAMP. Since neuroblastoma cells differentiate when treated with B2cAMP, understanding the protein changes induced by B2cAMP may help to understand cellular differentiation in neural tissue.


Synthesis and micellar formation of spin-labeled lysolecithin. A spin-labeled lysolecithin, 1-[12'-(N-oxyl-4",4"-dimethyloxazolidine)-stearoyl]-sn-glycero-3-phosphorylcholine, has been synthesized in which the spin is covalently attached to its fatty acyl chain. The electron spin resonance spectra of this lysolecithin is an aqueous solution generally showed sharp three resonance lines superposed on a broad resonance line. Investigation of changes in the signal intensity of these spectra against the concentration of lysolecithin led to the inference that the sharp lines are due to monomers of lysolecithin while the broad one to micelles. The critical micellar concentration was consistent with that evaluated from the spectral shift of a dye. In the electron spin resonance spectra obtained from spin-labeled lysolecithin solutions with various amounts of dimyristoyllecithin, the line width of broad signal arised from micellar spin-labeled lysolecithin broadened as increase of dimyristoyllecithin. The line-broadening thus observed was briefly discussed.


Effect of temperature and relative humidity on variola virus in crusts. The viability of variola virus in crusts under different conditions of temperature and relative humidity was studied for 16 weeks. At the ambient temperature of 25.8-26.4 degrees C and 85-90% relative humidity, the virus survived only 8 weeks but at lower temperatures and relative humidities the survival time was considerably prolonged.


[Cytotoxicity of human T-lymphocytes sensitized by Epstein-Barr virus. Role of HLA antigens at the surface of target cells infected by the virus]. Peripheral T-lymphocytes from patients with infectious mononucleosis are cytotoxic towards target cells from Epstein-Barr virus-genome carrying human lumphoblastoid lines. Thus, these T-cells appear to be sensitized to viral coded determinants. Daudi cells, that carry EBV-genome, but lack HLA antigens are resistant to specific cytolysis in this model. These results suggest direct HLA involvement in the target structure recognized by birus-sensitized cytolytic T-lymphocytes in human.


Formation and properties of lactate dehydrogenase inhibitors in NADH. We describe some characteristics of the mode of formation of inhibitors of lactate dehydrogenase from commercial NADH. Inhibitor formation is time- and concentration-dependent and also varies with the commercial source of NADH. At least two inhibitory components can form in concentrated NADH solutions. One of these can be separated from NADH by chromatography on either diethylaminoethyl-celluose or diethylaminoethyl-Sephadex; the second cannot. The NADH-associated inhibitor appeared to be present in each of the three commercial NADH preparations studied. The 260 nm/340 nm absorbance ratio was of no help in locating this inhibitor during chromatography.


Lactate dehydrogenase inhibitors in NADH preparations. The presence of a new lactate dehydrogenase inhibitor on the trailing edge of the NADH peak from chromatography on diethylaminoethyl-celluose [Loshon et al., Clin. Chem., this issue] was verified. It was resolved from the NADH by high-performance liquid chromatography on muBondapak C18. When the new inhibitor was present in a reaction mixture to the extent that, of the initial 260-nm absorbance, about 5% was contributed by the inhibitor, the rate of NADH oxidation by lactate dehydrogenase decreased by 65%. The inhibitor absorbs at 260 and 340 nm, and is different from the Strandjord-Clayson inhibitor [J. Lab. Clin. Med. 67, 144 (1966)] by both types of chromatography. Because this new inhibitor has ultraviolet properties similar to those of NADH and chromatographs with the NADH on DEAE-cellulose, the high-performance liquid chromatographic method must be used to ensure its absence in preparations of NADH used for clinical assay.


A fetal intestinal-type alkaline phosphatase in hepatocellular carcinoma tissue. We examined 19 hepatoma tissues for alkaline phosphatase isoenzyme and found that six have both the Kasahara isoenzyme and an alkaline phosphatase with a unique electrophoretic mobility, in addition to the liver-type enzyme. From two of six carcinoma tissues, the abnormal enzyme was partly purified and subjected to a detailed analysis, which clarified that the abnormal enzyme resembled a fetal intestinal alkaline phosphatase in most of its enzymic and immunologic properties and also in properties that reflect enzyme structure. This fetal intestinal-type alkaline phosphatase was not found in 24 specimens of normal liver from adults. The relevance of fetal intestinal-type alkaline phosphatase to Kasahara isoenzyme and adult intestinal alkaline phosphatase is discussed. The fetal and adult intestinal alkaline phosphatases differ in electrophoretic mobility, heat stability, and reactivity with concanavalin A. The adult-type enzyme has two components; only the electrophoretically slower, neuraminidase-resistant one is described here.


Improved direct determination of serum cholesterol in low-density lipoproteins with use of polycations. The previously described procedure of direct determination of LDL-cholesterol after selective extraction of VLDL and HDL with polycations and a polycation-exchange resin has been improved by using dodecoylated poly(ethyleneimine) instead of poly(ethyleneimine). The acyl derivative has a higher affinity for lipoproteins. Disc-electrophoretic patterns of lipoproteins separated by this procedure correspond to patterns of apolipoproteins in VLDL and HDL, a finding that corroborates the conclusion that the mechanism of the selective binding of certain lipoproteins to lipophilic water-soluble polycations is based on hydrophobic interaction between the polymer and lipids located at the surface of lipoprotein particles. The procedure offers a simple possibility for direct LDL-cholesterol and LDL-triglyceride determination and for the isolation of VLDL and HDL. The mechanism of the reaction has a biochemical correlate in the action of phospholipase A2 on phospholipids.


Lactate dehydrogenase isoenzymes linked to beta-lipoproteins and immunoglobulin A. A clinically asymptomatic individual had an abnormal electrophoretic pattern for lactate dehydrogenase, seven isoenzyme bands being present. This atypical pattern was a result of an interaction of lactate dehydrogenase with immunoglobulin A and beta-lipoprotein. Lactate dehydrogenase enzymes LD2 and LD3 were the isoenzymes responsible for the formation of the complexes.


Familial hyper-alpha-lipoproteinaemia. Further studies on serum lipoproteins and some serum enzymes. Some new data are given concerning a family whose members are characterized by high levels of alpha-(HDL)-cholesterol. Data from the new members of the family, not previously studied, confirm that the defect is not associated with any cardiac or neurological defect nor with xanthomata. Studies performed to assess the polypeptide composition of HDL of the affected members resulted in completely normal results. The post-heparin lipolytic activity (PHLA), the liver lipase and the lecithin-cholesterol acyltransferase (LCAT) activities also proved to be within the normal limits.


The composition of low (LDL) and very low (VLDL) density lipoprotein subfractions in type III hyperlipoproteinaemia: comparison with normal subjects. The lipid and protein composition of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) subfractions (Sf greater than 100, 60--100 and 20--60 VLDL and Sf 10.4--20, 5.7--12 and 3.5--6.5 LDL) in six subjects with type III hyperlipoproteinaemia (HLP) was compared to that of 12 normal subjects. In type III HLP all VLDL subfractions contained increased concentrations of cholesterol and triglycerides and were relatively enriched in cholesterol. VLDL of Sf 20--60 also contained and increased concentration of B-protein. The tetramethylurea (TMU) soluble apolipoproteins of the VLDL subfractions were separated by polyacrylamide disc gel electrophoresis. In the subjects with type III HLP the proportion of arginine rich protein (ARP) was increased in all subfractions. The concentrations of cholesterol and triglycerides were increased in the LDL subfraction of Sf 10.4--20 and cholesterol was decreased in LDL of Sf 5.7--12, but the ratios of cholesterol to triglycerides were not significantly different from those in the LDL subfractions of the normal subjects and the protein composition was also similar. These results provide further evidence that in type III HLP abnormalities are not confined to the stage of conversion of VLDL to LDL, but occur throughout the VLDL spectrum.


Measurement of the apoproteins of human serum lipoproteins by rocket immunoelectrophoresis. A method has been developed for assay of apolipoproteins A, B and C in human serum, using rocket immunoelectrophoresis. The technique is reasonably simple, sensitive and precise. The concentrations of apolipoproteins A, B and C in these classes of serum lipoproteins were measured in 116 healthy subjects. Concentrations of apoproteins within very low and low density lipoproteins increased with age but those of high density lipoproteins showed no consistent age trend.


The relationship between thyroid function and the renal response to parathyroid hormone. The effect of thyroid status on renal adenosine 3', 5'-cyclic monophosphate (cyclic AMP) metabolism was assessed by measuring the increase in plasma cyclic AMP concentration following the intravenous administration of parathyroid hormone (PTH) to five hypothyroid and six hyperthyroid patients and seven euthyroid control subjects. PTH caused a rapid, transient increase in the concentration of plasma cyclic AMP which was similar in magnitude and time course in the euthyroid and hyperthyroid groups, but reduced in magnitude in the hypothyroid patients. The magnitude of the plasma cyclic AMP response to PTH was positively related to the free thyroxine index. The results suggest that the development of hypothyroidism may impair the renal response to PTH by affecting the turnover of cyclic AMP in the renal cortex.


Diagnostic value of serum bile acids.

Diagnostic value of serum bile acids. With the development of simplified methods of bile acid analysis, a new era has drawned in the evaluation of hepatobiliary disease. 1. A total serum bile acid particularly in the postprandial periods is more sensitive than either BSP or ICG for the detection of minimal liver disease and will become a useful screening method. 2. The ratio of chenodeoxycholate to cholate in serum together with the total concentration can often distinguish hepatitis and cirrhosis from intrahepatic and extrahepatic cholestasis with normal liver cell parenchyma. However, in practice this is usually of less value than the total serum bile acid level. 3. Changes in serum bile acids throughout a 24 hour cycle reflect the enterohepatic circulation of bile acids and the capacity of the liver to transport them. These patterns are most useful in judging the severity of cholestasis and response to resin therapy. They also provide new insights into the pathophysiology of bile acid metabolism and excretion in different diseases of the liver.


Phagocytosis of immune complexes by polymorphonuclear leucocytes in patients with Felty's syndrome. The possible role of phagocytosis of circulating immune complexes by neutrophils in the production of the neutropenia of Felty's syndrome has been investigated. Normal neutrophils phagocytosed massive inclusions from the sera from twelve of fifteen patients with Felty's syndrome when incubated with these sera. Such inclusions were phagocytosed from only three of fifteen patients with seropositive RA who did not have Felty's syndrome. Normal neutrophils were more effective than patient neutrophils with regard to phagocytosis of inclusions from the patients' serum suggesting a defect in phagocytic function of Felty's neutrophils. The titre of granulocyte-reactive antinuclear antibodies did not appear to be related to the degree of neutropenia. The data suggest that phagocytosis of circulating immune complexes by neutrophils may interfere with the function of these cells in combating infection and also render them susceptible to removal from the circulation thus leading to the development of neutropenia.


The effect of acute and prolonged administration of prednisolone and ACTH on lymphocyte subpopulations. The effect of acute and prolonged three week administration of prednisolone and ACTH on the numbers and function of T- and B-lymphocyte subpopulations in patients requiring corticosteroid therapy was studied. Prednisolone caused severe reduction in E-rosette-forming lymphocytes, phytohaemagglutinin response, EAC-rosette-forming lymphocytes and surface-membrane mu-positive B-lymphocytes maximal at 4--6 hr after administration with reversal sometimes to supernormal levels by 24 hr. Prolonged administration resulted in a similar pattern of response. Acute but not prolonged prednisolone administration caused a reduction in the percentage of E-rosette-forming lymphocytes maximal at 4 hr. ACTH caused moderate reduction in these parameters at 4 and 6 hr which remained low at 24 hr after prolonged administration.


Antibodies against cytomegalovirus-induced early antigens (CMV-EA) in immunosuppressed renal-allograft recipients. Antibodies against cytomegalovirus-specific early antigens (CMV-EA) were followed in sera, obtained from fifteen immunosuppressed renal-allograft recipients. Eight patients (sixty-two sera) showed seroconversion 47--137 days post-transplantation. Five patients (forty sera) with CMV antibodies at the moment of renal implantation all showed CMV-EA antibody rises. Two patients remained seronegative until 4 years after transplantation. Thus, in immunosuppressed patients, antibodies against CMV-EA remained a high titres during many years (4--8 years) after transplantation as distinct from the apparently transient nature in acutely infected previously healthy adults with CMV mononucleosis or post-perfusion syndrome. These results support the view that CMV-EA antibodies reflect an active viral proliferation in the host.


Antibody-dependent cellular cytotoxicity (ADCC) against Epstein-Barr virus-determined antigens II. Induction of ADCC by FC-FC receptor bridging. Four different methods to test antibody-dependent cellular cytotoxicity (ADCC) against Epstein-Barr virus (EBV) determined antigens were analysed. It was found that optimal ADCC was obtained if viral antigens were present during the cytotoxicity reaction and that killing probably was mediated by EBV-related immune-complexes forming Fc-Fc receptor bridges between Fc receptor-bearing effector cells and Fc receptor-bearing target cells. Only lymphoid target cells with a high expression of Fc receptors were found to be susceptible in this particular system.


Bilirubin inhibition of protein kinase: its prevention by cyclic AMP. The effect of bilirubin on protein kinase activity has been studied. Bilirubin inhibits the binding of cyclic AMP to protein kinase. It also inhibits the phosphorylation of histone. The inhibitory action of bilirubin is competitive, and increased amounts of cyclic AMP relieved the inhibition.


Mapping AK1, ACONs, and AK3 to chromosome 9 in man employing and X/9 translocation and somatic cell hybrids. The adenylate kinase 1 (AK1), adenylate kinase3 (AK3), and aconitaseS (ACONS) genes have been assigned to chromosome 9 in man by employing an X/9 translocation segregating in man-mouse somatic cell hybrids. Segregation was controlled by taking advantage of the HAT/8-azaguanine selection-counterselection strategy directed at the X-linked HPRT locus. Assignment of AK1 to chromosome 9 has suggested the assignment of the ABO blood-group locus and the nail-patella (Np) locus to 9, since both loci are linked to AK1 by family studies.


Synteny of the genes for thymidine kinase and galactokinase in the mouse and their assignment to mouse chromosome 11. We have studied the expression of mouse galactokinase in human-mouse somatic cell hybrids segregating mouse chromosomes. Since concordant segregation of the expression of mouse galactokinase and the presence of mouse chromosome 11 were observed in the hybrid clones, we conclude that the gene for mouse galactokinase is located on mouse chromosome 11. We have also investigated the expression of mouse galactokinase in somatic cell hybrids between thymidine kinase-deficient Chinese hamster cells and mouse peritoneal macrophages. The results of this study indicate that the expression of mouse galactokinase and thymidine kinase segregates concordantly, and, therefore, we infer that the gene for mouse thymidine kinase is also located on mouse chromosome 11.


Search for inhibitors against herpes simplex virus type-I in cell extracts derived from human lymphoblastoid cell lines. Cell extracts obtained from KB cells and 5 human lymphoblastoid cell lines including 2 from Burkitt's lymphoma (P3HR-1 and Raji), one each from nasopharyngeal carcinoma (no.223), acute lymphatic leukemia (MOLT-4) and a healthy person (NC-37) were tested for their inhibitory effects on the growth of herpes simplex virus type-1 (HSV-1) in green monkey kidney (GMK) cells by the plaque titration method. The relationship between the production of HSV-1 inhibitors and the degree of Epstein-Barr virus (EBV) genome repression in lymphoblastoid cells were also examined. Among the cell lines used P3HR-1 and no.223 cells produced a few EBV particles, Raji and NC-37 cells contained EBV genomes only, and MOLT-4 as well as KB cells were EBV genome-negative. The results revealed that P3HR-1 cell extract showed a tendency to inhibit HSV-1 growth in GMK cells but the other 4 lymphoblastoid cell lines and KB cells did not produce HSV-1 inhibitors, indicating that EBV genomes governing the formation of EBV structural antigens were not related to the production of HSV-1 growth inhibitors. The extracts from MOLT-4 cells, which are only a T lymphocyte cell line used in this study, stimulated HSV-1 growth in GMK cells significantly.


Isolation and identification of Marek's disease virus by fluorescent antibody technique. Cell-associated herpesvirus related to Marek's disease (MD) was isolated from the direct culture of kidney cells of naturally infected chickens at Taoyuan or by inoculation of clinical specimens to chick kidney (CK) and chick embryo fibroblast cells. The virus isolates replicated in CK or chick embryo kidney cell cultures were identified to be MD by the fluorescent-antibody technique.


[Serum lipids in experimental atherosclerosis in guinea pigs treated with protein hydrolysate]. The authors carried out studies on 223 male guinea pigs, weighting from 350 to 500 gm, fed with a diet containing 0,3% of cholesterol, disolved in cow butter. A part of the animals were injected with protein hydrolysate every other day for a period of 140 days. The level of serum lipids-total cholesterol, beta-lipoproteins, phospholipids and total lipids was examined. They found significantly higher values in the animals, receiving cholesterol in comparison with the control animals-292.4:84.9; 139.4:59.3; 457.1:241.2; 105.5:62.0 respectively for, beta-lipoproteins, total cholesterol, total lipids and phospholipids. The administration of protein hydrolysate in a dose of 0,5 ml/100 gm of body weight affected favourably fat metabolism. The increase of serum fat-lipid components was less manifested in comparison with the nontreated atherosclerotic animals. The greatest difference was found in beta-lipoproteins (292.4:143.8) and total, lipids (457.1:300.8). Statistically significant difference was established in total cholesterol as well. (139.4:81.9). There was an impression that this effect was due to the protective action of aminoacids of the protein hydrolysate on the normal functions of liver and the favourable effect on the tunover of the administered cholesterol.


An improved bioassay for ACTH determination. An improved method for the bioassay of ACTH has been developed using short-term culture of adrenal cell suspensions derived from intact rats. Six separate pools of adrenals were used and cells derived from the same pool were compared after acute dispersion and overnight culture. The ED50, defined as the concentration of ACTH required to produce half maximal steroidogenesis, for cultured cells was 35.4+/-2 pg/ml compared to 240+/-60 pg/ml for cells used immediately after dispersion. The minimum concentrations of ACTH necessary to produce a response significantly above basal levels were 0.98+/-.10 pg/ml and 12.1+/-3 pg/ml respectively. Response characteristics of the cultured cells were highly reproducible. The incubation volume employed in this study was 0.25 ml, so the ED50 expressed as dose of ACTH per tube was actually 8.8 pg for the cultured cells. Such sensitivity provides requisite methodology for the measurement of ACTH under varying physiological conditions.


Characteristics of thyrotropin binding to bovine thyroid plasma membranes and the influence of human IgG. The characteristics of binding of 125I-labeled bovine thyrotropin to bovine thyroid plasma membranes were studied. At pH 7.5, 0 C, specific binding was maximal in 3 hr and was progressively inhibited by Na+, Mg++, Ca++ and (NH4)2 SO4 in concentrations of 25 mM and greater. Affinity constants of l.047 X 10(8)M-1 and 0.57 X 10(6)M-1 were obtained for two binding sites and Hill plots provided a mean slope of 0.86, suggesting negative cooperativity. Addition of 1-thyroxine or 1-triiodothyronine at 0.5 ng to 50 microgram per ml had no significant effect but propylthiouracil (10(-7) to 10(-5)M) and KC104 (10(-7) to 10(-4)M) had biphasic effects, first enhancing and then inhibiting binding. Normal human serum IgG, particularly two fractions (3 and 4) obtained by chromatography on columns of cellulose phosphate, and an alpha-globulin - rich fraction from chromatography of serum proteins on diethylaminoethyl cellulose, inhibited binding at concentrations of 0.5 mg per ml or greater. Thus two possibilities are that normal human serum globulins either compete with thyrotropin for its receptor on thyroid plasma membranes or, in binding to the membranes, cause such perturbation as to affect adversely the conformation of the receptor for thyrotropin.


Stimulation of corticosteroid biosynthesis by angiotensin I [des-asp1]angiotensin I, angiotensin II and [des-asp1]-angiotensin II in bovine adrenal fasciculata cells. The effect of angiotensin I (AI), angiotensin II (AII), [des-asp1]AI, [des-asp1]AII and [des-asp1-arg2]AII on corticosteroid production in isolated fasciculata cells from bovine adrenals has been studied. AII and [des-asp1]AII in concentrations ranging from 10(-9)M to 10(-6)M had a potent stimulatory effect on steroid biosynthesis. The dose-response curves for both peptides were identical. AI was about 3 times less potent than AII and [des-asp1]AII. The effect of AI was not due to its conversion to AII. [Des-asp1]AI was as active as AI. No significant conversion to [des-asp1]AII was observed. [Des-asp1-arg2]AII had only a minimal effect on steroidogenesis. The structural analog [sar1,-ala8]AII inhibited all angiotensins specifically and competitively. The affinity of the cellular binding site was higher for AII and [des-asp1]AII than for [sar1,ala8]ALL, but lower for AI and [des-asp1]AI than for the inhibitor. Combination of submaximal doses of AI and AII resulted in an additive effect on steroid production. By contrast, combination of maximal doses of both peptides had the same effect as AII alone. These data demonstrate a potent steroidogenic activity for AII as well as AI, [des-asp1]AI and [des-asp1]AII in bovine adrenal fasciculata cells. A common receptor site for all four peptides is suggested.


Functional heterogeneity in somatotrophs isolated from the rat anterior pituitary. Somatotrophs in suspensions of anterior pituitary cells from adult male rats can be separated into 2 fractions by density gradient centrifugation. In addition to their different densities, somatotrophs in these 2 fractions can be distinguished morphologically by their staining characteristics and ultrastructure. Somatotrophs of lesser density (type I; approximately 1.068 g/cm3) have fewer secretory granules and a more extensive Golgi apparatus than the somatotrophs of greater density (type II; approximately 1.073 g/cm3). Responsiveness of type I and type II cells to secretory agents (i.e., dibutyryl cyclic adenosine monophosphate, somatostatin, thyroxine, and hydrocortisone) was evaluated by GH radioimmunoassay. Type I cells were consistently more responsive (% GH release) than type II cells. During 7 days in culture, type I cells produced  more (approximately 200%) GH than they initially contained, whereas type II cells did not show evidence of increased GH production. Hydrocortisone significantly stimulated GH production in type I, but not type II cells. These results support the hypothesis that at least 2 functionally distinct populations of somatotrophs are present in the anterior pituitary gland of the adult male rat.


In vivo evidence for the intermediary role of 3',5'-cyclic AMP in parathyroid hormone-induced stimulation of 1alpha,25-dihydroxyvitamin D3 synthesis in rats. Teh stimulatory effect of parathyroid hormone (PTH) on renal 1alpha-hydroxylation of 25-hydroxyvitamin D3 (25-OH-D3) was studied in thyro-parathyroidectomized (TPTX), vitamin D-deficient rats into which bovine PTH, theophylline, cAMP or dibutyryl cAMP (dbcAMP) was constantly infused. The accumulation in plasma of 1alpha,25-dihydroxy-vitamin D3 [1alpha,25-(OH)2-D3], produced from 25-OH-D3, was enhanced by infusion of either cAMP (0.9 MUMOL/H) OR DBCAMP (1 mumol/h) to a level similar to the maximum obtained by PTH (i95--7.5 U/h) infusion. A submaximal dose (1 U/h) of PTH caused a similar extent of stimulation, when infused with theophylline. When either 2 mumol/h of cAMP or 7.5 U/h of bovine PTH was infused starting 18 h after TPTX, the accumulation of 1alpha,25-(OH)2-D3 in plasma was similarly restored withing 6 h to the level found in the sham-operated animals. These results strongly support the concept that cAMP plays an important intermediary role in the stimulation of 1alpha,25-(OH)2-D3 production induced by both exogenous and endogenous PTH.


Solubilization of a growth hormone-specific receptor from rabbit liver. Membrane preparations from rabbit liver, known to possess GH-specific binding sites, have been solubilized with Triton X-100 and the binding characteristics of [125I]-human GH (hGH) and [125I]-bovine GH (bGH) subsequently studied. Specific binding of the hGH and bGH by the solubilized preparation was demonstrated of bound and free hormone by either polyethylene glycol precipitation or by Sephadex G-100 chromatography. Binding of hGH was both rapid and reversible and was displaced only by other growth hormones (bovine and ovine) and not by lactogenic hormones (ovine and human prolactins, human placental lactogen). As shown by Scatchard analysis specific binding of [125I]-bGH exhibited a lower binding affinity and capacity than did [125I]-hGH. Overall, the characteristics of the binding reaction for hGH were not significantly different from those reported for the particulate membrane preparation. The solubilization process did not appear to alter the binding protein(s) therefore, and permits a further study of the isolation, purification and properties of the binding protein(s) itself.


Phenytoin, electric, ionic, and metabolic responses in cortex and spinal cord. Post-tetanic potentiation (PTP) of monosynaptic reflex was estimated in spinal cords in the drug-free state after the administration of a convulsant dose of penicillin and after the administration of phenytoin. There was no apparent correlation between the degree of depression of PTP and the efficacy of controlling seizure activity by phenytoin. Extracellular potassium levels were measured with ion-selective microelectrodes. The post-stimulation clearing of [K+]0 was not accelerated by phenytoin, and frequently it was slowed. Post-stimulus undershooting of [K+]0 was diminished. Oxidation of NADH in cortex and of cytochrome a, a3 in spinal cord were measured by optical methods. Stimulus-evoked transient oxidation responses evoked by electrical stimulation were depressed by phenytoin. It is concluded that systemic administration of phenytoin in therapeutic doses does not stimulate Na+-K+-activated membrane ATPase in cortex and spinal cord. Unlike other depressants, phenytoin did not cause a reduction of "resting" redox levels of respiratory enzymes. The local regulation of blood flow remained unaltered after phenytoin administration. Phenytoin caused a moderate but consistent depression of the stimulus-evoked responses of potassium activity, electric potential, and oxidative enzymes, consistent with diminished outflow of potassium from cells, owing either to lesser activation of cells or to a lesser exchange of ions.


Phenytoin: effects on calcium flux and cyclic nucleotides. Previous studies have demonstrated that phenytoin alters calcium conductance in isolated presynaptic nerve endings (synaptosomes) from rat or rabbit brain. Drug concentrations of 0.08 mM (20 microgram/ml) or higher inhibit stimulated calcium influx into synaptosomes depolarized by high concentrations of potassium (69 mM) by 7-58%. Calcium transport into undepolarized synaptosomes is only inhibited by 0.4 mM or greater concentrations of phenytoin. Recent investigations show that in mouse brain slices, phenytoin inhibited elevations of cyclic GMP and cyclic AMP produced by ouabain or veratridine. In contrast, elevations of the two cyclic nucleotides produced by high concentrations of potassium were not inhibited by phenytoin, suggesting that the anticonvulsant suppresses depolarization-induced elevation of cyclic nucleotide levels in brain slices by inhibiting influx of sodium into cells. These data indicate that phenytoin inhibits both sodium and calcium influx into cells during cellular depolarization and alters regulation of brain cyclic nucleotide levels. Both of these actions may be important for the antiepileptic effect of phenytoin.


Effects of phenytoin on the cyclic nucleotide system in the motor nerve terminal. The effects of phenytoin on the motor nerve terminal were evaluated on the in vivo cat soleus nerve muscle preparation. Phenytoin, 10 mg/kg, reduced the repetitive aftercharges in motor nerve endings due to tetanic conditioning. It also reduced the repetitive activity due to adenylate cyclase activation with NaF, or to exogeneous dibutyryl cyclic AMP. These effects of phenytoin could be reversed by administering theophylline, a phosphodiesterase inhibitor, or by increasing the extracellular concentration of calcium. The effects of phenytoin could also be reversed by 3-aminopyridine, but not by tetraethylammonium chloride. Verapamil, a calcium current antagonist, produced effects that were identical to phenytoin. It is concluded that phenytoin blocks a cyclic nucleotide-mediated calcium influx that is associated with transmitter release. This calcium flux also appears to control a slow potassium current that is responsible for post-tetanic hyperpolarization.


Effect of salt loading in the rat on adenylate cyclase and phosphodiesterase activity in kidney cortex, medulla and papilla. Adenylate cyclase (AC) and phosphodiesterase (PDE) activities were studied in the cortex, medulla and papilla of the rat kidney. Sodium loading in vivo for 14 days resulted in a decrease of AC activity in the cortex, a small increase in the medulla and a substantial increase of AC activity in the papilla. Sodium loading caused reciprocal effects on PDE activity: an increase in kidney cortex and a decrease in kidney papilla. Loading of glucose in vivo or chronic administration of antidiuretic hormone in vivo did not cause the changes in AC or PDE observed after sodium loading. The possible significance of these findings is discussed.


Unspecific arginine kinase of molecular weight 150 000. Amino acid composition, subunit structure and number of substrate binding sites. The amino acid composition of unspecific arginine kinase of molecular weight 150 000 of Sabella pavonina muscle has been determined. If was found to be very similar to that of the phosphagen kinases previously studied. The subunit structure of the enzyme has been investigated by physical and chemical means. The data obtained from ultracentrifugation studies in 6 M guanidine hydrochloride and from molecular sieving and disc electrophoresis in 8 M urea, as well as the tryptic peptide mapping, suggest that Sabella muscle kinase is composed of four non-covalently linked polypeptide chains, with similar molecular weights. The number of binding sites for the nucleotide substrate ADP-Mg2+ has been estimated, using differential spectrophotometry and gel filtration on Sephadex columns. By both methods it was demonstrated that the enzyme contains two catalytic sites per protein molecule of molecular weight 150 000. Thus, arginine kinase from Sabella muscle, of molecular weight 150 000, consists of four similar polypeptide chains, but possesses only two substrate binding sites per tetrameric molecule.


2-O-(beta-D-glucuronyl)-7-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-heptose: a constituent of the Bordetella pertussis endotoxin. The presence of bound D-glucuronic acid in the endotoxin of Bordetella pertussis was demonstrated. The branched chain trisaccharide named in the title was isolated after hydrolysis of the endotoxin with 3 M HCl for 2 h at 100 degrees C. Its structure was established by chemical and enzymic degradation.


Characterization of a soluble simian-virus-40 transcription complex. We have previously described the isolation, from nuclei of monkey cells infected with Simian virus 40 (SV40), of a nucleoprotein complex which is able to achieve viral transcription. This complex contains SV40 DNA and RNA polymerase II molecules which have initiated transcription during the viral development. We show here, by molecular hybridization experiments, that most of the templates active in SV40 transcription can be dissociated from host DNA. In conditions where supercoiled SV40 DNA form I sediments at 21 S, the transcription complex has a sedimentation coefficient of about 25 S. Inhibition of viral DNA synthesis by cytosine arabinonucleoside or chloroquine does not affect the activity of the transcription complex, which suggests that replicating molecules are not required for viral RNA synthesis and that SV40 DNA form I could serve as template for late SV40 transcription. A large fraction of the RNA synthesized in vitro remains associated with the SV40 DNA template in cesium sulfate density gradient. The RNA chains produced by the complex are heterogeneous in size, most of them being as large or larger than the viral genome.


Determination of the syn-anti equilibrium of some purine 3':5'-nucleotides by nuclear-magnetic-relaxation perturbation in the presence of a lanthanide-ion probe. The aqueous solution conformation of four purine 3':5'-nucleotides varying in their substituents at C-6 and C-8 has been studied using gadolinium(III) to perturb the proton relaxation times. The ribose conformations are inferred. All the nucleotides are best described as being in a dynamic equilibrium between syn and anti conformations and the position of this equilibrium is not dramatically affected by changing the substituent at C-6. These nucleotides in their neutral base form slightly favour an anti conformation. In the presence of a bulky methylthio group at C-8 the equilibrium is shifted towards a dominance from the syn conformation due to steric repulsion factors.


Incorporation of the 1-pro-R and the 1-pro-S hydrogen atoms of ethanol into steroids and phosphatidylcholines in vivo. The transfer of deuterium from chiral 1-monodeuteroethanols to various metabolites formed in the liver was studied in order to investigate the coupling of metabolic reductions to the alcohol dehydrogenase and the aldehyde dehydrogenase reactions. The ethanols were administered to female bile fistula rats for 10 h. The hydrogen at C-2 in the glycerol moiety of newly formed phosphatidylcholine molecules in bile, liver and plasma was derived to 22-25% from the 1-pro-R position and to 5-6% from the 1-pro-S position in the ethanol. sn-Glycerol 3-phosphate isolated from liver had a lower deuterium content at C-2. The ratio between the contributions from the two positions in ethanol to C-2 of free sn-glycerol 3-phosphate was the same as in the phosphatidylcholines. This indicates that the higher degree of labelling of this position in phosphatidylcholines is not due to a specific coupling between alcohol dehydrogenase and the formation of a phosphatidylcholine precursor. Cholesterol and chenodeoxycholic acid in bile became increasingly labelled, and the ratio between the incorporations from the 1-pro-S and the 1-pro-R positions of ethanol was about 0.37 in cholesterol and 0.46 in chenodeoxycholic acid. Thus, these NADPH-dependent reactions utilized hydrogen from the 1-pro-S position to a larger extent than NADH-dependent reactions.


Preparation of N6-[N-(6-aminohexyl) carbamoyl]-adenine nucleotides and their application to coenzymically active immobilized ADP and ATP, and affinity adsorbents. Reaction of ADP with hexamethylene diisocyanate in hexamethylphosphoramide followed by treatment in an acidic medium afforded three new adenine nucleotide analogues, N6-[N-(6-aminohexyl)carbamoyl]-ADP, N6-[N-(6-aminohexyl)carbamoyl]-ATP, and N6-[N-(6-aminohexyl)carbamoyl]-AMP in yields of 13%, 12% and 17%, respectively. The occurrence of the ATP analogue may be interpreted in terms of the equilibrium, 2ADP = ATP + AMP. Coenzymic activities of the ADP analogue against acetate kinase and pyruvate kinase were 82% and 20%, respectively, relative to ADP and those of the ATP analogue against hexokinase and glycerokinase were 63% and 87%, respectively, relative to ATP. These analogues were bound to CNBr-activated soluble dextran through their terminal amino group to give an immobilized ADP and an immobilized ATP, each of which was recycled in a system comprising acetate kinase and hexokinase, and when placed in a membrane reactor together with the enzymes, functioned as an immobilized coenzyme continuously yielding glucose 6-phosphate. A series of chemically defined affinity adsorbents were obtained by coupling these analogues to CNBr-activated Sepharose, and were used to separate the enzymes in a mixture of hexokinase, pyruvate kinase, phosphoglycerate kinase, lactate dehydrogenase, and alcohol dehydrogenase.


Identification of 2-methyl-3-hydroxydecanoic and 2-methyl-3-hydroxytetradecanoic acids in the 'lipid X' fraction of the Bordetella pertussis endotoxin. The 'lipid X' fraction, released from the Bordetella pertussis endotoxin upon treatment with trifluoroacetic acid of pH 3 at 50 degrees C, was shown to contain, in addition to 3-hydroxydecanoic, 3-hydroxydodecanoic, 3-hydroxytetradecanoic, and tetradec-2-enoic acids, 2-methyl-3-hydroxydecanoic-, and 2-methyl-3-hydroxytetradecanoic acids. The structure of these was established by gas-liquid chromatography/mass spectrometry.


Impaired responsiveness to the effect of glucagon on plasma adenosine 3':5'-cyclic monophosphate in normal man. Small doses (10-150 microgram; 3-45 nmol) of glucagon caused a dose-dependent increase in plasma adenosine 3':5'-cyclic monophosphate (cyclic AMP) concentration when injected into man. Infusion of glucagon (75 ng min-1 kg-1) for 2 h into normal subjects resulted in an initial increase in plasma cyclic AMP concentration, then a decline despite continuation of the hormone infusion and maintenance of high concentrations of circulating immunoreactive glucagon. When an injection of glucagon was given at the termination of such an infusion, the subsequent increase in plasma cyclic AMP concentration was markedly reduced when compared to that observed after a control injection which had not been preceded by a glucagon infusion. When the glucagon was injected at the end of an infusion of 1000 MRC units of bovine parathyroid hormone (BPTH) over 2 h, the plasma cyclic AMP response was normal. Conversely, after infusion of glucagon the response to injected BPTH was normal. This impairment of response was therefore specific to the hormone that had been administered and was not due to altered metabolism of circulating cyclic AMP. This phenomenon may be important in the regulation of the hormonal response by the target tissue.


Lipoprotein-X: a substrate for lecithin: cholesterol acyltransferase. The action of lecithin:cholesterol acyltransferase (LCAT) was studied on an abnormal lipoprotein (LP-X) rich in phosphatidylcholine and cholesterol from the plasma of patients with obstructive liver disease. 60 mg LP-X isolated free of other lipoproteins and subsequently labelled with 3H-cholesterol were incubated with 1 mg highly purified enzyme in the presence of albumin. After 45 h at 37 degrees C, the incubation mixture was subjected to zonal ultracentrifugation. 3H-cholesterol and 3H-cholesteryl esters were quantified in each fraction of the zonal gradient. More than 95% of the lipoproteins in this mixture banded in the density range of LP-X with no change in size distribution, but did contain 593 nmoles of newly formed cholesteryl esters. Agarose electrophoresis revealed an alpha-migrating band in addition to the original beta-band. Also on agar, the typically cathode migrating LP-X was changed to anode moving material. These studies indicate that LP-X can serve as a substrate for LCAT.


The actions of three diaminopyridines on the chick biventer cervicis muscle. The effects of 2,3-, 2,6- and 3,4-diaminopyridine were investigated on the isolated chick biventer cervicis muscle preparation. All three compounds reversed tubocurarine blockade and augmented twitch height in indirectly stimulated preparations. Less twitch augmentation was observed in directly stimulated preparations. 3,4-Diaminopyridine was the most effective of the compounds in facilitating neuromuscular transmission, but exhibited less convulsant activity than 4-aminopyridine. 3,4- and 2,3-diaminopyridine in high concentrations caused contractures that were inhibited by erabutoxin b or by beta-bungarotoxin. It is suggested that the diaminopyridines increase both evoked and spontaneous acetylcholine release.