Cyclic nucleotide-dependent phosphorylation of rat intestinal microvillus and basal-lateral membrane proteins by an endogenous protein kinase. Both the microvillus and basal-lateral membrane components of intestinal epithelial cells were found to contain endogenous cyclic nucleotide-dependent protein kinases and their endogenous protein substrates. The phosphorylation of either membrane component using [gamma-32P]ATP as substrate, occurred very rapidly, reaching maximal levels at 1 min. Both cyclic AMP and cyclic GMP were shown to stimulate the phosphorylation of the microvillus and basal-lateral membranes; the approximate concentrations of cyclic AMP and cyclic GMP required for half-maximal stimulation of phosphorylation were 2 x 10(-7) M and 1.7 x 10(-8) M, respectively, for the basal-lateral membranes, and 2 x 10(-7) M and 3.2 x 10(-8) M, respectively, for the microvillus membranes. Although both membrane components were phosphorylated by an endogenous protein kinase, the microvillus membrane was consistently phosphorylated to a greater extent at maximally effective concentrations of either cyclic nucleotide. The microvillus and basal-lateral membranes were also found to contain a phosphoprotein phosphatase; however, the rate of removal of [32P]phosphate from the microvillus membrane was found to be more rapid. Neither cyclic AMP nor cyclic GMP altered the activity of the enzyme in either membrane. The present results together with earlier studies are compatible with the possibility that the regulation of water and electrolyte transport in the small intestine by cyclic AMP and cyclic GMP may be mediated through modulation of the phosphorylation of protein components of the microvillus and basal-lateral membranes.


[Initial experience with a tocolysis ward (author's transl)]. Premature labour is best treated in a special ward, the tocolysis ward. From 1972 to 1975 there were around 8% premature deliveries. During the first six months of 1976 there were still around 8% premature deliveries. During the second six months of 1976 the premature rate was lowered to 6% in the tocolysis ward. This is a decrease of the 25%.


Genetic studies of acridine-induced mutants in Streptococcus pneumoniae. The mutagenic properties of acridines on pneumococcus are described. All seven acridines tested were mutagenic at the amiA locus conferring a resistance to 10(-5) M aminopterin. The effects of quinacrine were more specifically investigated. It was observed that: mutants can be obtained only by treatment of exponentially growing cells; a sharp maximum mutagenic effect occurs at a concentration slightly lower than the bacteriostatic value; and the amount of quinacrine required to yield the maximum mutagenic effect decreases with the pH of the medium. Moreover, the number of mutants detected after quinacrine treatment varies from locus to locus. The majority of quinacrine-induced mutants are readily reverted by quinacrine, but not by nitrosoguanidine treatment. This suggests that in pneumococcus quinacrine induces mainly frameshift mutations. A further study of the revertants obtained by quinacrine treatment of quinacrine-induced mutants strengths this interpretation: most of the revertants result from a mutation at the same site; some partial revertants exhibiting an intermediate resistance to aminopterin were found to contain two very closely linked mutated sites, each mutation conferring the maximum level of resistance to aminopterin. Thus, the majority of quinacrine-induced mutants at the amiA locus of pneumococcus consists of frameshift mutations. Nearly all of the isolated mutants induced by quinacrine as well as other acridines belong to the low efficiency class of transformation. It was concluded that the mismatch resulting from the pairing between the wild type and the frameshift-containing sequence is recognized by the excision-repair system involved in the discrimination function in a way similar to that in which it recognizes mismatched base pairs between a transition mutation and the wild-type sequence.


[Effects of psychotropic drugs on the spinal ventral root reflexes in cats. A comparison of the depressant actions in intact and spinal preparations (author's transl)]. Effects of the typical psychotropic drugs such as neuroleptics, tricyclic antidepressants and benzodiazepines on the monosynaptic reflex (MSR) and polysynaptic reflex (PSR) were investigated using intact and spinal cats. Drugs used were chlorpromazine, levomepromazine, perphenazine, prochlorperazine, droperidol, haloperidol, amitriptyline, imipramine, diazepam and flurazepam. Neuroleptics depressed the ventral root reflexes markedly to slightly in both preparations. The inhibitory effects of these drugs on MSR of intact cats were stronger than those on MSR of spinal cats, and the activity was slightly higher than that of PSR depression in intact preparations. In intact cats, those depressants effects of neuroleptics on PSR produced various characteristic action patterns in comparison with MSR inhibition. Tricyclic antidepressants were similar in the mode of action to the dimethylamino side-chain groups in phenothiazines, but the inhibitory effects of ventral root reflexes were distinctly weaker than that of neuroleptics. Benzodiazepines depressed only PSR without MSR inhibition, especially in spinal cats. Thus, it is suggested that the structure-activity relationship considered, a classification of the psychotropic drugs can be given according to the inhibitory activities on spinal ventral root reflexes in intact and spinal cats.


[Behavioral and EEG effects of triazolam in comparison with those of diazepam (author's transl)]. Triazolam was 4 to 5 times as potent as diazepam in reducing hyperemotionality of either septal-lesioned or olfactory bulbectomized rats (O.B. rats), and in suppressing muricide in O.B. rats. This agent was equipotent with diazepam in inhibiting fighting behavior of long-term isolated mice, but was longer in duration of action. Triazolam was approximately 4 times more potent than diazepam in preventing pentetrazol convulsion, but was 10 times less potent in inhibiting maximal electroshock convulsion in mice. The muscle relaxant effect of triazolam as assessed by the inclined screen test was 34 times, and the effect on rotarod performance was 17 times more potent than that of diazepam in mice. Triazolam (0.2 approximately 0.5 mg/kg i.v.) changed the EEG to a drowsy pattern in unanesthetized rabbits with a chronic electrode implant, and suppressed the EEG arousal response to auditory stimulation and electrical stimulation given to either the mesencephalic reticular formation or posterior hypothalamus. The limbic afterdischarges induced by either hippocampal or amygdaloid stimulation were also markedly inhibited by triazolam. These EEG effects of triazolam were qualitatively similar to, but were 4 to 5 times more potent than those of diazepam. These results indicate that triazolam is a potent tranquilizer with a longer duration of action, and the muscle relaxant effect is considerable as compared with diazepam.


[Effects of triazolam on conditioned behavior in rats (author's transl)]. Effects of triazolam on various types of conditioned behavior were investigated and compared mainly with diaepam in rats. The active conditioned avoidance response of the rat in a Shuttle box was inhibited by triazolam and diazepam only at large doses. The passive avoidance response in a step-down method was not affected by either triazolam or diazepam, but was markedly suppressed by chlorpromazine. The low rate response of hypothalamic self-stimulation behavior was markedly increased by triazolam at doses ranging from 2 to 40 mg/kg p.o., but was suppressed at doses over 80 mg/kg p.o. The high rate response was unaffected by triazolam even at doses of 40 approximately 180 mg/kg p.o. The low rate response was increased by diazepam at doses of 1 approximately 10 mg/kg p.o. and was suppressed at 80 mg/kg p.o. The high rate response was reduced by diazepam at 180 mg/kg p.o. In the conflict situation of the rat subjected to food reward and foot-shock punishment, the lever press response in the unpunished period was reduced by triazolam at doses of 1 approximately 5 mg/kg p.o., whereas that in the punished period was markedly increased. Similar effects were observed with diazepam at doses of 15 approximately 20 mg/kg p.o. Triazolam appeared to be 10 approximately 15 times more potent than diazepam in this anticonfluct effect. Thus, triazolam appears to be a potent antianxiety agent.


D-Amino acids of the amino acid pool and occurrence of racemase and D-amino acid oxidase activities in Escherichia coli B. Less than 20% of the amino acid content of the amino acid pool of Escherichia coli B exists in the D-form. Alanine, glutamic acid, and valine were shown by gas- chromatography to be partially in the D-form. Only D-alanine was formed by racemization in the crude extract of this organism. Alanine racemase was easily released from the membranes or vesicles but D-alanine oxidase activity remained firmly bound to the membrane. Most protein amino acids stimulated proline uptake into the vesicles, and the oxidative deamination activities were verified by the proline uptake stimulating amino acids. It is concluded that the obligatory pathway of L-amino acid--D-amino acid--oxo acid which exists in the oxidation of L-alanine does not exist with other L-amino acids. It is likely that other D-amino acids in the pool are formed in the presence of D-amino acid oxidase or D-amino acid aminotransferase.


[Absorption in the human small intestine relative to the intraluminal milieu]. The bulk of water and electrolyte absorption takes place in the human jejunum from isotonic solutions, and is determined largely by special transport mechanisms for different monosaccharides, amino acids and dipeptides. This is of considerable significance for regaining the large volumes of fluid delivered to the small intestine during the digestion of food. Small changes in intraluminal pH do not significantly influence the absorptive function of the jejunum and are rapidly compensated by the buffering capacity of the gut. The maintenance of an isotonic as well as neutral intraluminal milieu seems to be essential to the physiological processes of intestinal absorption.


[Psychotropic drugs as tools for clinical research into schizophrenia (author's transl)]. There is considerable similarity between paranoid schizophrenia and psychoses provoked by dopaminergic overstimulation in the central nervous system. The fact that neuroleptics are able to block dopaminergic neural activity has led to the hypothesis that there might exist a common biochemical substrate for schizophrenia and e. g. the amphetamine psychoses. Dopaminergic overstimulation may be elicited by different drugs interacting with the dopamine metabolism e. g. dopamine-beta-hydroxylase inhibition (disulfiram, fusaric acid); monoamine-oxidase-inhibition (phenelzine, tranylcypromine); dopamine release (amphetamine); stimulation of postsynaptic dopamine receptors (bromocriptine, apomorphine). Resulting psychotic symptoms consist of ideas of reference, delusions, visual and acustic hallucinations in a clear setting of consciousness. Psychoses occur usually in subjects, who have suffered from various psychiatric illnesses, which have apparently in common a reduced monoamine-oxidase activity in platelets. It is concluded from various biochemical findings, that psychoses resulting from dopaminergic overstimulation and schizophrenia have different biological substrates.


[Indicated and non-indicated administration of antibiotics in surgery (author's transl)]. Considerations and own experience regarding prophylactic administration of antibiotics and therapeutic customs in surgery are discussed. Based on own and long-term studies recommendations are given for effective and purposeful use of chemotherapy in surgery.


Epstein-Barr virus infection following bone-marrow transplantation. A 12-year-old patient with acute lymphoblastic leukemia received a bone-marrow transplant (BMT) from a matched sibling donor. Nine weeks prior to transplant the donor experienced Epstein-Barr virus (EBV)-induced infectious mononucleosis. The bone-marrow recipient was EBV-negative at the time of transplant; however, 4 weeks post transplant the recipient developed clinical symptoms of graft-verus-host disease (GVHD) coincident with serological evidence of acute EBV infection. In addition, a lymphoblastoid cell line positive for Epstein-Barr nuclear antigen was established from a bone-marrow sample obtained at the onset of symptoms compatible with GVHD. Sera obtained from the recipient over the ensuing 2 months showed the appearance of antibodies to specific EBV antigens consistent with a primary immune response to EBV infection. This association of acute EBV infection with symptoms of GVHD in a BMT recipient suggests a need for further investigation of the epidemiology of EBV infections in human bone-marrow transplantation and the relationship between EBV infection and GVHD.


Rate constants of absorption versus physico-chemical parameters. We examined the velocity constants of buccal absorption in the case of 10 drugs. The measurements tending towards what is the coherence between the velocity constants and the physico-chemical parameters of the drugs. On the basis of our investigations one can see that the dipol moment offers more reliable information for the size of the velocity of absorption than the lipid-water distribution ratio, molecular radii or diffusion constants. Our opinion is that it can be useful in modifying and planning the drug molecules.


Photolytic inhibition and labeling of proteins with aryl diazonium compounds. In the course of preparing aryl azide derivatives for use as photoprobes, we have observed significant light sensitivity in the precursor aryl diazonium compounds. The photosensitive properties of this class of compounds are of interest since they will seek out cationic binding sites in biological targets, and can be employed to inhibit complementary targets at acid pH. The relationship between photolytic change in the structure of diazonium compounds and the corresponding change in function of a biological target are presented. Experiments are described in which the dark and light sensitive properties of a model diazonium compound, diazobenzene sulfonate (DBS), were determined. The ultraviolet spectra were used to evaluate the dark stability and light sensitivity of DBS. Chymotrypsin and trypsin served as functioning targets for further evaluation of the photochemical properties. Both enzymes are stable to the probe in the dark at acid pH. A rapid loss of enzyme activity was observed following flash photolysis of DBS-enzyme solutions. Photolytic incorporation of radioactive DBS into chymotrypsin was observed. Aryl diazonium salts can be employed to probe the availability of complementary sites in biological targets at different acid pH values.


The denaturation of covalently inhibited swine pepsin. Studies are reported on the denaturation of freshly prepared, intact swine pepsin, which was inactivated by reaction with diazoacetylglycine ethyl ester, to prevent autolysis. Denaturation about pH 6 was found to involve a small expansion of the molecular domain with some loss of organized secondary structure. On the other hand, increasing concentrations of guanidine hydrochloride induced cooperative transitions in both the native and alkali denatured forms to give a cross-linked random coil. No conditions could be found in which these reactions were reversible. Removal of denaturing conditions usually resulted in aggregation and precipitation of protein. From these studies, it would seem that the active conformation is largely predetermined in the zymogen.


Cornea endothelial bicarbonate fluxes following preservation in solutions of varying composition. Numerous modifications have been made in MK solution, primarily concerned with alterations in both bicarbonate concentration and PCO2. Variations of the solutions from high bicarbonate to low bicarbonate and either high or low PCO2 were made which resulted in a decreased net bicarbonate flux across the endothelium after a 3-day storage period. Even with drastic changes in the storage solution there was a relatively small change in the net bicarbonate flux. Higher passive fluxes were found at a pH above 8, and a higher net flux with a bicarbonate concentration of 17.5 nM or higher. Variation in PCO2 made little difference to the net endothelial fluxes; when a high PCO2 was used, there was a concurrent elevation of PO2 in the storage solution, but no beneficial effect was found on the endothelial transport system. It was apparent that a good storage solution contained a buffer (bicarbonate was better than phosphate) and 5% dextran and was at a neutral pH (between 7 and 8).


Thymic humoral factor in the assessment of T lymphocytics in a patient with T cell chronic lymphocyte leukemia. Peripheral blood lymphocytes from a patient with T cell chronic lymphocytic leukemia were examined by a combination of cell markers (E and M rosettes, and surface Ig), the graft-vs.-host reaction, thymic humoral factor (THF) and scanning electron microscopy. It was not possible, at first, to determine by conventional methods whether the leukemic cells were of the B or T type, but the THF and graft-vs.-host reaction showed that T cell precursors were present. These cells were incompetent immature T cells that underwent maturation following treatment with THF. Three months later, the T cell nature of the disease was clearly demonstrated by the E rosette technique, although at this time the cells were no longer influenced by THF.


Detection of lipopolysaccharide in suspected bacteriuric urine using a carbocyanine dye. Currently practiced methods for the detection of gram negative bacteriuria require culturing and overnight incubation. Such an approach to bacteriuria detection is unacceptable for any screening program which requires rapid presumptive evidence of infection. In this study, the lipopolysaccharide-dependent formation of a unique dye absorption spectra of the cationic carbocyanine dye, 1-ethyl-2-[3-(1-ethylnaphtho[1,2d]-thiazolin-2-ylidene)-2-methylpropenyl] naphtho[1,2d]-thiazolium bromide, was used to detect bacteriuria caused by gram negative organisms in a hospitalized population. In an evaluation of 168 first morning and randomly collected suspected bacteriuric urines, the dye test detected 66% of the loop plate positive urines with false positive and false negative values of 28% and 34%, respectively. However, 37% of the false positive results occurred in urines containing less than 10(5) gram negative bacteria/ml and an additional 24% of the false positives were seen for patients currently receiving antibiotic treatment. Urine specimens were also evaluated using the limulus lysate assay for lipopolysaccharide.


[Conservative drug therapy of neurogenic bladder disorders]. Today neuropharmacas are a helpful part in the conservative treatment of the neurogenic bladder disorders. They are, or course, no "wonder-drugs" and usually lead to an improvement only of the troubles, but rarely to complete cure. If monotherapy does not lead to the results wanted, one should combine drugs of the same of similar effects but with different pharmacologic targets. A real progress was reached through alpha-receptor-blockers, whose use has, especially in children with myelomeningocele, changed the therapeutic concept in favour of a largely conservative treatment. We already know a number of substances that in one way or other influence the muscles of the bladder and the bladder outlet. If only part of them will reach clinical usage, it can be assumed that the pharmacotherapy will become even more meaningful in the treatment of neurogenic bladder disorders.


[Quantitative determination of phosphates in urinary calculi and their matrices]. The question whether the phosphate participates actively in the calculogenesis or whether it is only an accidental enclosure in the stone material can, in our opinion, only be answered by the quantitative determination of phosphorus in the stone material resp. in the organic stone matrix. The high enrichment of phosphorus in the stone material, in comparison with the urine, as well as its additional significant enrichment in the stone matrix, compared with the (crystalline) stone material, both deduced from our analytical data, nearly exclude a passive role of the phosphate in the calculogenesis. On the basis of our investigation we suppose that the phosphate crystalluriae, occurring spontaneously or induced, at moderate pH-values of urine, especially by Ca-oxalate, sometimes by urate crystalluriae, contribute to the crystal "density" in the urine, the sufficient crystal "density" being one of the preconditions for the formation of compact crystal aggregates; the other precondition is the presence of the reactive organic cement substance in a sufficient concentration. This active crystal agglomerating component is represented, in our conception, by partially hemolysed blood cells resp. by the cholesterol of their damaged membranes.


[Current considerations on male secretory sterility]. The problems of male sterility are considered in the light of fundamentals underlying the embryology and histophysiology of spermatogenesis and its hormonal control. Some forms of infertility are now understood but no treatment for them has yet been found. The mechanisms of other forms still elude firm definition: this is the case of, among others, some oligoasthenospermias in which, according to our experience, excessive estrogen production appears to play a physiopathologic role. In the prevailing uncertainty about therapeutic methods, the best short-term solutions in this field are in-vitro enhancement of the sperm's fecundating power and the improvement of artificial insemination techniques.


Determinants of oxygen uptake during sodium bicarbonate infusion. Steady-state passive hyperventilation alkalosis produces a predictable increase in oxygen uptake (VO2) proportional to the change in arterial pH (pHa) while variable changes in VO2 have been reported during alkali infusion. To compare metabolic with respiratory alkalosis 17 dogs were anesthetized with halothane and their VO2 response to respiratory alkalosis evaluated by hyperventilation. The pHa measured during this phase was duplicated during the later continuous infusion of NaHCO3 at which time either 1) ventilation was held constant at the control level, allowing arterial carbon dioxide tension (PaCO2) to rise as a consequence of the bicarbonate dissociation, or 2) PaCO2 was held constant by servo control of ventilation. Hyperventilation (pHa 7.6, PaCO2 13 Torr) produced an average increase in VO2 of 24%. During the bicarbonate infusion at constant ventilation (pHa 7.6, PaCO2 45 Torr) VO2 increased only 7%; however, when PACO2 was held constant by servo ventilation VO2 increased 21% above control. We conclude that respiratory and metabolic alkalosis produce similar increases in VO2 when steady-state acid-base conditions are achieved.


Intracellular pH and bicarbonate concentration in human muscle during recovery from exercise. Eight subjects exercised on an ergometer until exhaustion. Femoral venous blood was analyzed for lactate, pyruvate, protein, electrolytes, and acid-base parameters. Muscle samples taken during the recovery period from m. quadriceps femoris were analyzed for water, electrolytes, lactate, and acid-labile CO2. Water content in the muscle biopsy sample was increased after exercise to 78.7 +/- 0.5% compared with the normal 76.7 +/- 0.8% at rest. The distribution of water between the extra- and intracellular space was calculated by the chloride method. In spite of elevated PCO2 in femoral venous blood the content of acid-labile CO2 was decreased in muscle after exercise. One minute after termination of exercise muscle CO2 was about half of the normal content at rest. During the recovery period muscle CO2 increased but was 20 min after termination of exercise still significantly below the value at rest. Intracellular pH (pHi) and bicarbonate concentration ([HCO3-]i) in muscle have been calculated. The validity of the assumptions underlying the calculations are thoroughly discussed. pHi decreased from the normal value at rest, 7.00 +/- 0.06 (mean +/- SD), to about 6.4 after exercise. [HCO3-] decreased from 10.2 +/- 1.2 mmol/l at rest to about 3 mmol/l after exercise. The changes are the greatest so far reported for an in vivo situation. After 20 min recovery pHi was almost the same as at rest, whereas bicarbonate was still well below.


On the governance system of university schools of nursing. We find ourselves as schools of nursing in a period in which external pressures are increasing on the school and the university as the public and its representatives require better accounting for the resources as assigned to the university and access to the decisions as to the use of the resources. At the same time, our systems of governance are not fully functioning and in many cases the dysfunctions consume faculty time rather than releasing time to the developmental tasks. This situation thus should not be ignored but must be dealt with to permit the growth to proceed.


Professional interaction in academic health centers. A survey was made of 106 medical schools to determine the presence of the academic health center concept in the United States. Five centers were selected for an in-depth study of the attitudes and behavior of health center students, faculty members, and administrators in the professions of dentistry, medicine, nursing and basic science. The results were based on the return of 2,399 questionnaires which investigated the attitudes and behavior of the groups described relative to the health center concept. It was found that attitudes and behavior relative to the health center concept were different among professions, among various statuses of people within professions and among those persons functioning within the various health center administrative structures.


A systematic approach to module development. The primary purpose of this article has been to outline and discuss the fourteen steps of the systematic approach to modular development utilized by the author. In addition, examples of each step have been included in an effort to illustrate each point. The article ends with a list of useful resources for the beginning module developer and a statement about the value of each resource.


Time-dependent increase in the stability of collagen fibrils formed in vitro. I. Effects of pH and salt concentration on the dissolution of the fibrils. The time-dependent increase in stability, as measured in terms of the rate of dissolution, of collagen fibrils formed in vitro from pepsin-treated collagen was significantly affected only by temperature, and not by either ionic strength or pH. This is in contrast with collagen fibril formation, a process which is greatly affected by ionic strength and pH. Within the range of temperature 29-37 degrees C, lower temperature caused slower fibril formation and faster fibril stabilization. These results suggest that the intermolecular interactions involved in stabilizing collagen fibrils are entirely different from those involved in fibril formation. Based on kinetic analysis of the dissolution and stabilization of the fibrils, it is proposed that collagen molecules first form unstable fibrils which become gradually stabilized on prolonged incubation, without necessarily introducing covalent cross-links.


Chymotrypsin inhibitors from hemolymph of the silkworm, Bombyx mori. Three new protease inhibitors were isolated and purified about 200-fold from hemolymph of silkworm larvae, Bombyx mori, using ion-exchange and affinity chromatography. Two of the three inhibitors were basic proteins (SCI-I had pI 9.4 and SCI-II had pI 9.6) and one was acidic (SCI-III had pI 4.0). The molecular weight of each inhibitor was determined to be 7,000 by the sedimentation equilibrium method. The amino acid composition of the inhibitors were similar except for the contents of Asp, Glu, Ile, Leu, and Lys. Val, His, and Trp were not present in the inhibitors and Met appeared only in SCI-III. The CD spectra of the inhibitors were all similar and indicated a low content of alpha-helical structure (10% at most). Each inhibitor could inhibit the protease and esterase activities of bovine alpha-chymotrypsin at a one-to-one molar ratio, and the dissociation constants were 3.1 X 10(-9)M for SCI-I and II and 1.3 X 10(-8)M for SCI-III. Only SCI-II showed a weak inhibitory activity against bovine trypsin. Subtilisin BPN' and papain were not inhibited by these inhibitors.


An isoelectric focusing study of acid phosphohydrolases in rat liver lysosomes. The differentiation of rat liver lysosomal acid phosphatase, acid ATPase, acid phosphodiesterase, acid ribonuclease, and acid deoxyribonuclease was studied by isoelectric focusing. To prevent autolytic digestion, inhibitors of cathepsins and neuraminidase were used. The proportion of acidic forms of acid phosphatase, acid ATPase and acid phosphodiesterase was increased by the use of extraction medium containing 0.05% Triton X-100. To investigate the identity of acid ATPase and acid phosphodiesterase, the relative activities among the multiple forms of these enzymes, the acid phosphodiesterase/acid ATPase ratio at each activity peak, and the degree of enzyme inhibition by p-chloromercuriphenyl sulfonic acid were estimated. The results suggest that acid ATPase is not identical with acid phosphodiesterase. With extraction medium free of Triton X-100, acid ribonuclease appeared in two forms. However, in addition to these forms, a new form of this enzyme with a more acidic pI (4.22) emerged when extraction medium containing 0.05% Triton X-100 was used. The major peak of acid deoxyribonuclease with pI=8.40-9.39 was obtained regardless of the extracting method.


Mechanisms of desorption and adsorption of liver cytosolic fumarase to cellular membranous components. The cytosolic fumarase [EC 4.2.1.2[ of rat liver was bound, after dialysis, to the microsomal membrane in vitro. Binding of the enzyme was dependent on pH, and was facilitated in the pH range below 7.5. The binding reaction was completely inhibited by 0.5 mM fumarate, aurintricarboxylate or colchicine. The bound fumarase was released from the membrane by the substrates, isocitrate, citrate or 2,3-diphosphoglycerate at low concentrations. Desorption of the enzyme by metabolites was also dependent on pH, and was more rapid in the alkaline pH range. The enzyme desorption curves were sigmoidal, and kinetic studies suggested a biphasic cooperative mechanism for the action of the metabolites. The apparent desorption constants (concentrations necessary for 50% desorption of the enzyme) estimated at pH 7.3 for isocitrate, 2,3-diphosphoglycerate, L-malate, oxalacetate, fumarate, citrate, succinate, and KCl were 0.073, 0.074, 0.22, 0.39, 0.56, 2.9, and 19 mM, respectively. The bound fumarase showed little enzymatic activity, and its Km and Vmax values were fivefold and 31%, respectively, of those of the free enzyme.


Ribonuclease H from rat liver. I. Partial purification and characterization of nuclear ribonuclease H1. A ribonuclease H, an enzyme that specifically degrades the RNA moiety of RNA-DNA hybrid, has been partially purified from rat liver nuclei and characterized. Neither native or denatured DNA, nor single or double-stranded synthetic polyribonucleotides were degraded by the enzyme. The enzyme possesses a molecular weight of about 36,000 and requires alkaline pH, magnesium ions, and ammonium sulphate for maximum activity. The enzyme acts on the hybrid as an endonuclease, resulting in oligonucleotides with 3'-hydroxyl termini. The properties of this enzyme were distinct from those of the rat liver cytosol enzyme reported by Roewekamp and Sekeris in many respects, such as molecular weight, optimal pH and requirements for divalent cations. Preliminary experiments suggest that the nuclear enzyme is localized in the nucleoplasm and nucleoli. These results indicate that multiple forms of ribonuclease H exist in different regions of rat liver cells.


Ribonuclease H from rat liver. II. Partial purification and characterization of cytosol ribonuclease H1. We have detected in rat liver cytosol three enzymes (termed C-1, C-2, and C-3) which cleaved the RNA moiety of RNA-DNA hybrid. These enzymes were separated from each other by DEAE-Sephadex and Sephadex G-200 chromatography. C-1 and C-2 specifically act on the RNA moiety of RNA-DNA hybrid, while C-3 degrades single-stranded RNA as well as the RNA of the hybrid. The molecular weights of C-1, C-2, and C-3 are about 110,000, 35,000 and 110,000 daltons, respectively, and their activities are absolutely dependent on divalent cations such as Mg2+ and Mn2+. Cleavage by C-1 and C-2 is endonucleolytic, producing mostly oligonucleotides and a small amount of mononucleotides which possess 3'-hydroxyl termini. It seems likely that C-2 is originally present in the nucleus and is released into cytosol because of its loose binding to the nuclear components. As for biochemical properties, C-1 is very similar to the cytosol ribonuclease H initially reported by Roewekamp and Sekeris, and C-2 is very similar to the nuclear ribonuclease H reported by us in the preceding paper.


Multiple attack in porcine pancreatic alpha-amylase-catalyzed hydrolysis of amylose studied with a fluorescence probe. 1. A large fluorescence enhancement of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) observed in the presence of amylose was utilized to monitor quantitatively the time course of porcine pancreatic alpha-amylase [EC 3.2.1.1] (PPA)-catalyzed hydrolysis of amylose with a number-average degree of polymerization of 16.8. 2. The slope of the plot of decrease in the relative fluorescence intensity of the TNS-amylose system (termed as the fluorescence value) versus the number of linkages hydrolyzed (reducing value) (Kondo, H. et al. (1977) Agric. Biol. Chem. 41, 631-634) in the course of PPA-catalyzed hydrolysis was shown to be useful to describe the degree of "multiple attack," which is defined by the number of reattacks on a long chain substrate molecular per one encounter of the enzyme and the substrate. A parameter gamma was defined as the ratio of the reciprocal of the slopes obtained at each pH to that at pH 10.5, where the multiple attack is not operating. 3. The gamma versus pH profile gave an apparent pK value of about 9, indicating that some ionizable groups participate in the multiple attack mechanism. 4. Based on a reaction scheme involving a "sliding" of the substrate molecule on the enzyme, which may contribute to the multiple attack mechanism, besides binding, dissociation, and cleavage steps of the substrate, and on the assumption of the steady state for the enzyme-substrate complex, rate equations were obtained to describe the time course of hydrolysis of a linear substrate. The product distribution with the progress of the reaction can be calculated theoretically, and is dependent on the number of multiple attack and the mode of sliding. The number of multiple attack can be estimated from this distribution, and the fluorescence value can be calculated theoretically by combining the product distribution with the relative efficiency of fluorescence intensity of each maltooligosaccharide (Nakatani, H. et. al. (1977) Biopolymers 16, 2363-2370). By comparing the experimental data with the theoretical ones, it was suggested that the multiple attack occurs through the sliding by maltose unit of the retained fragment on the enzyme, which is one of the fragments produced by the initial cleavage of the substrate molecule. 5. It was found that anions (chloride, bromide, and nitrate ions) which critically affect the enzyme activity have no effect on the degree of multiple attack.


Subunit structure of islets-activating protein (IAP), a new protein isolated from the culture media of Bordetella pertussis. The subunit structure was studied of islets-activating protein (IAP), a new protein recently isolated from the culture media of Bordetella pertussis and possessing a unique action, i.e., potentiating insulin secretory responses of animals, IAP dissociated into three subunits, F-1, F-2, and F-3, when incubated in 8M urea. Three subunits isolated by chromatography on CM-Sepharose and DEAE-Sepharose columns showed different molecular weights (F-1: 44,000, F-2: 20,000, F-3: 11,000) and different isoelectric points, but similar amino acid compositions. The F-1 subunit consisted of two polypeptide chains linked by S-S bonding(s), while the F-2 and F-3 subunits were single-chain peptides. These subunits, none of which was biologically active alone, associated upon incubation for 2 h at 37 degrees C and regained biological activities after association only when the F-3 subunit was present in the association product. Thus, the F-3 subunit was essential, and the F-1 and F-2 subunits were permissive, for the development of IAP activity in animals.


Purification and characterization of L-pyrrolidonecarboxylate peptidase from Bacillus amyloiliquefaciens. Microorganisms capable of producing L-pyrrolidonecarboxylate peptidase [L-pyrrolidonyl peptidase, EC 3.4.11.8] were screened and a strain of Bacillus amyloliquefaciens was chosen as one of the most potent producers of the enzyme. The enzyme was purified from lysozyme-lysate of the bacterial cells by salting out with ammonium sulfate, adsorption on DEAE-cellulose, covalent chromatography on PCMB-Sepharose and by gel filtration on Sephadex G-150. By these procedures, the enzyme was purified about 800-fold with an activity recovery of 9%, and the preparation was electrophoretically homogenous. The enzyme was most active and stable at pH 7-8. The presence of 2-mercaptoethanol and EDTA was effective for stabilizing the enzyme. The molecular weight was estimated to be 72,000 by the gel filtration method and to be 24,000 by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a subunit oligomer, presumably trimer. The enzyme was inactivated by the addition of PCMB, sodium tetrathionate, Hg2+ and Cu2+, but the activity lost was restored by the addition of 2-mercaptoethanol and EDTA. The purified enzyme split amide and ester linkages in L-pyroglutamyl derivatives of L-alanine, beta-naphthylamine, alpha-naphthol, and 4-methylumbelliferone, but was completely inert towards various peptides and esters used as substrates for usual amino- and carboxy-peptidases, and for endopeptidases such as trypsin, subtilisin and alpha-chymotrypsin.


The stereochemical course of acetate activation by yeast acetyl-CoA synthetase. The purified alpha-thiophosphate diastereoisomers of adenosine 5'-(1-thio)-triphosphate were used to study the stereochemical course of the reaction catalyzed by yeast acetyl-CoA synthetase. Asymmetrically labeled adenosine 5'-thiophosphate was formed from the "B" diastereoisomer of adenosine 5'-(1-thio)-triphosphate and [18O]acetate. The label was found to be in the opposite orientation from the leaving pyrophosphate group showing that the acetate activation step occurred with inversion of configuration at the alpha-phosphorus.


The reversible binding of oxygen to sulfhemoglobin. The O2 binding properties of sulfhemoglobin were studied. The oxygen tension required for half-saturation of sulfhemoglobin is more than 2 orders of magnitude higher than that for hemoglobin A. The binding of O2 exhibits an alkaline Bohr effect larger than that observed for hemoglobin, yet the Hill number is unity. From the Bohr titration curve, 0.68 proton is released during O2 binding at 0 degrees C. Sulfhemoglobin prepared from carboxypeptidase A-treated hemoglobin has an affinity for O2 which is about the same as that of sulfhemoglobin at the theoretical limit of the Bohr titration curve. Like its carboxypeptidase A-treated hemoglobin precursor, this sulfhemoglobin does not bind O2 cooperatively. Thus, sulfhemoglobin appears to be in a high affinity form at alkaline pH and a low affinity form at acid pH, similar to hemoglobin A. These results demonstrate that the magnitude of the Hill number is not always an indicator of the interaction between oxygen binding and other functions in a hemoglobin.


Sea anemone toxin and scorpion toxin share a common receptor site associated with the action potential sodium ionophore. Toxin II isolated from the sea anemone Anemonia sulcata enhances activation of the action potential sodium ionophore of electrically excitable neuroblastoma cells by veratridine and batrachotoxin. This heterotropic cooperative effect is identical to that observed previously with scorpion toxin but occurs at a 110-fold higher concentration. Depolarization of the neuroblastoma cells inhibits the effect of sea anemone toxin as observed previously for scorpion toxin. Specific scorpion toxin binding is inhibited by sea anemone toxin with KD approximately equal to 90 nM. These results show that the polypeptides scorpion toxin and sea anemone toxin II share a common receptors site associated with action potential sodium ionophores.


Crystallographic studies on D-amino acid oxidase. D-amino acid oxidase, a flavoprotein from hog kidneys, has been crystalized in two different forms. Orthorhombic prisms have been obtained from the enzyme.benzoate complex at pH 8.3; the space group is C2221 and the cell dimensions are a = 325A, b = 138.8 A, c = 200 A. At lower pH values, the enzyme crystallizes in trigonal prisms with a = b = 116.0 A, c = 399 A, space group P3112 or its enantiomorph. The two crystal forms have been obtained at 28 degrees C while at 4 degrees C only weak evidence of crystallization has been detected. In both crystalline modifications, the protein is highly associated.


Activation of soluble splenic cell guanylate cyclase by prostaglandin endoperoxides and fatty acid hydroperoxides. Purified prostaglandin endoperoxides (PGG2 and PGH2) and hydroperoxides (15-OOH-PGE2) as well as fatty acid hydroperoxides (12-OOH-20:4, 15-00H-20:4, and 13-OOH-18:2) were examined as effectors of soluble splenic cell guanylate cyclase activity. The procedures described (in the miniprint supplement) for the preparation, purification, and characterization of these components circumvented the use of diethyl ether which obscured effects of lipid effectors because of contaminants presumed to be ether peroxides which were stimulatory to the cyclase. Addition of prostaglandin endoperoxides or fatty acid hydroperoxides to the reaction mixture led to a time-dependent activation of guanylate cyclase activity; 2.5- to 5-fold stimulation was seen during the first 6 min. The degree of stimulation and rate of activation were dependent on the concentration of the fatty acid effector; when initial velocities (6 min) were assessed half-maximal stimulation was achieved in the range of 2 to 3 micrometer. However, by extending the incubation time to 90 min similar maximal increases in specific activity could be achieved with 3 or 10 micrometer PGG2 or PGH2. Activation of guanylate cyclase upon addition of prostaglandin endoperoxides or fatty acid hydroperoxides was prevented or reversed by the thiol reductants dithiothreitol (3 to 5 mM) or glutathione (10 to 15 mM). Na2S2O4, not known as an effective reducing agent of disulfides, prevented but was relatively ineffective in reversing activation after it had been induced by PGG2. Pretreatment of the enzyme preparation with increasing concentrations of N-ethylmaleimide in the range of 0.01 to 1.0 mM prevented activation by PGG2 without affecting basal guanylate cyclase activity. These observations indicate that fatty acid hydroperoxides and prostaglandin endoperoxides promote activation of the cyclase by oxidation of enzyme-related thiol functions. In contrast PGE2, PGF2a, hydroxy fatty acids (13-OH-18:2, 12-OH-20:4) as well as saturated (18:0) monoenoic (18:1), dienoic (18:2), and tetraenoic (20:4) fatty acids were ineffective in promoting cyclase activation in the range of 1 to 10 micrometer. Studies to identify the species of the rapidly metabolized prostaglandin endoperoxides that serve as effectors of the cyclase indicated that PGG2 but not 15-OOH-PGE2 (the major buffer-rearrangement product of PGG2) is most likely an activator. In the case of PGH2, a rapidly generated (30 s) metabolite of PGH2 was found which contained a hydroperoxy or endoperoxy functional group and was equally as effective as PGH2 as an apparent activator of the enzyme. The combined effects of PGG2 and dehydroascorbic acid, another class of activator, exhibited additivity with respect to the rate at which the time-dependent activation was induced. These results suggest that activation of soluble guanylate cyclase from splenic cells can be achieved by the oxidation of sulfhydryl groups that may be associated with specific hydrophobic sites of the enzyme or a related regulatory component.


Biochemical studies of isolated glial (Müller) cells from the turtle retina. A method has been developed for the preparation of large numbers of glial (Muller) cells from the turtle retina. After proteolytic dissociation of the retina, Muller cells were separated from retinal neurons by velocity sedimentation at unit gravity. Fractions containing >90 percent morphologically identifiable Muller cells were prepared by this procedure. Fractions containing only Muller cells were obtained by drawing selected cells individually into a micropipette under visual observation. Biochemical analyses of isolated Muller cells showed that (a) these cells did not synthesize and accumulate acetylcholine, gamma-aminobutyric acid, or catecholamines when incubated with appropriate radioactive precursors; (b) the specific activities of choline acetyltransferase (EC 2.3.1.6), glutamate decarboxylase (EC 4.1.1.15), and tyrosine hydroxylase (EC 1.14.16.2) in these cells were less than 2 percent of those found in the retina; (c) Muller cells, however, contained high activities of transmitter degrading enzymes-acetylcholinesterase (EC 3.1.1.7) and gamma-aminobutyrate- transamine (EC 2.6.1.19); and (d) the cells also possessed high levels of two presumably glial-specific-enzymes-glutamine synthetase (EC 6.3.1.2) and carbonic anhydrase (EC 4.2.1.1). These results, together with other findings, suggest that Muller cells are not capable of neurotransmitter syntheses but possess the enzymes necessary for two important roles in the retina: (a) the inactivation of certain transmitters after synaptic transmission by uptake and degradation, and (b) the maintenance of acid-base balance and the provision of a stable microenvironment in the retina by the removal of metabolic products such as carbon dioxide and ammonia.


Unusual responses of rat hepatic and renal peroxisomes to RMI 14, 514, a new hypolipidemic agent. RMI 14, 514 ([5-tetradecycloxy]-2-furancarboxylic acid) represents a new class of hypolipidemic agents which cause unusual ultrastructural changes in liver of male rats and in selected peroxisomal enzymes in liver and kidney of both sexes. Among the principal ultrastructural changes in peroxisomes of male rat liver were (a) cavitation and compartmentalization of the matrix, often giving the appearance of a peroxisome-within-a-peroxisome, and (b) narrow, dense extensions of canaliculi or cisterns from the  periphery of the peroxisome, forming partial circlets or surrounding irregular areas of cytoplasm. The unusual enzyme responses were (a) elevation of catalase activity in liver and kidney in female rats, (b) increased activity of three hydrogen peroxide-producing oxidases (urate oxidase, L-alpha-hydroxy acid oxidase, and D-amino acid oxidase) in the liver of both sexes, and (c) elevation of activity of the last two oxidases in male kidney. The peculiar ultrastructural changes in liver peroxisomes combined with the responses of selected peroxisomal enzymes represent unusual modulations or adaptations of these organelles to a hypolipidemic agent, the effects of which have not been reported extensively.


Aspects of turnover and biogenesis of synaptic vesicles at locust neuromuscular junctions as revealed by zinc iodide-osmium tetroxide (ZIO) reacting with intravesicular SH-groups. Retractor unguis nerve muscle preparations from the locust were subjected to the zinc iodide-osmium tetroxide reaction (ZIO) after pre-fixation in glutaraldehyde. Applied for 18 h at 4 degrees C in the dark, ZIO reacts at pH 4.2--4.0 fairly selectively with the matrix of synaptic vesicles. Approximately 53% of the vesicles are completely and 4% partially stained. The percentage of ZIO-positive vesicles is increased to nearly 90% and reduced to 4% or less by pretreatment with SH-protecting (dithiothreitol) or SH-blocking (N-ethylmaleimide, p-chloromercuriphenyl sulfonic acid) and SH-oxidizing (azodicarboxylic acid-bis-dimethylamide) reagents, respectively. Stimulation of the motor nerve at 20 Hz for 7 min, partially fatiguing synaptic transmission, reduces the number of vesicles per square micrometer of terminal area by approximately 52%; 2 min of rest restores this number of its pre-stimulation level. These changes are chiefly accounted for by changes in the number of completely ZIO-positive vesicles. 2 min after the end of stimulation, partially ZIO-positive vesicles are three times more frequent than before. With all experimental conditions, the average volume of vesicles was as follows: ZIO-negative less than partially ZIO-positive less than completely ZIO-positive. The average volume of ZIO-positive vesicles is almost unaffected by stimulation; that of ZIO-negative vesicles is decreased by 25% immediately after stimulation, increasing with subsequent rest to the initial level after 1 h. It is suggested (a) that ZIO demonstrates intravesicular protein(s) containing SH-groups and (b) that the completely ZIO-positive vesicles represent the mature ones ready to be used for transmitter release. How the ZIO reaction differentiates between different developmental stages of vesicles which could arise from the smooth endoplasmic reticulum is discussed.


Ionic interactions between bovine chymotrypsinogen A and chondroitin sulfate A.B.C.. A possible model for molecular aggregation in zymogen granules. The formation of large aggregates by ionic interactions between acidic glucosaminoglycans and cationic secretory proteins has been proposed as one of the critical steps in the concentration process in the condensing vacuoles of secretory cells. In this paper, this hypothesis was tested by studies on the interactions between bovine chymotrypsinogen A and chondroitin sulfate as a simplified model. Small amounts of chondroitin sulfate were found able to induce chymotrypsinogen precipitation. Like zymogen granules, the resulting aggregates were moderately sensitive to ionic strength and insensitive to osmolality. Moreover, their pH dependence was similar to that of isolated zymogen granules. When sulfated glucosaminoglycans isolated from the zymogen granules of the guinea pig pancreas were used instead of chondroitin sulfate, the same kind of interactions with chymotrypsinogen were obtained. Our data support the hypothesis that the strong ionic interactions between those sulfated glucosaminoglycans and cationic proteins could be responsible for the concentration process.


Red cell aging. II. Anomalous electrophoretic properties of neuraminidase treated human erythrocytes. Desialylation of human red blood cells (RBC) by Vibrio cholerae neuraminidase (VCN) was found to produce cells with electrophoretic properties which were inconsistent with the view of simple loss of N-acetylneuraminic acid (NANA) as the sole effect of VCN treatment. Modification of human RBC with 50--350 U VCN/10(10) RBC for one hour at 37 degrees C releases 90-100% of the NANA and produces a progressive decrease towards zero in their electrophoretic mobilities when measured in 0.15 M NaCl (pH 7.2) at 25 degrees C. The appearance of positive groups on the desialylated cells was indicated by the VCN-treated cells displaying positive mobilities below approximately pH 5.5 and increased negative mobilities at approximately pH 9 as well as substantial increases in their mobility at neutral pH following treatment with formaldehyde. Adsorption of about 95% of the VCN activity at 0 degrees C to the RBC did not produce any significant change in their electrophoretic mobilities thus indicating that the observed changes in the electrophoretic properties of the RBC following VCN treatment could not be attributable to adsorption of VCN. These studies indicate that the cationic charge groups which appear at the electrophoretic surface of the RBC after VCN treatment are probably of endogenous origin. It is suggested that this alteration rather than simple NANA release may operate to shorten the in vivo survival time of desialylated red cells.


Analysis of lorazepam and its glucuronide metabolite by electron-capture gas--liquid chromatography. Use in pharmacokinetic studies of lorazepam. This paper describes a rapid and sensitive method for analysis of lorazepam and its glucuronide metabolite in plasma and urine following therapeutic doses of lorazepam in humans. After addition of the structurally related benzodiazepine derivative, oxazepam, as the internal standard, 1-ml samples of plasma or urine are extracted twice at neutral pH with benzene (containing 1.5% isoamyl alcohol). The combined extracts are evaporated to dryness, reconstituted, and subjected to gas chromatographic analysis using a 3% OV-17 column and an electron-capture detector. Lorazepam glucuronide in urine is similarly analyzed following enzymatic cleavage with Glusulase. The sensitivity limits are 1--3 ng of analyzed following enzymatic cleavage with Glusulase. The sensitivity limits are 1--3 ng of lorazepam per ml of original sample, and the variability of identical samples is 5% or less. The applicability of the method to pharmacokinetic studies of lorazepam is demonstrated.


Isoelectric focusing of the phosphoprotein of rat-incisor dentin in ampholine and acid pH gradients. Evidence for carrier ampholyte-protein complexes. Rat-incisor phosphoprotein (RIP) has been subjected to isoelectric focusing at 4 degrees in (a) an Ampholine pH gradient of 2.5-4 and (b) an acid pH gradient created by electrolysis of a system of acids and acidic ampholytes and covering the pH range 0.5-3.5. In the Ampholine gradient, the RIP unexpectedly formed several adjacent and strongly opalescent bands in the pH range 2.5-3.1. These bands, which migrated slowly toward the anode on prolonged focusing, are interpreted as being the result of an interaction between the amino groups of the Ampholine and the numerous phosphate groups of the protein. In the acid pH gradient, the RIP focused into one narrow zone corresponding to an isoelectric pH of 1.1 at 4 degrees. This value is consistent with the amino-acid composition and the phosphate content of the protein,


Preparation of prereduced anaerobically sterilized media and their use in cultivation of anaerobic bacteria. Several modifications of the roll-tube method have made it simpler for routine use in the isolation and growth of anaerobic bacteria. These include use of a check valve for the production of prereduced anaerobically sterilized media; a Salvarsan tube under oxygen-free gas pressure for the dispensing of molten prereduced anaerobically sterilized agar medium; a Kelly infusion bottle with a graduated pipette side arm (also under gas pressure) for quantitative delivery of fluid prereduced anaerobically sterilized media; and screw-capped prescription bottles for the cultivation of anaerobes. Colonies of Bacteroides melaninogenicus were easily identified and counted by this method. Other anaerobic bacteria have also been cultivated successfully.


Immunoelectrophoresis for detection of polysaccharides in immune complexes. A procedure for detecting pneumococcal capsular polysaccharides in immune complexes is described. Separation of antigen from immune complexes is achieved by electrophoresis at 56 degrees C.


Primary isolation media for Legionnaires disease bacterium. Yolk sac suspensions infected with the Legionnaires disease bacterium (LDB) were plated onto 17 different bacteriological agar media. The LDB grew only on Mueller-Hinton agar supplemented with 1% Iso Vitale X and 1% hemoglobin (MH-IH). This medium was subsequently analyzed to determine the components required to support growth of the LDB. L-Cysteine hydrochloride can replace the Iso Vitale X reagent, and soluble ferric pyrophosphate can replace hemoglobin. A new medium, F-G agar, was formulated incorporating these chemicals. Different cultures conditions (oxygen tension, temperature, and pH) were also evaluated. The LDB grew optimally at 35 degrees C under 2.5% CO2 on the F-G agar adjusted to pH 6.9. When infected tissues were inoculated onto both F-G agar and MH-IH, the F-G agar produced colonies of the LDB more rapidly and in greater numbers than did MH-IH.


The stimulus-secretion coupling of glucose-induced insulin release. Metabolic and functional effects of NH4+ in rat islets. NH4+ caused a dose-related, rapid, and reversible inhibition of glucose-stimulated insulin release by isolated rat islets. It also inhibited glyceraldehyde-, Ba2+-, and sulfonylurea-stimulated insulun secretion. NH4+ failed to affect glucose utilization and oxidation, glucose-stimulated proinsulin biosynthesis, the concentration of ATP, AD, and AMP, and the intracellular pH. NH4+ also failed to affect the ability of theophylline and cytochalasin B to augment glucose-induced insulin release. However, in the presence and absence of glucose, accumulation of NH4+ in islet cells was associated with a fall in the concentration of NADH and HADPH and a concomitant alteration of 86Rb+ and 45Ca2+ (or 133Ba2+) handling. These findings suggest that reduced pyridine nucleotides, generated by the metabolism of endogenous of exogenous nutrients, may modulate ionophoretic processes in the islet cells and by doing so, affect the net uptake of Ca2+ and subsequent release of insulin.


Reproduction and breeding of goats.

Reproduction and breeding of goats. Reproduction and genetics of the goat are reviewed with a view of increasing their contribution to mankind. The goat contributes most in tropical regions (within 30 degrees of the equator). The most important product from the goat is milk with meat a close second. Other products are minor. Reproductive rate is a problem only with the Angora goat, but increased reproduction with any type of goat would contribute to improved efficiency. Also, a knowledge of the reproductive phenomenon is necessary for effective management. Genetic studies of goats are limited, but this should not limit improvement programs. Excellent genotypes for producing milk and fiber are available, but adaptation to tropical conditions is needed. Even within temperature regions, there is little evidence of progress in breeding for milk production. Little has been done on the development of the goat as a meat animal. Also, research on crossbreeding for milk or meat production is limited.


Efficacy  of intravenous pretesting and antihistamine prophylaxis in radiocontrast media--sensitive patients. Intravenous pretesting with radiocontrast media (RCM) was performed in 204 RCM-sensitive patients considered for repeat contrast radiography. Group 1 had vague histories of prior anaphylactoid reaction and negative pretests, and 2 of 41 (4.9%) had reactions upon contrast radiography. Groups 2 to 5 had definite histories of prior anaphylactoid reaction. Group 2 had the radiographic study cancelled: 18 of 21 (85.7%) had positive pretests. Group 3 had positive pretests and underwent contrast radiography, and 9 of 15 (60%) had reactions despite premedication. Group 4 (no premedication) and group 5 (diphenhydramine premedication) had negative pretests, but 11 of 53 (20.7%) and 3 of 71 (4.2%), respectively, developed reactions (p less than 0.001). The reaction frequency in group 3 (posivie pretest) of 12 of 18 (66.7%) was greater than that in groups 4 and 5 (negative pretest) combined (14 of 124 (11.3%), p less than 0.001). Intravenous pretesting identified a high-risk group and diphenhydramine premedication decreased the frequency of reaction in patients sensitive to radiocontrast media.


[Therapeutics basis of threatened abortions (author's transl)]. The diverse hormonal and vascular medications are studied in the way of their mechanism of action, of their efficiency and their side effects on the mother as well as on the fetus. Neither definite argument, nor important statistical study has been made after elimination of all genetics and chromosomic anomalies. beta-mimetic, alphablocking and progesterone medications are the most efficient and certainly the less dangerous for the fetus.


Frequencies of pneumococcal types causing serious infections in patients admitted to the Radcliffe Infirmary, Oxford, 1969-77. During a 7 1/2 year period pneumococci were isolated from body fluids of 124 patients at the Radcliffe Infirmary, Oxford -72 with pneumonia, 26 with meningitis and 26 with other serious infections. Eighty-one (65%) of the patients were over 50, and 33 (27%) were over 70 years old. Of the 124 pneumococcal strains 104 (84%), including 23 (79%) of those from patients who died, belonged to types included in the vaccines successfully used in South Africa and in Papua New Guinea. The relative frequencies of types in the Oxford series and in a larger British series agreed closely with those found in a recent survey of 3644 bacteraemic pneumococcal infections in 10 American cities. Any polyvalent pneumococcal vaccine licensed for use in the United States is thus likely to be relevant to the situation in Britain.


Effect of splenectomy on the expression of regulatory T cell activity. The effect of adult splenectomy on the expression of suppressor and amplifier T cell activity was examined with respect to the serum antibody response to Type III pneumococcal polysaccharide (SSS-III) by using a sensitive radioimmunoassay. Suppressor T cell activity, as measured by the degree of low-dose paralysis induced, was not impaired in the least by splenectomy; however, amplifier T cell activity was almost completely eliminated within 7 days after splenectomy. These findings indicate that suppressor T cell activity is not confined solely to the spleen, the major site of antibody synthesis after immunization with SSS-III, and that the spleen may be an important site for the generation and/or maintenance of amplifier T cell activity.


An immunoprecipitation-dissociation technique for large scale antibody purification and an antigen consumption electroimmunoassay for antibody quantitation. A model study with antibodies to pregnancy zone protein. A simple immunoprecipitation--dissociation technique for large scale purification of antibodies is described, which comprises selective denaturation of the antigen and recovery of the antibody fraction by exclusion chromatography at low pH. Its use is illustrated by the purification of antibodies to pregnancy zone protein. A purification factor of about 60 was achieved. An antigen consumption electroimmunoassay was also developed which permits quantitative determination of the antigen binding activity of antibodies with a given specificity. The methods have general application.


Organic solvents and temperature effects on desorption from immunoadsorbents. DNP-BSA anti-DNP as a model. Conditions for desorbing a DNP carrier from its immunoadsorbent were studied in various hydro-organic media at different pHs and temperatures, including the sub-zero range. It appeared that DNP--anti-DNP interaction is the result of a balance between various bonds and the best result (95% yield) was obtained in hydro-organic solvent at high pH and +30 degrees C.


The effect of calcium and magnesium on the spontaneous release of transmitter at insect motor nerve terminals. 1. The effect of the extracellular calcium and magnesium concentrations and calcium ionophore, X-537A, on the frequency of miniature excitatory post-synaptic potentials (MEPSPs) was studied in cockroach leg muscle fibres. 2. The frequency of MEPSPs increased as the calcium concentration was increased from 0.1 to 10 mM. In the presence of 10 mM magnesium, however, raising the calcium concentration from 0.1 to 1 mM slightly depressed the frequency. In saline containing elevated potassium (20.8 mM), increasing the calcium concentration produced a much higher frequency than that in the normal potassium saline (10.8 mM) in the absence of magnesium. Raising the extracellular potassium concentration was without effect unless the bathing solution contained calcium. 3. The frequency of the miniature potentials was reduced as the magnesium concentration was raised from 0 to 10 mM, depending on the presence of calcium ions. On the contrary, a slightly increased frequency was observed in the low calcium saline as the magnesium concentration was raised from 1 to 10 mM. The reciprocal relationship between calcium and magnesium and the time course of the effect suggest that both ions act at the same surface sites in the presynaptic membrane. 4. X-537A elicited a transient increase in frequency followed by a fall of the frequency to a very low rate. Further application of the ionophore was without effect. The effect of X-537A on the spontaneous release of transmitter at the insect neuromuscular junction was comparable with that on the spontaneous acetylcholine release in vertebrate neuromuscular junctions.


Secretion of lysosomal hydrolases by stimulated and nonstimulated macrophages. Peritoneal macrophages were obtained from untreated mice and from mice treated with thioglycollate medium (TA), proteose peptone medium (PP), or a suspension of streptococcus A cell wall material (SA). The biochemical and secretory properties of these cells in long term cultures (up to 2 wk) were compared. TA-elicited macrophages contained more protein, lactate dehydrogenase, lysosomal hydrolases, and in particular, more plasminogen activator than the other cells studied. All types of macrophages studied were found to release considerable amounts of lysosomal hydrolases (beta-glucuronidase, N-acetyl-beta-glucosaminidase, alpha-mannosidase, and acid phosphatase) into the medium. Release was independent of phagocytosis and must, therefore, be regarded as true secretion. In both elicited and nonelicited macrophages, the rates of lysosomal enzyme secretion were virtually identical in the presence and in the absence of serum, and they were not enhanced by increasing serum concentrations. Lysosomal enzyme secretion in macrophages appears to depend on protein synthesis, since it was blocked by low concentrations of cycloheximide which neither affected cell viability nor lowered the intracellular enzyme levels. The amounts of lysosomal hydrolases secreted were highest in TA-elicited macrophages. The rates of secretion of PP- or SA-elicited and of nonelicited macrophages were about one-fourth of that of the TA-elicited cells. This difference, although significant, is much smaller than that observed for the secretion of plasminogen activator which was 20-50 times higher in TA-elicited cells. Acid glycosidases were also found in the peritoneal lavage media used for cell harvesting from both treated and nontreated mice. This indicates that active secretion of lysosomal hydrolases may be an in vivo property of the macrophage.


T-lymphocyte heterogeneity in the rat: separation of functional subpopulations using a monoclonal antibody. W3/25 antibody is the monoclonal product of a hybrid cell resulting from the fusion of a mouse myeloma cell line with spleen cells from a mouse immunized with rat thymocytes. Pure clones have been derived, and segregants free of parental myeloma chains have been isolated. Previous studies have shown that this antibody recognizes a subpopulation of T cells among rat thoracic duct lymphocytes. In the work reported here, three T-cell functions were assayed after separating rat thoracic duct lymphocytes on the fluorescence-activated cell sorter on the basis of labeling with W3/25 antibody. Two of the functional activities appeared to be completely segregated by this procedure. Thus, helper cell activity for an anti-hapten plaque-forming cell response was confined to the labeled population, whereas the allogeneic suppressive effect produced in a parental vector F1 adoptive transfer was mediated by cells in the unlabeled fraction. The third function, graft-versus-host activity, was almost entirely contained within the labeled subpopulation. It is concluded that the antigenic determinant recognized by the monoclonal antibody W3/25 is a differentiation marker for T-cell functional subpopulations.


Demonstration of opsonic activity and in vivo protection against group B streptococci type III by Streptococcus pneumoniae type 14 antisera. The present studies demonstrate that antisera directed against Streptococcus pneumoniae type 14 is opsonic for group B streptococci type III in a neutrophile-mediated bactericidal assay. Specificity was demonstrated by the observations that group B streptococci type III and S. pneumoniae type 14 adsorbed the opsonic activity of anti-S. pneumoniae type 14 antisera. Group B streptococci strain 090R (devoid of type antigens) and S. pneumoniae type 3, did not remove the opsonic activity of anti-S. pneumoniae type 14 serum. In vivo studies using a suckling rat model of neonatal group B streptococcal type III sepsis demonstrated that antisera directed against S. pneumoniae type 14 was highly protective.


In vitro studies on lymphocyte differentiation. I. Long term in vitro culture of cells giving rise to functional lymphocytes in irradiated mice. In vitro cultures of mouse bone marrow cells, maintained for periods up to 7 wk, were shown to contain cells able to repopulate irradiated hosts with T and B lymphocytes. The lymphocytes were fully functional and there did not appear to be any gross restriction of their receptor repertoire. The cultured cells reconstituted irradiated semiallogenic hosts without evidence of graft-versus-host disease, suggesting that culture of donor marrow might be a useful preliminary to transplantation when tissue matching is incomplete.


Fat digestion in the stomach of premature infants. I. Characteristics of lipase activity. Lipolytic activity was studied in gastric aspirates of 13 premature infants of birth weight 1,050 to 1,786 gm. All infants received a diet of infant formula fed by gastric tube. Gastric aspirates were collected after irrigating the stomach with 2 to 5 ml sterile saline before regular feeding. Lipolytic activity, tested with doubly labeled 3H glyceryl-14 C tripalmitin substrate, was 55.6 +/- 11.7 n mol/min/ml (range 4.2 to 140). The lipolytic activity had a pH optimum of 5.4 and produced partial glycerides (mono and diglycerides), glycerol, and free fatty acids. Lipolysis was inhibited by bile salts. Our findings show that in premature infants, as in adults, digestion of dietary fat starts in the stomach. Since bile salt concentrations are low in premature infants, the amphiphilic reaction products formed (monoglyceride and FFA) could play a significant role in the stabilization of lipid emulsions.


Pharmacokinetics of flunitrazepam following single- and multiple-dose oral administration to healthy human subjects. Healthy human subjects received single and multiple oral doses of flunitrazepam. Absorption and disposition were first order and reproducible from administration. The oral doses were virtually completely available to the liver, and elimination from the body occurred entirely via metabolism. Assuming the liver to be the sole eliminating organ, hepatic blood clearance and extraction ratio were approximately 0.235 liter/hr/kg and 0.154, respectively. Steady-state blood volume of distribution averaged 3.76 liters/kg in the single-dose studies. Terminal exponential half-lives from the single- and multiple-dose studies (different subjects) averaged 13.5 and 19.2 hr, respectively; these differences were not due to clearance changes but were entirely attributable to variations in volumes of distribution.


Instability of digoxin in acid medium using a nonisotopic method. A selective nonisotopic assay was used to investigate the digoxin hydrolysis rates at 37 +/- 0.1 degrees over the pH 1.1--2.2 range. The colorimetric method adopted is based on the use of a xanthydrol reagent after extraction with chloroform. The spectrofluorometric method specified in the dissolution test for digoxin tablets was nonspecific because of digoxigenin interference. Digoxin hydrolysis followed specific acid hydrolysis, and K values of the apparent first-order reaction varied from 0.0357 to 0.0027 min-1 over the pH range used. The effect of the dissolution medium on digoxin stability during the dissolution tests of the tablets also was studied. Water (the BP medium) and 0.6% HCl (the USP medium) were compared using the fluorometric method and the xanthydrol method. In the USP medium (pH 1.3), no hydrolysis was revealed by the fluorometric estimation whereas the xanthydrol method showed about 74% hydrolysis. In water, the two methods revealed no hydrolysis. The extent of hydrolysis after 1 hr in the USP medium was studied using three brands of digoxin tablets of differing dissolution characteristics. The fast dissolving brand showed relatively more hydrolysis than the slow dissolving tablets.


Ditheophylline succinate: transfer of theophylline across everted rat intestinal sacs. The cumulative theophylline transfer rate across 10-cm everted rat intestinal sacs incubated at 37 degrees in pH 7.4 Krebs phosphate buffer was determined. A suspension of ditheophylline succinate ( a potential prodrug of theophylline) and a solution of theophylline at equimolar concentration were evaluated to determine the magnitude of the difference between the cumulative theophylline transfer rates from the two preparations. A linear concentration dependency for the rate across the intestinal wall was evidenced. The theophylline formation rate from ditheophylline succinate suspended in pH 7.4 Krebs buffer at 37 degrees followed apparent zero-order kinetics. The observed difference (fourfold) between the cumulative transfer rates estimated for the theophylline solution and the ditheophylline succinate suspention was attributed to the prevailing theophylline concentration in the mucosal solutions. The biopharmaceutical implication of these observations are discussed.


Distribution of camazepam in rats and mice. Camazepam, 5 mg/kg iv, was injected in rats and mice to study its distribution in the blood and brain. Peak blood levels were about 0.9 microgram/ml in rats and 0.6 microgram/ml in mice. Peak brain levels were about 1.5 microgram/g in rats and 0.8 microgram/g in mice. The apparent blood half-life of camazepam was 9 min in mice and 20 min in rats.


Inhibition of epinephrine oxidation in weak alkaline solutions. The effect of sodium metabisulfite and cysteine on the oxidation of epinephrine in weak alkaline solutions was studied. The fluorescent intensity of the reacting mixture also was measured. Sodium metabisulfite in 2 x 10(-3) M NaOH at concentrations lower than 3 x 10(-5) M had no effect on epinephrine oxidation; however, in concentrations from 5 x 10(-5) to 3 x 10(-4) M, it greatly accelerated this process. Its inhibitory action appeared only in concentrations greater than 5 x 10(-4) M. The mechanism of these reactions also was explained. Cysteine in 2 x 10(-3) M NaOH inhibited the oxidation of epinephrine even in concentrations of 7 x 10(-5) M.


The effect of neuroleptics on acetylcholine concentration and choline uptake in striatum: Implications for regulation of acetylcholine metabolism. It has previously been shown that neuroleptic drugs block an apparently inhibitory influence of dopamine on cholinergic interneurons in striatum, thereby increasing acetylcholine turnover. In this study, systemic administration of the neuroleptic, fluphenazine, decreased the acetylcholine content in the striatum but not the neocortex of rats killed by focussed microwave irradiation. The effect was observed with doses of fluphenazine as low as 0.05 mg/kg, and was also seen after two other neuroleptics, spiroperidol (1 mg/kg) and haloperidol (4 mg/kg). In contrast, neither fluphenazine nor haloperidol pretreatment had any effect on the high affinity accumulation of choline by striatal synaptosomes. These observations suggest that after administration of dopamine receptor antagonists the release and metabolism of acetylcholine in the striatum is increased, but that a compensatory increase in choline uptake does not occur, thereby resulting in a temporary decrease in acetylcholine concentration. On the basis of these findings, we conclude that acetylcholine synthesis is regulated differently in the striatum than in other brain regions.


The effects of several narcotic analgesics on brain levels of 3-methoxy-4-hydroxyphenylethylene glycol sulfate in the rat. The acute administration of levorphanol, morphine, anileridine, methadone, cyclazocine and pentazocine was found to increase brain levels of 3-methoxy-4-hydroxyphenylethylene glycol sulfate (MOPEG-SO4) in rats. Drug-induced increases in brain levels of this norepinephrine metabolite were dose-dependent and peak drug effects generally occurred 1 hr after intraperitoneal injection. Six to 8 hr after opiate agonist or partial agonist administration, MOEG-SO4 levels returned to control values or below. The narcotic effect appeared to be stereospecific, since high doses of d-methadone and dextrorphan produced either no change or only minimal increases in brain MOPEG-SO4 levels when compared with saline-treated controls. The relative potency of the analgesics tested in producing increases in brain MOPEG-SO4 levels appeared to correlate reasonably well with the ability of these drugs to produce other characteristic pharmacological effects. The drug-induced increases in brain MOPEG-SO4 levels were antagonized by naloxone. The rate of disappearance of MOPEG-SO4 from the brains of animals treated with pargyline was not decreased by the opiate agonists or partial agonists tested indicating that these drugs did not inhibit the acid transport process. These results indicate that the production of an increase in brain norepinephrine turnover is a specific component of the pharmacological actions of narcotic analgesics.


In vivo desensitization to beta receptor mediated bronchodilator drugs in the rat: decreased beta receptor affinity. When tracheas were isolated from rats pretreated with isoproterenol (ISO) or terbutaline, they were found to be considerably less sensitive to the relaxant action of ISO than tracheas which were isolated from saline-pretreated rats. The dissociation constant (Kb) for the propranolol-beta receptor complex was determined to be up to 400-fold larger in the tracheas isolated from beta agonist-pretreated rats (1.1 +/- 0.1 X 10(-6) M) than in tracheas isolated from saline-pretreated rats (3.0 +/- 0.3 X 10(-9) M). The longer the duration of pretreatment and the higher the dose of ISO or terbutaline used, the more attenuated was the response of tracheal smooth muscle to ISO, and the greater was the Kb for propranolol-beta receptor complex. These findings provide strong evidence which shows that desensitization, which occurs as a result of in vivo pretreatment with beta agonist drugs, results from pronounced reduction in this affinity of the beta receptors for beta agonist drugs. We observed that the in vivo treatment of rats with aminophylline (Amino), a phosphodiesterase inhibitor, did not affect the responsiveness of their isolated tracheas to either ISO or Amino. In addition, the responsiveness to Amino was determined in tracheal preparations taken from rats desensitized to ISO in vivo. The response to ISO was attenuated and the Kb for the propranolol-beta receptor complex was elevated (1.1 +/- 0.1 X 10(-6) M); however, Amino was half as effective in these tissues as in the saline control tissues. It is postulated, therefore, that the intracellular enzymes controlling the levels of cyclic adenosine monophosphate may be affected by the ISO-induced desensitization process, but are not affected by pretreatment with Amino.


Post-tetanic enhancement of stimulus-induced muscarinic afterdischarge in the rat superior cervical ganglion. The enhancement of asynchronous muscarinic ganglionic firing which follows a preganglionic nerve stimulus volley by high frequency repetitive conditioning stimuli was studied in the rat isolated superior cervical ganglion. Muscarinic afterdischarge which occurred in chlorisondamine-blocked ganglia was enhanced for up to 1 hr after a 40 Hz conditioning volley lasting 7.5 to 30 sec. Enhancement did not occur when release of acetylcholine was blocked by reducing the calcium in the modified Krebs' solution or when magnesium or manganese chlorides were added to the saline during the conditioning period. Metabolic inhibitors, dinitrophenol, sodium azide and ouabain, blocked the enhancement process but not the asynchronous muscarinic firing. Phenytoin, 10(-6) M, did not reduce the enhancement of firing. Dopamine, 5 X 10(-5) M, had no effect on muscarinic afterdischarge, but dibutyryl cyclic adenosine 3':5'-monophosphate, 5 and 10 X 10(=3) M, caused a 43 and 53% increase in the maximum amplitude of afterdischarge. Dibutyryl cyclic guanosine 3':5'-monophosphate had no effect on post-tetanic enhancement or afterdischarge in concentrations up to 1 X 10(-3) M. A working hypothesis is proposed: following a conditioning stimulus, a prolonged postsynaptic change occurs which results in an increased responsiveness to muscarinic agonists, and this change probably involves a metabolic reaction since it is reduced by metabolic inhibitors. Evidence to support the concept that dopamine is a modulator of enhancement in the rat superior cervical ganglion was not obtained.


Effect of luminal pH on acid secretion from Heidenhain pouches evoked by topical and parenteral stimulants. 1. An apparatus for intragastric titration has been devised and its validity tested. Both when attached to a beaker simulating a pouch and when attached to a pouch whose secretion was suppressed by infusing cimetidine, the apparatus accurately measured added acid when the endpoint setting was between pH 3.0 and 9.0. At pH 2.0 and 1.0 with liver extract and at pH 1.0 with saline, the amount of acid added was markedly underestimated.2. In dogs with vagally denervated pouches, during stimulation by I.V. infusion of histamine or pentagastrin, the rate of acid secretion as measured by intrapouch titration was uninfluenced by changes in luminal pH between 2.0 and 9.0. The apparent decrease in acid secretion at pH 1.0 could be shown to be due entirely to artifact in that no change in acid secretion was found when the gain in mass of acid was simultaneously measured by using a non-absorbable dilution indicator to measure volume gain and titration of samples to pH 7.0 to measure acid concentration.3. During stimulation of acid secretion by solutions of liver extract or of L-histidine instilled into the pouch, the rate of acid secretion was found to increase markedly as pH was increased from 3.0 to 9.0 thus confirming our earlier findings.4. We conclude that while stimulation of acid secretion by topical stimulants is highly dependent on luminal pH, secretion increasing as pH increases, stimulation by parenteral agents such as histamine and pentagastrin is not influenced by luminal pH in the range from pH 1.0 to 9.0.


Renal bicarbonate reabsorption in the new-born dog. 1. Renal bicarbonate reabsorption was measured in thirty new-born dogs 2-27 days of age. Plasma bicarbonate was varied in the puppies by exchanging their blood with blood containing high levels of bicarbonate and low levels of chloride.2. The exchange transfusion resulted in increases of plasma pH, P(CO2) and bicarbonate in the puppies without changing plasma sodium and potassium or glomerular filtration rate (g.f.r.) and body weight.3. There was no tubular reabsorption maximum (T(m)) for bicarbonate and reabsorption values as high as 50 muequiv/ml. g.f.r. could be attained. No animals excreted bicarbonate at plasma levels below 25 mM and some animals had plasma bicarbonate threshold values in excess of 40 mM.4. Bicarbonate reabsorption increased as arterial P(CO2) rose (r = 0.62) but this was due to the rise of filtered bicarbonate since (a) there was no correlation between arterial P(CO2) and bicarbonate reabsorption when factored by filtered bicarbonate and (b) lowering arterial P(CO2) by mechanical hyperventilation did not reduce bicarbonate reabsorption corrected for filtered load.5. Inhibition of renal carbonic anhydrase by acetazolamide (50 mg/kg) resulted in an inhibition of bicarbonate reabsorption of only 4.5 muequiv/ml. g.f.r. (less than 20% of the total). Even with renal carbonic anhydrase inhibited, there was no bicarbonate T(m) and bicarbonate reabsorption values as high as 40 muequiv/ml. g.f.r. could be attained.6. There was good correlation (r = 0.82) between inhibition of sodium and bicarbonate reabsorption during renal carbonic anhydrase inhibition. However, with carbonic anhydrase inhibited, there was no correlation between arterial P(CO2) and bicarbonate reabsorption.7. These results demonstrate that tubular bicarbonate reabsorption mechanisms in the new-born dog are as efficient as those reported for the adult as long as body fluid and plasma sodium and potassium levels are carefully maintained.8. The results are also consistent with a bicarbonate reabsorptive mechanism explained either by direct ionic bicarbonate reabsorption or by hydrogen ion secretion with diffusion of carbonic acid.


Glutamine and glutamic acid uptake by rat renal brushborder membrane vesicles. Glutamine uptake by rat renal brushborder vesicles occurred via two distinct saturable processes with Km values of 0.145 and 8.5 mM which were stimulated by both ionic and sodium gradients with a pH optimum of 6.8--7.1. Glutamic acid uptake also occurred by a two-component system with Km values of 0.016 and 3.60 mM. Both components were stimulated specifically by a sodium gradient. The low Km system for glutamic acid had a pH optimum of 7.2--7.4. Glutamine entry at 0.06 mM was inhibited by a variety of amino acids at 3 mM, including dibasic amino acids, glycine, valine, and phenylalanine. Glutamic acid entry at 0.06 mM was inhibited 20--30% by 3 mM phenylalanine, valine, alpha-aminoisobutyric acid, and glutamine. No metabolic alteration of glutamic acid was observed on incubation with membrane vesicles, but glutamine was significantly hydrolyzed to glutamic acid upon prolonged incubation. Hydrolysis of glutamine was negligible at 15 sec incubation which was employed for measurement of initial rate of entry. These studies provide support for the existence of an uptake system in the brushborder of the renal proximal tubule cell capable of handling the reabsorption of glutamine normally present in glomerular filtrate.


Cryptorchidism and testicular neoplasia. A retrospective review of 20 cases of testicular neoplasia in cryptorchid patients is presented. These cases serve to emphasize the need for more effective diagnosis and management of the undescended testis. The various clinical presentations, diagnostic modalities, prognostic indicators, and types of management are reviewed. Due to its inaccessibility and array of clinical presentations, the intraabdominal testies tumor continues to be a diagnostic challenge. Strict attention to diagnostic cues and adherence to the management practices presented here would hopefully result in an overall decrease in testis tumors in the post-pubertal male.


Complement activation and hematologic, hemodynamic, and respiratory reactions early after soft-tissue injury. The effect of soft-tissue trauma was studied in dogs. Following injuries to the hind leg an aggregation of thrombocytes in blood and trapping in the lung was noted. Injury was initially followed by leukopenia and later by leukocytosis. Early hemolysis of red cells was observed. The injury was accompanied by complement activation. Its possible relation to hemolysis, leukopenia, thrombocytopenia, and increased insufflation pressure is discussed.


The congenitally dilated prostatic utricle. A case of utricular dilatation in a child with recurrent urinary tract infection and associated genitourinary anomalies is presented. The embryology of the utricle and the relationship of the dilated utricle to the müllerian duct regression factor are discussed. Clinical sequelae, diagnosis and management are outlined briefly.


Cardiovascular effects of isoproterenol and possible antagonistic actions of propranolol and perhexiline. A beta-adrenergic blocking activity of perhexiline was compared with that of propanolol. In the Langendorff's preparation of rabbits, canine heart-lung preparations, open-chest dog preparations and hind-limbs of dogs, perhexiline failed to block isoproterenol-induced changes in cardiovascular functions, which were effectively blocked by propranolol. These data suggest that perhexiline is not a beta-adrenergic receptor blocking agent.


[Gamma-glutamyl transpeptidase activity in ischemic heart disease]. The blood plasma gamma-glutamyltranspeptidase (GGTP) activity was studied in 133 patients with macrofocal myocardial infarction, in 40 patients with microfocal myocardial infarction, in 30 patients with angina pectoris, and in 75 patients with cardiosclerosis and congestive cardiac failure. The activity of the enzyme increased in most patients with macrofocal myocardial infarction and in less than half of those with microfocal myocardial infarction beginning with the 3rd or 4th day, reached maximum by the 6th to 8th day of the disease, and then returned to normal levels in various lengths of time. In all patients with angina pectoris and acute left-ventricular failure the activity of the enzyme remained normal. It may be assumed from the results of the study that determination of GGTP activity in dynamics may be mainly employed in the diagnosis of macrofocal myocardial infarction, particularly after the first days of the disease. The enzyme test is hardly suitable for differential diagnosis between microfocal myocardial infarction and angina pectoris.


Physicians' assistants on a university cardiothoracic surgical service. A five-year update. In 1973 two physicans' assistants (P.A.'s) were employed on a cardiothoracic surgical service at Emory University Hospital. In 1974 our initial experience with these paramedical personnel was presented to this Association. Since that time eight additional P.A.'s have been added to our service. They are now employed in four hospitals of the Emory University Woodruff Medical Center. New guidelines and regulations have been imposed at both the state and federal levels regarding P.A.'s, and their role in our center has become rather well defined. With over 1,700 cardiac cases and 600 thoracic cases per year to cover on our service, the P.A. has assumed a position of increasing importance both in operating room assistance and in preoperative and postoperative care. Since the university has maintained a constant number of residents and fellows during this interval, P.A.'s have filled needs of expanded clinical service in the various hospitals. In the pediatric and community hospitals associated with a university, the P.A. now functions as a junior house officer. In our university center, with a large resident staff, their role has become narrowed with definite guidelines. A Credentials Committee now governs the hiring of all P.A.'s by the University. When properly utilized and supervised, the P.A. can be a vital member of the cardiothoracic team. This report details our experience with P.A.'s for the past 5 years--culminating in a staff of ten P.A.'s working on our service in four types of hospitals within our university medical center.


Postperfusion lung syndrome. Comparison of Travenol bubble and membrane oxygenators. To examine the role of the oxygenator in the postperfusion lung syndrome, we studied 16 patients undergoing aorta-coronary bypass with a bubble oxygenator and 14 similar patients with a membrane oxygenator both before and for 2 days after the operation. To maintain the same pulmonary artery occluded pressure and hemoglobin level at the end of the surgical procedure, significantly more blood was required in the bubble than in the membrane group. Postoperative pulmonary dysfunction in the bubble group was characterized by increased pulmonary vascular resistance (PVR) and lung water. The increase in lung water was present after bubble oxygenation on three successive measurements, whereas there was no increase in lung water above control value at any measurement time in the membrane group. The bubble group had a significantly greater increase in PVR at the immediate postoperative study time than did the membrane group. PVR returned to control value for the duration of study. These differences in lung water and PVR measurements may be related to greater blood component trauma with a Travenol bubble oxygenator than with a membrane lung.


Protection of the ischemic myocardium. Volume-duration relationships and the efficacy of myocardial infusates. In studies in the isolated rat heart that were designed to optimize the composition of the infusion conditions for a cardioplegic protective solutuin, we have observed a complex relationship between the duration and volume of infusion and the extent of tissue protection. Our results would indicate that solutions, such as that formulated at St. Thomas' Hospital, which are based on extracellular electrolyte content, afford (after a brief equilibration period) a constant degree of protection, irrespective of infusion volume or duration. In contrast other solutions, such as the Bretschneider solution, which have extremes of electrolyre concentration, are associated with a complex dose-response relationship. In the latter instance, infusion of small volumes for short durations affords an increasing degree of protection against ischemia. Increasing the infusate volume may result in a progressive loss of protection. Excessive infusion may lead to an exacerbation of ischemia-induced damage. Our studies suggest that the relative patterns and rates of re-equilibration of various ions, especially sodium and calcium, during infusion may play a major role in determining the efficacy of the infusate.


New health professional practice patterns. Career patterns and practice correlates of 143 New Health Professionals (NHPs) educated at one institution are studied to determine similarities among different types of practitioners (Health Associates and Nurse Practitioners). Employment since graduation has been high with low job turnover. Nurse Practitioners (NPs) are found most often in metropolitan areas, while over 50 per cent of Health Associates (HAs) are employed in rural settings. More NPs work in hospitals than HAs, who have a higher rate of employment in private practices, HAs are also involved in a wider range of medical specialties than NPs. In terms of patient care, HAs see more patients than NPs and also have larger primary care caseloads. From a functional perspective, only minimal differences are found in comparing the activities of the practitioners. Most NHPs have high job satisfaction, although HAs anticipate a high rate of job turnover; no differences were found in levels of perceived responsibility nor in the amount of supervision in patient care. Although there were only small differences in the activities of the two groups, numerous contextual and practice correlates were found to differentiate the NHPs, a finding which argues against the current practice of conceptualizing NHPs as a single group.


[Biochemical and pathophysiological aspects of alcohol metabolism (author's transl)]. The metabolism of ethanol to acetaldehyde proceeds in the liver via alcohol dehydrogenase (ADH) and the microsomal ethanol oxidizing system (MEOS), whereas catalase plays no significant role. ADH is localized in the cytosol, required required NAD+ as cofactor and exhibits a pH optimum in the alkaline range and a Km of less than 2 mM for ethanol. Conversely, the MEOS resides in the endoplasmic reticulum, requires NADPH and O2, is inhibited by CO, and exhibits a Km of about 10 mM for ethanol. The microsomal system also metabolizes higher aliphatic alcohols such as butanol which is not a substrate for catalase. Moreover, it could be separated from ADH and catalase by column chromatography. The MEOS exhibits a variety of properties similar to those of other microsomal drug metabolizing enzymes and is characterized by inducibility of its activity following chronic alcohol consumption, which suggests the involvement of the microsomal system in the adaptive enhancement of ethanol clearance commonly observed in alcoholics.


The regulations of renal ammoniagenesis in the rat by extracellular factors. I. The combined effects of acidosis and physiologic fuels. Substrate oxidation by rat kidney slices regulates renal ammoniagenesis from glutamine. At concentrations close to those expected in plasma, lactate alone, or combined with other renal fuels, inhibits ammoniagenesis markedly; glucose and citrate decrease ammoniagenesis slightly. However, lactate, citrate, and glucose inhibit ammoniagenesis of kidney slices from acidotic rats less than ammoniagenesis of kidney slices from control rats. Lesser inhibition of ammoniagenesis is seen also when acidotic slices rather than control slices are incubated in the presence of all the tested substrates combined in the same medium. In addition to decreasing the ammoniagenesis of renal slices from control rats, the presence of lactate causes an augmented accumulation of glutamate. In contrast, adding lactate to acidotic slices does not increase glutamate accumulation nearly as much. When glutamate is substituted for glutamine in the medium, lactate still decreases ammonia production, but to a lesser extent with acidotic slices. Changes in medium pH from 7.0 to 7.8 have no, or only small, overall effects on net renal slice ammonia production from glutamine under any of the circumstances tested. We conclude that lactate alone and combined with other substrates decreases ammoniagenesis primarily at the glutamate dehydrogenase step and that slices from acidotic rats are relatively resistant to substrate mediated inhibition.