A pharmacological study of the angiotensin receptor and tachyphylaxis in smooth muscle. The interaction of angiotensin with its receptor has been studied on the basis of the tachyphylaxis shown by the rat uterus towards angiotensin II when pH and Ca2+ concentration are below physiological levels. 14C-Angiotensin binding and 45Ca2+-uptake investigations suggest tachyphylaxis to be due to increased binding at low pH and Ca2+ concentration. Studies with alkylating (affinity labeled) angiotensin derivatives containing the N-mustard chlorambucil suggest a "Charnière type" inhibition at the Ca-binding site of receptor and an irreversible inhibition at an anionic site. Angiotensin inhibitors containing chlorambucil do not alkylate tissue but are competitive inhibitors suggesting that the aromatic side chain in angiotensin may induce conformational changes in the receptor. The results obtained lead to a logical model for the angiotensin receptor allowing for normal activation by the hormone as well as for production of tachyphylaxis.


5-Hydroxytryptamine and narcotic-analgesics interactions in the intestine. The effects of 5-hydroxytriptamine (5-HT), 5-HT blocking agents, morphine, narcotic-antagonists and ganglionic blocking agents were tested in the dog intestine by close intra-arterial injection. Morphine, as 5-HT, induced an immediate increase in the intestinal tonus followed by phasic contractions. When 5-HT was injected immediately after cessation of morphine induced phasic contractions, a significant potentiation of the 5-HT induced intestinal contraction could be observed. The potentiation could be demonstrated for other narcotic-analgesics like dextromoramide and it is specific for 5-HT. 5-HT blocking agents like LSD, BOL, and cyproheptadine did not block either 5-HT or morphine. However, bufotenidine, a neural tryptaminergic blocking agent, blocked the effects of both 5-HT and morphine. The effects of 5-HT and morphine upon intestinal motility were also diminished by ganglionic depolarizing agents such as nicotine and DMPP. This effect could, however, be prevented by the pretreatment with hexamethonium. These results seem to confirm the hypothesis of a 5-HT mediator role in the intestinal contractile effect induced by the narcotic-analgesics. On the other hand, narcotic-antagonists such as nalorphine and cyclazocine, not only lacked the 5-HT potentiation effect but also prevented the 5-HT potentiation induced by morphine. Cyclazocine also showed a long lasting 5-HT blocking effect. These results seem to show that the 5-HT potentiating effect of morphine in vivo is very specific and characteristic of the narcotics and thus could be implicated in some of their central effects.


The effect of cold storage on the sensitivity to alpha and beta agonists in the isolated rabbit kidney. Rabbit kidneys were perfused at 25 degrees C, and the effect of alpha and beta agonists was studied, before and after 24 hr of cold storage. Vascular and in vitro functional parameters were evaluated. We concluded that cold storage impairs partially the vascular alpha receptor. We could not find beta receptors under these conditions. An electron microscope analysis of these kidneys has shown some degree of autolysis of the tubular cells and good preservation of the vascular smooth muscle cells and membranes. These results are very important for kidney preservation before transplantation.


Effect of colchicine and vinblastine on crayfish neuromuscular junction. 1. The effect of the microtubule disrupting agents colchicine and vinblastine was studied on crayfish excitatory neuromuscular transmission. 2. Colchicine, in concentrations of 1-3 mM, brought about a decrease in the amplitudes of intracellularly recorded excitatory junctional potentials (ejps). Vinblastine, in concentrations of 0.08-0.32 mM, increased the amplitudes of ejps. 3. Drug-induced alterations in ejp amplitude resulted from changes in quantal content of the excitattory nerve as determined by extracellular recording. The effect could not be explained by changes in quantal size. 4. The effect of colchicine and vinblastine does not appear to be related to an action on microtubules.


Accumulation of chlorporomazine by subcellular fractions of rat brain. 1. The study of the in vitro accumulation of [3H] chlorpromazine that a state of rapid equilibrium was reached for subcellular particles from cortex, midbrain and hindbrain of rats. 2. This accumulation was not saturable by increasing the concentration of chlorpromazine, and the relationship between accumulation and concentration was not modified by temperature, preincubation with high concentration of unlabelled chlorpromazine or by lowering the fraction concentration. 3. The highest accumulation of radioactivity was seen in the midbrain region and the lowest in the hindbrain. 4. In the cortex and midbrain the membrane fraction showed the highest accumulation. 5. The effect of pH on the accumulation indicated that a significnat rise was seen between pH 8 and 5. 6. This factor could also be explained in terms of solubility of the drug in membranes. 7. Studies on the metabolic fate of the drug during incubation indicated that there was a fairly rapid breakdown during incubation, the highest being seen with mitochondrail fractions and the least with membrane procedures. 9. It is concluded that accumulation of chlorpromazine by subcellular fractions in vitro may be associated with solubility in membranes and that the picture  may be complicated by the presence of breakdown products.


[Effect of mutagenic metabolic activation of N-nitrosomorpholine on microorganisms]. Almost similar sensitivity is demonstrated of different methods of studying mutagenic metabolic activation of N-nitrosomorpholine (NM) in Salmonella typhimurium TA 1950 (host-mediated assay and the system of metabolic activation with rat liver homogenate). The role of correlation of certain ingredients of NM metabolic activation system with homogenates (homogenate and cofactor NADPH concentrations) is studied. The fact of influence of microsome protein activator (phenobarbital) on the NM mutagen activation effect is established.


Mechanisms of arrhythmias in chronic obstructive lung disease. Because of the close anatomic and physiologic relationship between the heart and lungs, patients with chronic obstructive lung disease are at special risk of arrhythmias. Effective therapy hinges on identifying the mechanisms of the arrhythmias--hemodynamic, metabolic, or drug-induced. Impulsive use of antiarrhythmic agents may result only in a more complex and dangerous rhythm disorder. Extremes of pH are a major cause of arrhythmias in these patients. Respiratory alkalemia usually originates with inappropriate ventilation, often during mechanical respiration, while metabolic alkalemia generally can be traced to diuretic or bicarbonate therapy. Lidocaine or diphenylhydantoin are of little use, since the alkaline pH inside and outside heart muscle cells hampers drug distribution and activity. At the other extreme, the arrhythmias of acidemia strike patients who have severe respiratory failure with carbon dioxide retention or severe cardiac failure with shock and lactic acidemia. Arrhythmias may develop if vagal restraint is lost, which is especially likely in patients with potassium depletion. Irritant receptors along the bronchopulmonary tree can trigger arrhythmias if stimulated by cough, microembolism, or mechanical irritation, which is a hazard with endotracheal or tracheostomy tubes.


Social work training of new health professionals. The Child Health Associate program provides a model for using social workers in the training of new health professionals. Administratively, social workers have contributed to curriculum development in the behavioral sciences. They have influenced decisions regarding student selection and have developed and implemented a faculty adviser program. Because of their practical experience as primary care practitioners, social workers have been able to define effectively the skills needed by the new professionals to meet the social and emotional needs of their patients. Social workers have made use of their contacts in hospitals and communities to design and implement courses and clinics that combine in a meaningful manner theoretical material and practical experiences in patient contact. Evaluative research in the area of interviewing skills and crisis management demonstrates that the teaching methods applied by the social work profession are effective in increasing the level of students' interviewing skills in clinical practice and help prepare them to recognize familiy crises.


[Experimental study on prediction of drug toxicity. Drug-induced hemolysis and lysosomes lysis in vitro]. The effect of drugs on isolated rat liver lysosomes, erythrocytes and some erythrocyte enzymes was studied in vitro. Many tranquilizers, antihistaminics and antidepressants, which are known to have the greatest number of adverse reactions in clinical use, had a lytic effect on the particle membranes at concentrations above 2 X 10(-4) M. The present study revealed a good correlation between the p.o. LD50 (rat and mouse) and H30 (molar concentration of drug causing 30% hemolysis). It is suggested, therefore, that the labilizing effect of drugs on rat erythrocytes is useful for testing the lytic action of drugs in vivo.


[Action mechanisms of the contracting drugs, K, acetylcholine, histamine and Ba and of the antispasmodics, isoproterenol and papaverine in the isolated guinea pig ileum, particularly in relation to Ca]. Shapes of the contractions induced by K, acetylcholine (AHc), histamine and Ba consisted of the phasic contraction (PC) and the subsequent tonic contraction (TC). PCs by K, ACh and histamine are initiated by the release and the passive influx of Ca, whereas that by Ba is only initiated by the release of Ca. TCs by K, ACh and histamine are maintained by the active influx of Ca, whereas that by Ba is maintained by the active influx and the release of Ca. Storage sites of Ca in the cell membrane of this preparation can be divided into three; the first, the second and the third, which contain the loosely, the less loosely-, and the tightly-bound Ca, respectively. K releases Ca to elicit contraction from the first division, ACh or histamine does so from the first and second divisions, and Ba does so from all of the three divisions. Based on the influence of high K-depolarizing bath solution on the relaxations by isoproterenol (Iso) and papaverine (Pap) and the effects of Iso and Pap on the shapes of contractions by K, ACh, Ba and exogenous Ca, the following assumptions were made: antispasmodic action of Iso is produced by inhibition of cell membrane (inhibition of release and influx of Ca), whereas that by Pap is due to this inhibition followed by inhibition of the muscle contractile system with the increase of concentrations. The effects of Iso and Pap on the concentration-action curves of the contractions by K, ACh, Ba and exogenous Ca(Table II) suggest that the parallel shift to the right of the curves of K, ACh and Ba is due to the functional antagonism between the antispasmodics and the mobilization of Ca produced by the contracting agents.


[Action mechanism of dibenamine on the tonus and inhibition of drug-induced contraction of the isolated guinea pig ileum, with special reference to its relationship to Ca]. Dibenamine (DB) produced contraction due to influx and release of Ca in normal medium, whereas it produced relaxation of the K-induced contraction due to depression of the activity of the muscle cell membrane. DB inhibited active influx, passive influx and release of Ca induced by ACh in this order as the concentrations were increased and also inhibited the contraction by histamine selectively as compared with the contractions by ACh, K and Ba, the inhibition of the ACh-, K- and Ba-contractions being almost to the same degree. In addition, DB inhibited to much the same degree the phasic contraction(PC) and tonic contraction(TC) by histamine, whereas it inhibited TC in preference to PC induced by ACh, K and Ba. Irreversible inhibition by DB of ACh-, K- and Ba-induced contractions were protected by Ca, whereas those of histamine-induced contraction were selectively protected by histamine and antihistamine, but not by Ca. These results indicate that the antagonism of DB and its irreversibility against histamine may be due to blockade of the histaminergic receptor, whereas those against ACh, K and Ba may be due to inhibition of the Ca-site. Evidence has been obtained suggesting that the irreversible parallel shift to the right of the log concentration-action curve of histamine after washout of DB may be due to spare receptors, whereas that of ACh, K or Ba may be due to inhibition of the Ca-site.


Effect of hypophysectomy and insulin on lipogenesis in cockerels. Hypophysectomy increases hepatic lipogenesis in cockerels. In an attempt to find the possible hormonal mechanism for this we have examined the effects of hypophysectomy, insulin and pituitary homogenates on hepatic lipogenesis. Insulin (5 U/kg) injected intravenously, simultaneously with glucose-14C tracer, increased the amount of 14C found in liver lipids at 30 min after the injection. Similarly, insulin injected 5 min before killing, increased the incorporation of glucose-14C and acetate-14C by liver slices during a 30 min incubation. Hypophysectomy increased lipogenesis within 24 hours. The effect of insulin was most pronounced in hypophysectomized cockerels. The activity of the lipogenic enzyme, acetyl CoA carboxylase was similarly affected. A homogenate of chicken pituitaries added to the medium reduced lipid synthesis from glucose-14C by liver slices. This effect was larger in liver slices in which lipogenesis had been stimulated by insulin. The increased rate of hepatic lipgenesis in hypophysectomized cockerels may be caused partly by increased hepatic sensitivity to the lipogenic action of insulin or by the removal of a direct inhibition by pituitary hormones.


Effects of lithium on inducible enzymes of rat liver. Chronic lithium administration to female rats results in elevation of serum corticoids, lowering of serum glucose and altered induction of liver enzymes. The cortisol induction of tyrosine transaminase is increased selectively over that of tryptophan oxygenase. The glucose induction of glucokinase following a fast is increased by chronic lithium treatment but diminished by acute treatment. These results indicate that lithium may alter the glucose metabolic set-point in rats.


[Metabolism of nicotinic acid in plant cell suspension cultures: II; Isolation, characterization and enzymology of nicotinic acid N-alpha-arabinoside (author's transl)]. A very hydrophilic compound was isolated from parsley cell suspension cultures in high yield after application of nicotinic acid. Using chemical, chromatographic and spectroscopic procedures the structure of this new plant constituent has been elucidated as nicotinic acid N-alpha-L-arabinopyranoside. This structure has been proved by chemical synthesis. An arabinosyltransferase was isolated from parsley cell suspension cultures and purified about 19-fold. The enzyme converted nicotinic acid N-alpha-arabinoside with UDP to nicotinic acid and UDP-arabinose. pH-Optimum (pH 7.0-8.0), Km value for nicotinic acid N-alpha-L-arabinoside (2.2 X 10(-4) mol/l) and mol. wt. (app. 70 000) of the transferase were measured. Function and biosynthesis of the arabinoside in cell cultures are discussed.


Chemical modification of two tryptophan residues abolishes the catalytic activity of aminoacylase. 1) The reaction of 1 H-diazotetrazole and N-bromosuccinimide with aminoacylase was studied under different conditions. A tenfold molar excess of 1 H-diazotetrazole (2 X 10(-4) M) at pH 5.5 abolishes the catalytic activity of the enzyme while modifying only two tryptophan residues. No other amino acid reacted under these conditions as tested by amino acid analysis. 2) With a 40-fold molar excess of N-bromosuccinimide (8 X 10(-4)M) at pH 5.0, two tryptophan residues of the enzyme were oxidized with complete loss of activity. Under these conditions no significant cleavage of the polypeptide chain was observed. Neither tyrosine nor histidine was modified by this reagent, up to a 100-fold molar excess. 3) Substrates and reversible (N-tosylalanine) and irreversible (TosPheCH2Cl) inhibitors of the enzyme do not protect the two reactive tryptophans against the modification reagents. Under more drastic conditions, lysine, tyrosine and histidine residues are also modified by the reagents.


Studies on ribonucleic acid metabolism using nuclear columns. Release of rapidly labeled RNA from rat liver nuclei. A method is described to study the effect of successively changing incubation conditions on the release of rapidly labeled RNA from isolated nuclei. Nuclear columns containing immobilized rat liver nuclei isolated after in vivo application of labeled orotic acid are perfused with different non-radioactive media. Within the course of one perfusion, the rate of RNA release can be repeatedly altered by variation of temperature, acidity and concentrations of nucleoside triphosphates, complexing agents, sodium chloride and manganese chloride. RNA release can be started and stopped, indicating that the reaction does not result from damage to nuclei. During 60 min perfusion the same product, labeled ribonucleoprotein (sigma = 1.43 g/cm3 in CsCl), is released. High release rates depend on the ratio of nucleoside triphosphate to divalent cation concentration, not on the concentration of the agents per se. Ribonucleoside and deoxyribonucleoside triphosphates exert the same effect as ATP. The SH reagents iodoacetamide and iodoacetate only slightly affect the ATP-induced reaction. In contrast, p-chloromercuribenzoate, after an initial stimulation, causes inhibition of RNA release.


[Hydroxylation of testosterone and 5alpha-dihydrotestosterone--isolation of 2alpha-hydroxytestosterone (author's transl)]. The hydroxylation at C-2 of testosterone and 5alpha-dihydrotestosterone is measured by isolation of HTO after incubating "metabolically labile" tritium labeled steroids with the enzymes. After hydroxylation of testosterone in rat liver 2alpha-hydroxytestosterone was isolated and identified by gaschromatography and mass spectra.


The activator of cerebroside sulphatase. Lysosomal localization. 1) An activator protein necessary for the enzymic hydrolysis of cerebroside sulphate could be partially purified from unfractionated rat liver. This activator, which is similar to that of human origin, proved to be a heat-stable, non-dialyzable, low molecular weight protein with an isoelectric point of 4.1. Its activity could be destroyed by pronase. 2) For elucidation of the subcellular localization of the activator, rat liver was fractionated by differential centrifugation. The intracellular distribution of the cerebroside sulphatase activator was compared to the distribution patterns of marker enzymes for different cell organelles and found to coincide with the lysosomal arylsulphatase, thus indicating a lysosomal localization. 3) This was confirmed using highly purified secondary, i.e. iron-loaded, lysosomes. After disruption by osmotic shock, these organelles hydrolyzed cerebroside sulphate when incubations were performed under physiological conditions with endogenous as well as exogenous sulphatase A as enzyme. 4) After subfractionation of the disrupted secondary lysosomes into membrane and lysosol fractions by high speed centrifugation, it was found that the activator protein was exclusively associated with the lysosol, whereas the acid hydrolases were distributed differently between the two fractions. 5) The lysosol was further fractionated by semi-preparative electrophoresis on polyacrylamide gels. Two protein fractions were obtained: a high molecular weight fraction, containing the activator-free acid hydrolases, and a low molecular weight fraction, containing the enzyme-free activator of cerebroside sulphatase. 6) The significance of these findings for the hydrolysis of sphingolipids in the lysosomes is discussed.


Blastogenic response of human lymphocytes to oral bacterial antigens: comparison of individuals with periodontal disease to normal and edentulous subjects. Cell-mediated immunity in humans to antigens derived from oral plaque bacteria was investigated by using the lymphocyte blastogenesis assay. Subjects with varying severities of periodontal disease including normal, gingivitis, periodontitis, and edentulous were compared. Mononuclear leukocytes were separated from peripheral blood and cultured with antigens prepared by sonication of Actinomyces viscosus (AV), Actinomyces naeslundii (AN), Veillonella alcalescens (VA), Leptotrichia buccalis (LB), Bacteroides melaninogenicus (BM), and homologous dental plaque (DP). The lymphocyte response of subjects with gingivitis or periodontitis was significantly greater than that of normal subjects to antigens of AV, AN, and DP, but did not differ from the response of edentulous subjects. Periodontitis subjects were significantly more reactive than edentulous and normal subjects in response to VA, LB, and BM. These findings suggest that the tested gram-negative bacteria and the host response they evoke are associated with advanced periodontal destruction.


Prevention of benzo(a)pyrene-induced mutagenicity by homogeneous epoxide hydratase. Benzo(a)pyrene and benz(a) anthrancene which, in contrast to the K-region epoxides benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, are not mutagenic to Salmonella typhimurium TA 1537 in the absence of mammalian enzyme preparations, were activated by liver microsomes from C3H mice, which had not received any pretreatment, to mutagens reverting this tester strain to histidine prototrophy. Addition of epoxide hydratase inhibitors greatly increased this mutagenicity and addition of pure epoxide hydratase reduced it by more than 95% down to the range of spontaneous mutations as observed in absence of any added mutagen. This demonstrates than the metabolic pathway responsible for the mutagenicity of both polycyclic hydrocarbons observed in this system proceeds entirely via an epoxidation pathway and that the responsible metabolites are epoxides or species arising from them. Moreover, further metabolism by epoxide hydratase does not lead to produce contributing to the mutagenicity observed with the tester strain used. Finally, the epoxides relevant for the observed mutagenicity are substrates for epoxide hydratase; indeed, modest amounts of the pure enzyme can prevent the mutagenic effect.


The intraocular pressure response of conscious rabbits to clonidine. A study has been made of the time courses of the pupillary and intraocular pressure responses of conscious rabbits to clonidine administered either topically or intravenously. Topical unilateral application of clonidine caused transient pupil dilatation and a biphasic intraocular pressure response; an initial hypertensive response preceded a hypotensive phase lasting several hours. Pupillary and hypertensive responses were absent in the untreated eye, but there was a rapid decrease of intraocular pressure. Intravenous administration of clonidine caused an immediate and large decrease of intraocular pressure in both eyes. Phenoxybenzamine given intravenously inhibited the pupillary dilatation and the hypertensive responses to clonidine. The role of efferent adrenergic neuronal activity in mediating the local biphasic pressure response was studied in rabbits with unilateral precervical and postcervical sympathotomy. The results showed the hypotensive response to be dependent on an intact adrenergic innervation of the ocular tissues.


The effect of miotics on the intraocular pressure of conscious owl monkeys. The intraocular pressure of conscious, unsedated owl monkeys (Aotus trivirgatus) was measured with an applanation tonometer. Untreated eyes of the conscious animals were found to have higher values than those reported for owl monkeys anesthetized with pentobarbitone. Locally applied pilocarpine, carbachol, and oxotremorine gave concentration-related reduction in pressure, oxotremorine being the most potent and having longer duration of effect than the other compounds. Slight reductions were also observed with aceclidine and R. S. 86. These results are discussed in relation to the effects of miotics in man.


Interaction of Listeria monocytogenes and influenza in an animal model. This study was designed to investigate the effects of viruses in the pathogenesis of Listeria monocytogenes. The organisms used in this study were: Listeria monocytogenes Type 1 isolated from a local fatal case; Mouse adapted influenza A/PR8/34 (HONI); Streptococcus pneumoniae Group B (U.M. Med. Ctr.) and poliovirus Type 2 MEF--G3M2. Balb-C mice were inoculated intraperitoneally with one LD50 of Listeria monocytogenes. Ten days later, the survivors were challenged intransally with 10 LD50 of influenza virus and observed for 14 days. Another set of Balb-C mice was inoculated intranasally with one LD50 of influenza virus and the survivors challenged 14 days later intraperitoneally with 10 LD50 of Listeria monocytogenes. Controls consisted of similar inoculation and challenge methods in mice using Streptococcus pneumoniae and polio virus with Listeria monocytogenes and influenza virus. Cross protection was observed only between Listeria monocytogenes and influenza virus. Cellular immunity may play a role in this interaction. This findings seem to agree with reports from others who showed cross protection between Listeria monocytogenes and other intracellular bacteria and parasites.


[The symptomatic treatment of seasonal allergic rhinitis (author's transl)]. The aetiology, pathophysiology and symptomatology of seasonal rhinitis are briefly presented. Symptomatic therapy with antihistamines, nasal decongestants and local and systemic corticosteroids is critically discussed. The results of a trial with Disodium cromoglycate are reported. This is shown to be a valuable drug in the symptomatic management of seasonal rhinitis. It should be tried before resorting to steroid therapy.


Male pseudohermaphroditism, cryptorchism, and Sertoli cell neoplasia in three miniature Schnauzers. A syndrome of male pseudohermaphroditism, cryptorchidism, and testicular neoplasia was diagnosed in 3 Miniature Schnauzers. The dogs had clinical signs of hyperestrogenism and were either unilateral or bilateral cryptorchids. At surgery, it was discovered that the dogs were male pseudohermaphrodites, having intra-abdominal testes containing Sertoli cell tumors and uteri that had undergone endometrial cystic hyperplasia.


Observations on immobilization of Père David's deer. Etorphine and xylazine were found to be a safe and reliable drug combination for the immobilization of Père David's deer, whether excited or unexcited. Excited deer had a longer preimmobilization period, when compared with unexcited deer at comparable dosages. Generally, the acid-base status of Père David's deer during immobilization was not seriously altered. Deer that had been excited and exercised experienced mild respiratory problems; the unexcited, relatively calm deer experienced minimal acidosis. Significantly high pH and PO2 and significantly lower PCO2 and bicarbonate values were found in the excited deer, when compared with the unexcited deer. Rapid physiologic changes occurred after the intravenous administration of the antagonist, diprenorphine.


The proliferation of transplanted haematopoietic cells derived from bone marrow and fetal liver. The proliferation of transplanted murine haematopoietic cells, derived from the femoral bone marrow of young adults and from the liver on the sixteenth day of gestation, has been compared under standardized conditions. Suspensions of bone marrow cells contained 9-6+/-0-9 CFU per 10(5) cells (sm) and suspensions of fetal liver cells contained 3-7+/-0-5 CFU per 10(5) cells (Sl).Sl/Sm = 0-4. The mean diameter of colonies established by cells derived from bone marrow was 1-3+/-0-3 mm (established mean volume, Vm = 1-2 mm3) and the mean diameter of colonies established by cells derived from fetal liver was 1-7+/-0-1 mm (estimated mean volume, Vl ;-6 mm3). Vl/Vm ;-2. The relationship between the weight of the spleen and the number of bone marrow cells injected into lethally irradiated recipients 10 days previously has been confirmed, and a similar relationship between the weight of the spleen and the number of fetal liver cells injected has been demonstrated. An arbitrarily defined difference of 50 mg between the weight of the spleen in untreated irradiated controls and the weight of the spleen in irradiated recipients of haematopoietic cells has been observed 10 days after the administration of 2-2 x 10(6) bone marrow cells (Dm) or 3-0 x 10(6) fetal liver cells (Dl). Dm/Dl = 0-7. The calculated values of Dm/Dl (relative proliferative capacity), Sl/Sm (relative stem cell content) and Vl/Vm (relative clone size) are in good agreement with the values estimated using the equation Dm/Dl = Sl/Sm x Vl/Vm.


Metabolism of DL-(+/-)-phenylalanine by Aspergillus niger. A fungus capable of degrading DL-phenylalanine was isolated from the soil and identified as Aspergillus niger. It was found to metabolize DL-phenylalanine by a new pathway involving 4-hydroxymandelic acid. D-Amino acid oxidase and L-phenylalanine: 2-oxoglutaric acid aminotransferase initiated the degradation of D- and L-phenylalanine, respectively. Both phenylpyruvate oxidase and phenylpyruvate decarboxylase activities could be demonstrated in the cell-free system. Phenylacetate hydroxylase, which required reduced nicotinamide adenine dinucleotide phosphate, converted phenylacetic acid to 2- and 4-hydroxyphenylacetic acid. Although 4-hydroxyphenylacetate was converted to 4-hydroxymandelate, 2-hydroxyphenylacetate was not utilized until the onset of sporulation. During sporulation, it was converted rapidly into homogentisate and oxidized to ring-cleaved products. 4-Hydroxymandelate was degraded to protocatechuate via 4-hydroxybenzoylformate, 4-hydroxybenzaldehyde, and 4-hydroxybenzoate.


Microbial oxidation of methane and methanol: crystallization and properties of methanol dehydrogenase from Methylosinus sporium. Obligate methylotrophs are divisible into two types on the basis of ultrastructural biochemical characteristics. Both groups possess a soluble phenazine methosulfate (PMS)-dependent methanol dehydrogenase. In addition, particulate PMS-dependent methanol dehydrogenase and PMS-independent methanol oxidase have been found in the type I membrane group. A procedure was developed for the crystallization of methanol dehydrogenase from the soluble fraction of the type II obligate methylotroph Methylosinus sporium. This is the first report of a crystalline methanol dehydrogenase from a methylotrophic bacterium. The crystallized enzyme is homogeneous as judged by ultracentrifugation and by acrylamide gel electrophoresis. In the presence of an electron acceptor (phenazine or phenazinium compound) and an activator (ammonium compound), the crystallized enzyme catalyzed the oxidation of primary alcohols and formaldehyde. Secondary, tertiary, and aromatic alcohols were not oxidized. The molecular weight of the enzyme as estimated by gel filtration is approximately 60,000, and as estimated by sedimentation equilibrium analysis it is 62,000. The sedimentation constant (S20,W) is 2.9. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 60,000. The amino acid composition and spectral properties of the enzyme are also presented. Antisera prepared against the crystalline enzyme are nonspecific, they cross-reacted and inhibited isofunctional enzymes from other obligate methylotrophic bacteria.


Mutant strains (nit) of Salmonella typhimurium with a pleiotropic defect in nitrogen metabolism. We have isolated mutant strains (nit) of Salmonella typhimurium that are defective in nitrogen metabolism. They have a reduced ability to use a variety of compounds including glutamate, proline, arginine, N-acetyl-glucosamine, alanine, and adenosine as sole nitrogen source. In addition, although they grow normally on high concentrations of ammonium chloride (greater than 1 mM) as nitrogen source, they grow substantially more slowly than wild type at low concentrations (less than 1 mM). We postulated that the inability of these strains to utilize low concentrations of ammonium chloride accounts for their poor growth on other nitrogen sources. The specific biochemical lesion in strains with a nit mutation is not known; however, mutant strains have no detectable alteration in the activities of glutamine synthetase, glutamate synthetase, or glutamate dehydrogenase, the enzymes known to be involved in assimilation of ammonia. A nit mutation is suppressed by second-site mutations in the structural gene for glutamine synthetase (glnA) that decrease glutamine synthetase activity.


Stable, inducible thermoacidophilic alpha-amylase from Bacillus acidocaldarius. Bacillus acidocaldarius Agnano 101 produces an inducible thermoacidophilic alpha-amylase. The enzyme production occurs during the stationary phase of growth in the presence of compounds with alpha-1,4-glucosidic linkages. The enzymatic activity is both present in the culture medium and associated with the cells; the enzymes purified from both sources show identical molecular and catalytic properties. The purified amylase has a single polypeptide chain of molecular weight 68,000 and behaves like an alpha-amylase with affinity constants for starch and related substances of 0.8 to 0.9 mg/ml. The pH and temperature optima for activity are 3.5 and 75degreesC, respectively. The amylase is stable at acidic pH (below 4.5). Its thermal stability is strictly dependent upon protein concentration; the half-life at 60degreesC of the amylase in a 70-mug/ml solution is about 5 days.


Size and transforming activity of deoxyribonucleic acid in Diplococcus pneumoniae during thymidine starvation. The transforming activity and the molecular structure of DNA from cells of Diplococcus pneumoniae during thymidine starvation have been analyzed and the effects of thymidine starvation have been compared with the effects of single-strand breaks produced by deoxyribonucleases in DNA of unstarved cells. The decrease in transforming activity of lysates from starved cells as a function of the size of DNA particles, measured by centrifugation in neutral and alkaline sucrose gradients, does not follow the kinetics observed after enzymatic degradation of DNA of unstarved cells. Moreover, a strain lacking exo- and endonuclease activities is not protected from thymineless death. These results suggest that the basic lethal mechanism of thymidine starvation might have an origin other than the activation of nucleases.


Carbon and ammonia metabolism of Spirillum lipoferum. Intact cells and extracts from Spirillum lipoferum rapidly oxidized malate, succinate, lactate, and pyruvate. Glucose, galactose, fructose, acetate, and citrate did not increase the rate of O2 uptake by cells above the endogenous rate. Cells grown on NH+/4 oxidized the various substrates at about the same rate as did cells grown on N2. Added oxidized nicotinamide adenine dinucleotide generally enhanced O2 uptake by extracts supplied organic acids, whereas oxidized nicotinamide adenine dinucleotide phosphate had little effect. Nitrogenase synthesis repressed by growth of cells in the presence of NH+/4 was derepressed by methionine sulfoximine or methionine sulfone. The total glutamine synthetase activity from N2-grown cells was about eight times that from NH+/4-grown S. lipoferum; the response of glutamate dehydrogenase was the opposite. The total glutamate synthetase activity from N2-grown S. lipoferum was 1.4 to 2.6 times that from NH+/4-grown cells. The levels of poly-beta-hydroxybutyrate and beta-hydroxybutyrate dehydrogenase were elevated in cells grown on N2 as compared with those grown on NH+/4. Cell-free extracts capable of reducing C2H2 have been prepared; both Mg2+ and Mn2+ are required for good activity.


Photosystem II regulation of macromolecule synthesis in the blue-green alga Aphanocapsa 6714. Polymers synthesized by heterotrophically growing (glucose as carbon source)  cultures of Aphanocapsa 6714 were compared with polymers synthesized in photosynthetically grown cultures. Loss of photosystem II by dark incubation, or inhibition of light-grown cells with the photosystem II-specific inhibitor dichlorophenylmethylurea, caused an 80 to 90% reduction in the rate of lipid and total ribonucleic acid synthesis, and more than a 90% reduction in the rate of protein synthesis. In contrast, glycogen synthesis was reduced only about 50% in dark cells and less than 30% in dichlorphenylmethylurea-inhibited cells. After longer heterotrophic growth, glycogen became the major component, whereas in photosynthetically grown cultures protein was the major constituent. 14C (from 14CO2 and/or [14C]glucose) assimilated into protein by heterotrophically grown cells was found in amino acids in nearly the same proportions as in photosynthetically grown cells. Thus, routes of biosynthesis available to autotropic cells were also available to heterotrophic cultures, but the supply of carbon precursors to those pathways was greatly reduced. The limited biosynthesis in heterotrophic cells was not due to a limitation for cellular energy. The adenylates were maintained at nearly the same concentrations (and hence the energy charge also) as in photosynthetic cells. The concentration of reduced nicotinamide adenine dinucleotide phosphate was higher in heterotrophic (dark) cells than in photosynthetic cells. From rates of CO2 fixation and/or glycogen biosynthesis it was determined that stationary-phase cells expended approximately 835, 165, and less than 42 nmol of adenosine 5'-triphosphate per mg (dry weight) of algae per 30 min during photosynthetic, photoheterotrophic, and chemoheterotrophic metabolism, respectively. Analysis of the soluble metabolite pools in dark heterotrophic cultures by double-labeling experiments revealed rapid equilibration of 14C through the monophosphate pools, but much slower movement of label into the diphosphate pools of fructose-1,6-diphosphate and sedoheptulose-1,7-diphosphate. Carbon did flow into 3-phosphoglycerate in the dark; however, the initial rate was low and the concentration of this metabolite soon fell to an undetectable level. In photosynthetic cells, 14C quickly equilibrated throughout all the intermediates of the reductive pentose cycle, in particular, into 3-phosphoglycerate. Analysis of glucose-6-phosphate dehydrogenase in cell extracts showed that the enzyme was very sensitive to product inhibition by reduced nicotinamide adenine dinucleotide.


Subunits of the alkaline phosphatase of Bacillus licheniformis: chemical, physicochemical, and dissociation studies. The alkaline phosphatase (orthophosphoric monoester phosphydrolase, EC 3.1.3.1) of Bacillus licheniformis MC14 was studied in an attempt to determine the number of subunits contained in the 120,000-molecular-weight native enzyme. Two moles of arginine was liberated per mole of native enzyme by carboxypeptidases A and B in the presence of sodium dodecyl sulfate. The effect on the native enzyme of progressively lowering the solvent buffer pH was monitored by determining the molecular weight by sedimentation equilibrium analysis, the sedimentation coefficient, the frictional coefficient, and the percent alpha-helix content of the enzyme. The alkaline phosphatase dissociates into two subunits around pH 4. At pH 2.8 a further decrease in S value, but no change in molecular weight, is observed, indicating a change in conformation. The frictional coefficients and percent alpha-helix content agree with this interpretation. A subunit molecular weight of 59,000 was calculated from sodium dodecyl sulfate gels.


Inorganic nitrogen assimilation by the photosynthetic bacterium Rhodopseudomonas capsulata. The photosynthetic bacterium Rhodopseudomonas capsulata lacks glutamate dehydrogenase and normally uses the glutamine synthetase/glutamate synthase sequence of reactions for assimilation of N2 and ammonia. The glutamine synthetase in cell-free extracts of the organism is completely sedimented by centrifugation at 140,000 X g for 2 h, is inhibited by L-alanine but not by adenosine 5'-monophosphate, and exhibits two apparent Km values for ammonia (ca. 13 muM and 1 mM).


Interactions on 3-deoxy and 6-deoxy derivatives of N-acetyl-D-glucosamine with hen lysozyme. The interactions of deoxy derivatives of GlcNAc, 6-deoxy-GlcNAc, and 3-deoxy-GlcNAc with hen egg-white lysozyme [EC 3.2.1.17] were studied at various pH's by measuring the changes in the circular dichroic (CD) band at 295 nm. It was shown that 6-deoxy-GlcNAc and 3-deoxy-GlcNAc bind at subsite C of lysozyme and compete with GlcNAc. The pH dependence of the binding constant of 6-deoxy-GlcNAc was the same as that of GlcNAc. On the other hand, the binding constants of 3-deoxy-GlcNAc were 3--10 times smaller than those of GlcNAc in the pH range from 3 to 9. X-ray crystallographic studies show that O(6) and O(3) of GlcNAc at subsite C are hydrogen-bonded to the indole NH's of Trp 62 and Trp 63, respectively, but the above results indicate that Trp 63, not Trp 62, is important for the interaction of GlcNAc with lysozyme.


Interactions of alpha- and beta-N-acetyl-D-glucosamines with hen and turkey lysozymes. The binding constants of alpha- and beta-GlcNAc to hen and turkey lysozymes [EC 3.2.1.17] were determined at various pH's using the method proposed by Ikeda and Hamaguchi (1975) J. Biochem. 77, 1-16). The pH dependence of the binding of beta-GlcNAc to hen lysozyme was essentially the same as that for turkey lysozyme. The pH dependence curves of the binding constants of beta-GlcNAc to hen and turkey lysozymes were interpreted in terms of the participation of Glu 35 (pK 6.0), Asp 52 (pK 3.5), Asp 48 (pK 4.5), and Asp 66 (pK 1.5). The binding constants of alpha-GlcNAc to hen and turkey lysozymes were the same below pH 3.5 but were different above this pH. The main participant residues in the binding of alpha-GlcNAc were Glu 35, Asp 48, and Asp 66 for hen lysozyme and Glu 35 and Asp 66 for turkey lysozyme. The results obtained here were well explained by the following assumptions: (1) above about pH 4, alpha-GlcNAc binds to hen lysozyme in both alpha- and beta-modes, which correspond to the binding orientation of alpha-GlcNAc and that of beta-GlcNAc, respectively, as determined by X-ray crystallographic studies, but it binds predominantly in the beta-mode below about pH 4, (2) beta-GlcNAc binds to hen and turkey lysozymes predominantly in the beta-mode above about pH 4 and in both alpha- and beta-modes below pH 4, and (3) alpha-GlcNAc binds to turkey lysozyme predominantly in the beta-mode over the whole pH range studied.


Binding of substrate analogs to hen egg-white lysozyme with an ester linkage between Glu 35 and Trp 108. The interactions of the substrate analogs beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which an ester linkage had been formed between Glu 35 and Trp 108 (108 ester lysozyme), were studied by the circular dichroic and fluorescence techniques, and were compared with those for intact lysozyme. The binding constants of beta-methyl-GlcNAc and (GlcNAc)2 to 108 ester lysozyme were essentially the same as those for intact lysozyme in the pH range of 1 to 5. Above pH 5, the binding constants of these saccharides to 108 ester lysozyme did not change with pH, while the binding constants to intact lysozyme decreased. This indicates that Glu 35 (pK 6.0 in intact lysozyme) participates in the binding of these saccharides. The extent and direction of the pK shifts of Asp 52 (pK 3.5), Asp 48 (pK 4.4), and Asp 66 (pK 1.3) observed when beta-methyl-GlcNAc is bound to 108 ester lysozyme were the same as those for intact lysozyme. The participation of Asp 101 and Asp 66 in the binding of (GlcNAc)2 to 108 ester lysozyme was also the same as that for intact lysozyme. These findings indicate that the conformations of subsites B and C are not changed by the formation of the ester linkage. On the other hand, the binding constants of (GlcNAc)3 to 108 ester lysozyme were higher than those for intact lysozyme at all pH values studied. This result is interpreted in terms of an increase in the affinity for a GlcNAc residue of subsite D, which is situated near the esterified Glu 35.


Near-UV circular dichroism of trypsin inhibitor of adzuki beans attributable to disulfide groups. The trypsin inhibitor of adzuki (Phaseolus angularis) beans shows a CD spectrum with a negative extremum at 280 nm and a positive shoulder around 245 nm. Since the inhibitor lacks tryptophan and tyrosine, the observed CD spectrum can be attributed to the six disulfide groups in the molecule. The CD features completely disappeared on reduction of the disulfide groups, and converged into a single negative extremum at 270 nm when the groups were modified to form mixed disulfides with glutathione. These observations of the CD properties of the inhibitor strongly suggest the presence  of disulfide groups constrained with respect to their dihedral angles.


Interaction of diiodo-L-tyrosine and triiodophenol with bovine serum albumin. Circular dichroism and fluorescence studies. As a model study to investigate the binding mechanism between thyroid hormones and carrier protein, the interaction of diiodo-L-tyrosine (DIT) and triiodophenol (I3phi) with bovine serum albumin (BSA) was investigated by circular dichroism (CD) and fluorescence methods. In both the DIT-BSA system and the I3phi-BSA system, induced Cotton effect was observed in the wavelength region near 320 nm. This induced Cotton effect was measured at various molar ratios of ligands to BSA (L/P). The value of the ellipticity at 319 nm, [theta]319, in the I3phi-BSA system was remarkably large compared with that of the DIT-BSA system, and [theta]319 at an L/P ratio of one was -1.96 X 10(4) (degree cm2 decimole-1) for the I3phi-BSA system and -0.1 X 10(4) for the DIT-BSA system. The binding constants for the combination of BSA with a single molecule of ligand, calculated by measuring the quenching of the fluorescence of the protein, were 1.33 X 10(5) M(-1) at 15 degrees for the DIT-BSA system and 1.6 X 10(9) M(-1) at 28 degrees for the I3theta-BSA system. These results suggest that the binding of I3theta to BSA is stronger than that of DIT and a cleft may exist more congruent with the molecular dimensions of I3theta than with those of DIT.


Renal angiotensin I-converting enzyme as a mixture of sialo- and asialo-enzyme, and a rapid purification method. Angiotensin I-converting enzyme [EC 3.4.15.1] was rapidly and highly purified from a particulate fraction of hog kidney cortex with 13% yield. The procedure, which was rapid, included fractionation on DEAE-cellulose and calcium phosphate gel, chromatographies on DEAE-Sephadex A-50 and hydroxylapatite columns, and gel filtration on a Sephadex G-200 column. The purified enzyme preparation gave two protein bands on standard disc gel electrophoresis, but showed a single protein component on the gel after treatment with neuraminidase [EC 3.2.1.18]. The data strongly suggest that the purified enzyme preparation was a mixture of sialo- and asialo-enzyme. Sialic acid residues apparently do not contribute to the catalytic activity of the enzyme. The enzyme was activated more by chloride ions than by other halide ions tested, using Bz-Gly-Gly-Gly as a substrate. The dissociation constant for chloride ions was determined to be 2.2 mM. Chloride did not protect the enzyme against heat or low pH. The enzyme was resistant to inactivation by trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1].


Some catalytic and molecular properties of threonine deaminase from Bacillus stearothermophilus. Threonine deaminase [EC 4.2.1.16] was highly purified from Bacillus stearothermophilus. The enzyme exhibited maximum activity at 65 degrees and at pH 9.2--9.6. It was inactivated on dilution and on storage at 4 degrees, but was protected by egg albumin. The enzyme was labile at 65 degrees, but became stable in the presence of egg albumin and isoleucine at pH 7.0. The substrate saturation curve for the enzyme reaction at 40 or 65 degrees was hyperbolic, but in the presence of isoleucine, the curve became sigmoidal (n = 2). The enzyme was more sensitive to isoleucine at 40 degrees than at 65 degrees, while valine slightly inhibited the enzyme at both 40 and 65 degrees. Inhibition of the enzyme by isoleucine was antagonized by valine at 40 and 65 degrees. These properties were essentially similar to those of the enzymes from mesophilic and thermophilic bacteria. The enzyme existed in two forms with different molecular sizes, 1.5-5 X 10(6) and 2 X 10(5) daltons, at pH 7.0 and at temperatures below 40 degrees. The larger component disaggregated into the small one at pH 8.5 or above, at temperatures above 50 degrees or in the presence of isoleucine and valine.


Temperature-jump studies on the interaction of benzeneboronic acid with chymotrypsinogen. The interaction of chymotrypsinogen A with benzeneboronic acid (BBA), a transition state along inhibitor of serine proteases, was investigated by the temperature-jump method using pH indicators. It was found that l/tau is dependent on BBA concentration, in contrast to the case of the alpha-chymotrypsin [EC 3.4.21.1]-BBA system in which l/tau is independent of BBA concentration. By examination of the pH dependences of the kinetic parameters, the acid dissociation behavior of His 57 in chymotrypsinogen, chymotrypsinogen-trigonal BBA complex and chymotrypsinogen-tetrahedral BBA complex was analyzed. The kinetic deuterium isotope effect was also examined and found to occur principally on the acid dissociation constants. The state of the catalytic residues in the zymogen molecule is discussed based on these results.


The structure and function of acid proteases. V. Comparative studies on the specific inhibition of acid proteases by diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(p-nitrophenoxy) propane and pepstatin. Comparative studies have been made on the effects of diazoacetyl-DL-norleucine methyl ester (DAN), 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and pepstatin on acid proteases, including those from Acrocylindrium sp., Aspergillus niger, Aspergillus saitoi, Mucor pusillus, Paecilomyces varioti, Rhizopus chinensis, and Trametes sanguinea, and also porcine pepsin [EC 3.4.23.1] and calf rennin [EC 3.4.23.4] for comparative purposes. These enzymes were rapidly inactivated at similar rates and in 1:1 stiochiometry by reaction with DAN in the presence of cupric ions. The pH profiles of inactivation of these enzymes were similar and had optima at pH 5.5 to 6. They were also inactivated at similar rates by reaction with EPNP, with concomitant incorporation of nearly 2 EPNP molecules per molecule of enzyme. The pH profiles of inactivation were again similar and maximal inactivation was observed at around pH 3 to 4. Some of the EPNP-inactivated enzymes were treated with DAN and shown still to retain reactivity toward DAN. All these enzymes were inhibited strongly by pepstatin, and the reactions of DAN and EPNP with them were also markedly inhibited by prior treatment with pepstatin. These results indicate that the active sites of these enzymes are quite similar and that they presumably have at least two essential carboxyl groups at the active site in common, one reactive with DAN in the presence of cupric ions and the other reactive with EPNP, as has already been demonstrated for porcine pepsin and calf rennin. Pepstatin appears to bind at least part of the active site of each enzyme in a simmilar manner.


In vitro stimulation by retinol of porcine pancreatic esterase activity toward esters of short-chain fatty acids. 1. Retinol exerted a remarkable stimulating effect (approx. 260% increase), essentially similar to that (300%) of phytol, on the so-called esterase activity displayed by crude pancreatic lipase [EC 3.1.1.3] toward true solutions of esters, but none of the typical lipase activity toward emulsions of water-insoluble esters. 2. Comparison of the stimulatory effects of retinol derivatives on the esterase activity revealed that retinyl acetate was the most active, being sustantially similar in effect to retinol; retinal was fairly active, while retinoic acid, retinyl palmitate, and beta-ionone were far less active. 3. With various isoprenoid compounds, the efficiency of stimulation increased with the carbon chain length, attaining a maximum at 15 to 20 carbon atoms. Above this chain length the efficiency decreased rapidly. 4. Comparison of the effects of retinol and phytol on the esterase activity of various other lipolytic enzymes indicated that this kind of activator may be relatively specific to porcine pancreatic esterase activity.


Cardiac myosin from pig heart ventricle. Purification and enzymatic properties. A method is described for the preparation of high purity myosin from the left ventricle of pig heart. The purified myosin was free from nucleic acid, actin, tropomyosin, troponin, the 150,000 molecular weight protein and other contaminants. Analyses of subunits in the purified myosin were carried out on 3.5% acrylamide gel with 0.1% SDS. Of the total protein present in myosin, 11.3% was in the light chains; light chain 1 (LC1), 5.9% and light chain 2 (LC2), 5.4%. Urea gel electrophoresis of the purified myosin showed three closely spaced bands corresponding to the 20,000 dalton, the charge-modified 20,000 dalton and the phosphorylated 20,000 dalton components. The properties of the Ca2+-activated and K+-activated ATPases [EC 3.6.1.3] of the purified myosin were also studied. The Km values were 27 and 55 muM and the Vmax values were 0.263 and 0.317 mumole P1/mg/min for the Ca2+-activated and K+-activated ATPases, respectively. The pH-activity profiles and the effects of SH modification were of the skeletal myosin type except that the activities were lower.


Inability of the smallest light chain to bind to fetal fast muscle myosin. 1. The smallest light chain of myosin, g3, was not transferred from adult HMM to fetal myosin in alkali (pH 10.5) under conditions when the light chains dissociated from myosin. 2. The g3 isolated from adult myosin did not bind to fetal myosin at either pH 7.8 or 10.5.


Differences in phosphofructokinase regulation in normal and tumor rat thyroid cells. The kinetic and molecular properties of a phosphofructokinase derived from a transplantable rat thyroid tumor lacking regulatory control on the glycolytic pathway were studied. The properties of the near-purified enzyme (specific activity 140 units/mg) were compared with those of phosphofructokinase from normal rat thyroid (specific activity 134 units/mg). The electrophoretic mobilities and gel elution behavior of these two enzymes were almost similar. The thyroid tumor phosphofructokinase showed, however, a greater degree of size and/or shape heterogeneity in the presence of ATP than the normal thyroid enzyme, as determined by gel filtration and sucrose density gradient centrifugation. Kinetic studies below pH 7.4 showed a sigmoid response curve for both enzymes when the velocity was determined at 1 mM ATP with varying levels of fructose-6-P. The interaction coefficient, however, was 4.2 and 2.6 for normal and tumor thyroid phosphofructokinase, respectively. Ammonium sulfate decreased the cooperative interactions with the substrate fructose-6-P in both enzymes. The thyroid tumor enzyme, however, was less sensitive to the inhibition by ATP and by citrate. The reversal of citrate inhibition by cyclic 3':5'-adenosine monophosphate was also less effective with the thyroid tumor phosphofructokinase, while the protective effect of fructose-6-P was stronger. The difference in citrate inhibition between tumor and normal thyroid enzyme was not strongly affected by varying the MgCl2 concentration up to 10 mM. It is concluded that the complex allosteric regulation typical of the normal thyroid phosphofructokinase is still present in the enzyme isolated from the thyroid tumor tissue. The latter, however, is more loosely controlled by its physiological effectors, such as ATP, citrate, and cyclic AMP.


Partial purification and characterization of a triglyceride lipase from pig adipose tissue. A triglyceride lipase was extracted from defatted pig adipose tissue powder with dilute ammonia and purified about 230-fold by a combination of ammonium sulfate fractionation, heparin-Sepharose 4B, DEAE-cellulose, and Sephadex G-150 column chromatographies and isoelectrofocusing electrophoresis. The enzyme was distinguishable in physical and kinetic properties from the two previously defined lipases in adipose tissue, lipoprotein lipase, and hormone-sensitive lipase. The purified enzyme was fully active in the absence of serum lipoprotein and was not stimulated by adenosine 3':5'-monophosphate-dependent protein kinase. In marked contrast to the already defined lipases, the enzyme was strongly inhibited by serum albumin. The enzyme had a molecular weigt of about 43,000, a pI of 5.2, and pH optimum of 7.0. The enzyme hydrolyzed triolein to oleic acid and glycerol, and did not exhibit esterase activity. The apparent Km for triolein was 0.05 mM. Physiological roles of this new species of lipase remained to be explored.


Compound X. An intermediate in enzymatic halogenation. Previous studies have shown that chlorite serves as a halogenation substrate for horseradish peroxidase. In its substrate role, chlorite serves both as a halogen donor and as a source of oxidizing equivalents in the chlorination reaction. We now show that a new spectral intermediate, which we have termed Compound X, can be detected as the initial product of the reaction of chlorite with horseradish peroxidase. The reaction of chlorite with horseradish peroxidase to form Compound X is a relatively fast reaction especially at acidic pH values. The second order rate constant (Kf) for the formation of Compound X at pH 4.5 (optimum pH) is 0.9 X 10(6) M-1 S-1. Compound X, in the absence of a halogen acceptor, decomposes to Compound I and chloride ion. The first order rate constant (Kd) for the decay of Compound X to Compound I is 0.2 s-1 at pH 4.5. The pH optimum for enzymatic chlorination with chlorite compares favorably with the pH profile for the lifetime of Compound X (Kf/Kd). These observations indicate that Compound X is the halogenating intermediate in the chlorite reaction and that the rate of enzymatic chlorination is directly related to the stability of Compound X. We propose an -OCl ligand on a ferric heme as the most likely structure for Compound X.


Studies on the regulation of chloroplast NADP-linked glyceraldehyde-3-phosphate dehydrogenase. Chloroplast NADP-linked glyceraldehyde-3-phosphate dehydrogenase was resolved into three forms that differed in molecular weight: (a) larger than or equal to 1.5 million; (b) 600,000; and (c) less than or equal to 100,000. After preincubation with an effector (ATP, NADPH, or Pi) the activity of forms a and c was unaffected, whereas the activity of b, the regulatory form, was increased 10-fold. Activation was accompanied by the exposure of previously hidden sulfhydryl groups. The rate of activation was slower than the rate of catalysis and resulted in a lag phase during the measurement of activity when the enzyme was preincubated in the absence of an effector. The addition of one of several compounds as a second effector (at a concentration which itself was nonactivating) in the presence of a first effector enhanced activation by lowering the concentration of the first effector required for half-maximal activation (Pi constant/ATP or NADPH varied; ATP or NADPH constant/Pi varied). Other combinations of effectors caused little change in activity (ATP constant/NADPH varied; NADPH constant/ATP varied). Glyceraldehyde 3-phosphate added as a second effector induced contrasting changes: an increase in the ATP-mediated activation and a decrease in the NADPH-mediated activation. The results are consistent with the view that the products of the photochemical reactions of chloroplasts, ATP, and NADPH, in conjunction with other metabolites, regulate the activity of glyceraldehyde-3-phosphate dehydrogenase in the photosynthetic assimilation of CO2.


Induction of lipogenesis during differentiation in a "preadipocyte" cell line. 3T3-L1 fibroblasts differentiate in culture into cells having adipocyte character. This transition is accompanied by a 40- to 50-fold  rise in the incorporation of [14C]acetate into triglyceride. The increase in lipogenic rate is exactly parallel to a coordinate rise in the activities of the key enzymes of the fatty acid biosynthetic pathway (ATP-citrate lyase, acetyl-CoA carboxylase, and fatty acid synthetase). Immunological studies indicate that the elevated acetyl-CoA carboxylase activity is the product of an increased cellular enzyme level.


Enzymatic synthesis of biopterin from D-erythrodihydroneopterin triphosphate by extracts of kidneys from Syrian golden hamsters. An enzyme system was found in either crude homogenates of dialyzed extracts of liver, kidney, lung, and brain from Syrian golden hamsters that catalyzed the synthesis of radioactive 6(L-erythro-1',2'-dihydroxypropyl)pterin (biopterin) from [U-14C]6(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropterin triphosphate (D-erythrolH2neopterin-PPP) preparation. The specific radioactivity of biopterin was found to be comparable to that of D-erythroH2neopterin-PPP. The enzyme system from hamster kidney was purified severalfold by fractionation with ammonium sulfate and with an Ultrogel AcA-34 column. It was demonstrated that (a) NADPH or NADAH was essential and that (b) Mg2+ was stimulatory for the enzymatic synthesis of biopterin from D-erythroH2-NEOPTERIN-PPP. Also GTP and nonphosphorylated neopterins were not converted to biopterin. Although 6-lactyl-7,8-dihydropterin (sepiapterin) was converted to biopterin in the presence of NADPH, sepiapterin was not detected from D-erythroH2neopterin-PPP in the absence of NADPH. A preliminary experiment was performed to identify dihydrobiopterin.


Purification and characterization of N-hydroxy-2-acetylaminofluorene sulfotransferase from rat liver. N-Hydroxy-2-acetylaminofluorene (N-OH-2-AAF) sulfotransferase is an enzyme that catalyzes the sulfate transfer from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to N-OH-2-AAF to form a highly reactive product acetylaminofluorene N-sulfate. It has been purified about 2000-fold with a yield of over 12% from adult Sprague-Dawley male rat livers by an eight-step procedure. The final preparation was homogeneous on analytrical disc gel electrophoresis. The purified enzyme had activity toward p-nitrophenol with an approximately 1600-fold increase in specific activity over the crude homogenate, but it had almost no detectable activity toward steroids such as estrone, beta-estradiol, testosterone, dehydroisoandrosterone, and corticosterone. There was also very little sulfation activity toward serotonin and L-tyrosine methyl ester. The optimal pH for the enzyme activity is approximately 6.3 when measured in sodium phosphate buffer. Mg2+ at 6 to 9 mM could increase the enzyme activity up to 30%. Mn2+ activated the enzyme only slightly at very low concentrations. Zn2+, Co2+, Cu2+, and Ni2+ were all strongly inhibitory, but Ca2+ had very little effect. Thiol compounds were found to have a stabilizing effect and thiol-blocking reagents were potent inhibitors for this enzyme. The pure enzyme was very unstable especially in diluet solutions. The isoelectric point (pl) of the enzyme is 5.66 +/- 0.07. The molecular weight of the native enzyme was 68,000 +/- 500 as estimated by Sephadex G-100 and G-200 gel filtrations. A single component with molecular weight of 38,250 +/- 1,350 was observed on sodium dodecyl sulfate gel electrophoresis in the absence and presence of 2-mercaptoethanol. Comparison of the enzyme activity in mail and female rat livers at each stage of purification revealed that there was only a trace amount of N-OH-2-AAF sulfotransferase present in the female rat liver.


Photochemical studies and ultraviolet sensitization of Escherichia coli thymidylate kinase by various halogenated substrate analogs. The effect of 5-iodo-2'-deoxyuridine monophosphate (IdUMP), various 5-halogenated-5'-azido-2', 5' -dideoxyuridine derivatives, 2'-deoxy-6-azauridine (AzdUrd), and its halogenated analogs on the ultraviolet sensitization of Escherichia coli thymidylate kinase has been investigated. Only those compounds iodinated in position 5 enhance the rate of ultraviolet inactivation of this enzyme. However, 5'-azido nucleosides with iodo, bromo, chloro, or fluoro substituents in position 5 neither protect nor sensitize thymidylate kinase to ultraviolet inactivation. Thymidine 5'-monophosphate partially protects the enzyme against ultraviolet inactivation either in the presence or absence of ultraviolet-sensitizing iodinated analogs. Magnesium ion does not enhance the ultraviolet inactivation of thymidylate kinase by 5-iodinated nucleoside analogs. The kinatic data support an active site-directed enhancement of the enzyme to ultraviolet inactivation by 5-iodo-2'-deoxyuridine monophosphate, since the concentration of IdUMP required to attain 50% maximal enhancement is 0.24 mM which is in good agreement with its Ki of 0.18 mM. When either [125I]IdUMP or [2-14C]IdUMP was irradiated with the enzyme, both radioactivities were associated with the enzyme, however only with the 14C analog was the amount bound at half-saturation essentially equal to the amount required to inactivate the enzyme by 50%. These data support the hypothesis that the active entity in the enhancement by IdUMP of thymidylate kinase inactivation during ultraviolet irradiation is the uridylate free radical which is formed photochemically from IdUMP. Photochemical studies of 6-azauracil (AzUra), 2'-deoxy-6-azauridine, and 5-iodo-2'-deoxy-6-azauridine (IAzdUrd) were performed. Photolysis of IAzdUrd in the presence of a hydrogen donor yields AzdUrd which upon further photolysis yields the photohydrate. The photohydrate of AzdUrd when incubated in the dark at pH 5.2 is 90% converted back to AzdUrd, whereas the photohydrate of AzUra is only partially (20%) converted to AzUra. The rate of deiodination of IAzdUrd is 2.1-fold greater than that of IdUMP. Although the Ki of IdUMP and IAzdUrd is similar, the increased photosensitivity of the aza analog accounts for the much greater enhancement of ultraviolet inactivation of thymidylate kinase. The ability of a compound to enhance the ultraviolet inactivation of deoxythymidylate kinase is correlated with the potential of the compound to produce a free radical rather than a photohydrate when the enzyme-substrate analog complex is irradiated.


Kinetic studies on the binding affinity of human hemoglobin for the 4th carbon monoxide molecule, L4. L4, the affinity of hemoglobin for the 4th CO molecule, has been determined for human adult hemoglobin (HbA) as a function of pH and the presence of organic phosphates by measuring the kinetic parameters for the reaction. l'4, the rate of combination of CO with the triliganded molecule, was measured by flash photolysis while l4, the rate of CO dissociation for the ligand-saturated molecule, was measured by ligand replacement. L4 is pH-dependent and affected by 2,3-diphosphoglycerate. Additionally, this pH dependence of the high affinity state is largely eliminated by carboxypeptidase A digestion. L4 for human fetal hemoglobin (HbF) in phosphate buffers was also determined and found to be pH-dependent. These results cannot be reconciled within the framework of the two-state allosteric model. Additional structures in the conformational equilibrium due to either intermediates in the T to R transition or two or more R states must exist.


Isolation and partial characterization of monophosphoglycerate mutase from human erythrocytes. Monophosphoglycerate mutase has been purified to homogeneity from outdated human erythrocytes as indicated by exclusion chromatography, polyacrylamide gel electrophoresis, and equilibrium centrifugation. Occasionally, the recommended purification procedure yields a small amount (3% or less) of a single extraneous protein which can be deleted from the enzyme preparation by employing an additional purification step. The native enzyme has a molecular weight of 54,000 to 56,000 as determined by equilibrium centrifugation and exclusion chromatography. Disc gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band with a molecular weight of 28,600, indicating that the native macromolecule is a dimer composed of subunits of similar mass. Homogeneous monophosphoglycerate mutase is free of diphosphoglycerate mutase, enolase, and nonspecific phosphatase activities; however, the enzyme manifests intrinsic 2,3-diphospho-D-glycerate phosphatase activity as shown by thermal denaturation studies. The diphosphatase activity is stimulated by PPi and glycolate-2-P, but is inhibited by Cl-, HSO3-, and Pi. The pH optimum for both the diphosphatase and the mutase is 6.8. The Km for 2,3-diphospho-D-glycerate in the phosphatase reaction is 82 muM at 37 degrees and pH 7.2. The amino acid composition of homogeneous monophosphoglycerate mutase is given.


Conversion of proparathyroid hormone to parathyroid hormone by a particulate enzyme of the parathyroid gland. The conversion of proparathyroid hormone (proparathormone) to parathyroid hormone (parathormone) by subcellular fractions of the bovine parathyroid has been investigated. The identification of the conversion product as parathormone was established by its elution postion during ion exchange chromatography and gel filtration, and by partial amino acid sequence analysis of its NH2-terminal region. Total homogenates and derived subcellular fractions (600 X g pellet, 5,000 X g pellet, 20,000 X g pellet, 190,000 X g pellet, and 190,000 X g supernatant) all catalyzed the conversion of exogenous [3H]- or [14C]prohormone. Over 60% of the converting activity was in the particulate fractions; the 190,000 X g particulate fraction contained the highest specific converting activity. The converting activity appeared to be an integral component of the membranes since it could only be partially removed by extraction with Triton X-100. The production of parathormone by the particulate converting enzyme increased with time and the concentration of enzyme protein. The optimum pH range was between 7 and 9, and the enzyme was inactive below pH 6. Conversion by the particulate enzyme was inhibited by benzamidine or chloroquine, but not by pancreatic trypsin inhibitor, indicating its dissimilarity to trypsin. When a mixture of [14C]proparathormone and [3H]parathormone was used as substrate, the particulate enzyme did not metabolize the hormone despite over 70% conversion of the prohormone to hormone and other peptides. There was a close correlation between the subcellular distribution of converting activity and that of newly formed parathormone found in the membrane fraction. These data suggest that the particulate converting activity is that concerned with the formation of parathormone in vivo.


Quantitative aspects of relationship between glucose 6-phosphate transport and hydrolysis for liver microsomal glucose-6-phosphatase system. Selective thermal inactivation of catalytic component in situ at acid pH. Studies of the thermal stability of rat liver glucose-6-phosphatase (EC 3.1.3.9) were carried out to further elevate the proposal that the enzymic activity is the result of the coupling of a glucose-6-P-specific translocase and a nonspecific phosphohydrolase-phosphotransferase. Inactivation was observed when micorsomes were incubated at mild temperatures between pH 6.2 and 5.6. The rate of inactivation increased either with increasing hydrogen ion concentration or temperature. However, no inactivation was seen below 15 degrees in media as low as pH 5 or at neutral pH up to 37 degrees. The thermal stability of the enzyme may be controlled by the physical state of the membrane lipids and the degree of protonation of specific residues in the enzyme protein. Microsomes were exposed to inactivating conditions, and kinetic analyses were made of the glucose-6-P phosphohydrolase activities before and after supplementation to 0.4% sodium taurocholate. The results support the postulate and the kinetic characteristics of a given preparation of intact microsomes are determined by the relative capacities of the transport and catalytic components. Before detergent treatment, inactivation (i.e. a decrease in Vmax) was accompanied by a decrease in Km and a reduction in the fraction of latent activity, whereas only Vmax was depressed in disrupted preparations. The possibility that the inactivating treatments caused concurrent disruption of the microsomal membrane was ruled out. It is concluded that exposures to mild heat in acidic media selectively inactivate the catalytic component of the glucose-6-phosphatase system while preserving an intact permeability barrier and a functional glucose-6-P transport system. Analyses of kinetic data obtained in the present and earlier studies revealed several fundamental mathematical relationships among the kinetic constants describing the glucose-6-P phosphohydrolase activities of intact (i.e. the "system") and disrupted microsomes (i.e. the catalytic component). The quantitative relationships appear to provide a means to calculate a velocity constant (VT) and a half-saturation constant (KT) for glucose-6-P influx. The well documented, differential responses of the rat liver glucose-6-phosphatase system induced by starvation, experimental diabetes, or cortisol administration were analyzed in terms of these relationships. The possible influences of cisternal inorganic phosphate on the apparent kinetic constants of the intact system are discussed.


Acid-induced phosphorylation of adenosine 5'-diphosphate bound to coupling factor 1 in spinach chloroplast thylakoids. Adenosine 5'-diphosphate, bound to coupling factor 1 (CF1) in spinach chloroplast thylakoids, is in part converted to adenosine 5'-triphosphate, upon injection of the thylakoids into strong acids in the dark. Bound phosphate serves as the phosphoryl donor for this uncoupler-insensitive conversion. Exposure of the thylakoids to heat or to urea prior to their injection into acid caused dissociation of ADP and prevents the apparent acid-induced synthesis of ATP. Conformational changes in CF1 may be elicited by acid denaturation which resemble those brought about by the proton electrochemical gradient across thylakoid membranes.


An in vitro investigation of the anodic polarization and capacitance behavior of 316-L stainless steel. Determinations were made of how the corrosion-resistant properties of the passive film on 316-L stainless steel are influenced by the material's mechanical and surface states, and the variable pH and PO2 conditions of the interstitial fluid. Cold-rolled and annealed specimens were surface-prepared, commercially and in the laboratory, respectively, as if for orthopedic implantation. Passive film behavior was studied by the anodic polarization and pulse-potentiostatic capacitance methods. The pH and PO2 of the Ringer's test solution were varied to include interstitial fluid values occurring postoperatively and onto recovery. The anodic polarization behavior of all specimens was found to be pH- and PO2-independent. Breakdown potentials of annealed specimens were 800-950 mV (SCE), in contrast to previously reported values of approximately 350 mV. This substantial increase is related to the influence of surface preparation and, in particular, to the optimization of electropolishing time which acts to produce a microscopically smooth surface, free of debris and disarrayed material. Capacitance behavior of annealed material for potentials greater than 400 mV was consistent with a model involving the entry of chloride and metal ions (mostly Fe) into the passive film. This entry is related to the onset of pitting.


Secretion of old versus new exportable protein in rat parotid slics. Control by neurotransmitters. The possibility that old and new secretory granules do not mix and that older exportable protein can be secreted preferentially was tested on parotid gland in vitro. Slices from fasted animals were pulse labeled for 3 min with L-[3H]leucine. Subcellular fractionstion showed that after 1 90-min chase period, the formation of new labeled secretory granules was mostly completed. The ratio of label in secretory granules to label in microsomes increased 250-fold during the period 5--90 min postpulse. After the 90-min chase, a submaximal rate of secretion was initiated by adding a low concentration of isoproterenol to the slices. Preferential secretion of old unlabeled exportable protein was evident from the finding that the percent of total amylase secreted was 3.5-fold greater than the percent of labeled protein secreted. Preferential secretion of old unlabeled exportable amylase was undiminished even when the chase period before addition of isoproterenol was extended to 240 min. Such long chase incubations were still meaningful due to the fact that the spontaneous rat of amylase release and radioactive protein release from the slices was negligibly low. A high isoproterenol concentration added to the slices after a 90-min chase produced the following results. An initial phase of preferential secretion of old unlabeled protein was soon replaced by secretion of a random mixture of new and old exportable protein. Electron micrographs indicated that high rates of secretion involved sequential fusion of secretory granules so that the lumen extended deep into the cell where the new labeled granules were presumably located. At low rates of secretion, the lumen showed no such deep extensions. Experiments were also conducted on slices from glands which had been largely depleted of old granules by prior injection of isoproterenol into the animals. Secretion of labeled protein from such slices stopped with the export of 80% of the labeled protein. This finding indicates that about 20% of the radioactive protein is cellular nonexportable protein and that the slices are capable of exporting the entire amount of secretory protein which was symthesized in vitrol. In addition to the beta-adrenergic receptor which mediates protein secretion, the parotid acinar cell also possesses an alpha-adrenergic and a cholinergic receptor both of which cause K+ release, vacuole formation, and water secretion. Activation of either of the latter two receptors in conjunction with the beta-adrenergic receptor increased randomization of the protein secreted. It is concluded that in the rat parotid acinar cell there is little spontaneous mixing between old granules near the luminal cell membrane and new granules coming up behind from the Golgi complex. The neurotransmitters which induce secretion produce the observed randomization.


The microsporidian spore invasion tube. The ultrastructure, isolation, and characterization of the protein comprising the tube. The extrusion apparatus of the microsporidian parasitic protozoan Nosema michaelis discharges an invasion (or polar) tube with a velocity suitalbe for piercing cells and injecting infective sporoplasm. The tube is composed of a polar tube protein (PTP) which consists of a single, low molecular weight polypeptide slightly smaller than chymotrypsinogen-A. Assembled PTP tubes resist dissociation in sodium dodecyl sulfate and brief exposures in media at extreme ends of the pH range; however, the tubes are reduced by mercaptoethanol and dithiothreitol. When acidified, mercaptoethanol-reduced PTP self-assembles into plastic, two-dimensional monolayers. Dithiothreitol-reduced PTP will not reassemble when acidified. Evidence is presented which indicates that PTP is assembled as a tube within the spore; that the ejected tube has plasticity during sporoplasm passage; and, finally, that the subunits within the tube polymer are bound together, in part, by interprotein disulfide linkages.


Adaptive enhancement of amino acid uptake and exodus by thymic lymphocytes: influence of pH. Entry of certain free amino acids (alpha aminoisobutyric acid (AIB), alanine and proline), but not of leucine into rat thymic lymphocytes increased progressively when the cells were incubated in amino acid deficient medium. Actinomycin D, cycloheximide, or a high concentration of AIB abolished the time-related increase in AIB accumulation, whereas exposure to a high concentration of leucine had no effect. This phenomenon could not be attributed to a progressive alteration in the nature of the incubation medium nor to reduced transinhibition of AIB uptake. The exodus of AIB also increased with time, but to a smaller degree than AIB entry. Initial rates of AIB entry and exodus increased with increases in the pH of the incubation medium over the range 6.5-8.0. The effects of pH on entry and exodus were time-related, increasing progressively oveb nullified the magnified time related increments in AIB transport caused by prolonged incubation at pH 8.0. The influence of a given pH on transport of AIB decreased rapidly when the cells were transferred to medium of another pH, but this tendency diminished the longer the cells were exposed to the initial pH. pH influenced the entry of alanine and proline in the same fashion as that of AIB, but did not affect leucine entry. These results indicate that thymic lymphocytes exhibit adaptive enhancement in the accumulation of free amino acids that are transported largley by the A or alanine-preferring system, and that the adaptive process involves both entry and exodus. Moreover, alterations in pH modify entry and exodus of these same amino acids, profoundly affect the magnitude of time-released increases, and may induce fundamental changes in the mechanism(s) serving amino acid transport.


Lysosomal hydrolase secretion by Tetrahymena: a comparison of several intralysosomal enzymes with the isoenzymes released into the medium. Tetrahymena pyriformis were grown in proteose-peptone medium and then washed and incubated in a dilute salt solution for one hour. The cells were then discarded and the lysosomal hydrolases that had been secreted were subjected to DEAE cellulose column chromatography. At least three isoenzymes of acid phosphatase, three of acid protease, and two of beta-N-acetylhexoseaminidase were found, as well as single peaks of alpha-mannosidase, beta-galactosidase, and beta-fucosidase. The latter two activities were not resolved by the DEAE column and could not be separated in a second chromatographic step on CM-cellulose. Cells were also grown under identical conditions and homogenized in 0.25 M sucrose in order to allow comparison of some of the intracellular lysosomal hydrolases with their secreted counterparts. Two lysosomal populations were resolved by sucrose density gradient sedimentation, a heavy lysosomal fraction, contered at a density of about 1.25 gm/cm3, and a light lysosomal fraction, centered at a density of about 1.16 gm/cm3. These two populations differed in that the light lysosomes did not appear to contain significant amounts of beta-fucosidase, beta-galactosidase, or acid protease, whereas all six of the hydrolase activities studied were present in the heavy lysosomes. The light lysosomal peak occurred in cells grown to transition phase, but was markedly reduced in cells from cultures grown to stationary phase. In addition to these two fractions a third very light particle, containing only alpha-mannosidase activity, was detected just inside the gradient. Measurements were made of the effect of heat (10 minutes at 66 degrees) and of a change in pH from 4.5 (standard assay condition) to 6.0 on the three acid phosphatases and two beta-N-acetylhexoseaminidase isoenzymes resolved by DEAE column chromatography of the secreted hydrolases and on these hydrolyases in the heavy and light lysosomal fractions on the sucrose gradient. Use of the thermostability and pH criteria permitted computation of the expected properties of the intralysosomal acid phosphatase and hexoseaminidase activities if these consisted of the respective isoenzymes in the proportions secreted. It was found that neither the intralysosomal acid phosphatase nor the intralysosomal hexoseaminidase had the properties expected if they consisted of the secreted mixture of the respective isoenzymes, indicating that modification of some of these isoenzymes may have occurred during the 1-hour starvation period or after secretion.


Synthesis of a liver enzyme in hybrid cells. Rat hepatoma cells were fused with cells of an established mouse lymphoma line, with normal diploid mouse macrophages, lymphocytes and fibroblasts and with normal diploid rat macrophages and lymphocytes. The liver-specific enzyme tyrosine aminotransferase was produced by almost all the hybrid cells, but usually at a lower level than in the parental hepatoma cells. Most of the hybrids also showed increased levels of this enzyme after exposure to dexamethasone. In the rat x mouse hybrids, the electrophoretic mobility of the enzyme indicated that only the rat hepatoma enzyme was produced. The findings are difficult to explain in terms of simple models involving a single diffusible repressor or activator of tyrosine aminotransferase synthesis.


[Zollinger-Ellison syndrome and parathyroid adenoma. Limits of Wermer's syndrome]. The authors report the case of a patient with pancreatic and parathyroid adenomatosis and discuss whether it belongs to Wermer's syndrome. After a study of the various possible endocrine involvements, the complexity of hormonal inter-relations is studied, the prognosis and classification of multiple endocrine tumours are then discussed.


Ion-pair chromatography of acidic drug metabolites and endogenic compounds. Liquid-liquid chromatographic systems based on ion-pair partition with silica microparticles as the support for the stationary phase have been used for the separation of anionic compounds of biochemical and pharmacological interest. A high separating efficiency can be obtained with both aqueous and organic mobile phases and the retention is easily regulated by the nature and the concentration of the quaternary ammonium counter ion, present in the aqueous phase. The influence of the composition of the liquid phases on the selectivity and separating efficiency has been studied, as well as equilibration methods and the stability of the systems. Examples are given of separations of sulphonamides, barbiturates, glucuronic and sulphuric acid conjugates of steroidal compounds and phenols glycine conjugates of carboxylic acids (hippuric, nicotinuric and salicyluric acid) and anionic metabolites of biogenic amines (indoleacetic, benzoic, mandelic and phenylacetic acid derivatives).


Analysis of the acidic fraction of marijuana smoke condensate by capillary gas chromatography-mass spectrometry. A method for the analysis of organic acids and phenols isolated from marijuana smoke condensate has been developed. Comparative analyses of standard tobacoo, Mexican, and Turkish marijuana smoke condensates carried out by means of capillary gas chromatography indicated both qualitative and quantitative changes in the constituents of chromatographic profiles. Samples were converted to volatile derivatives by methylation and trimethylsilylation; whereupon, 49 aliphatic acids, aromatic acids, and phenolic compounds were identified by means of capillary gas chromatography-mass spectrometry.


Electron capture and multiple ion detection of benzodiazepine esters in pharmacokinetic studies. A sensitive and highly specific gas-liquid chromatographic method is described for the determination of benzodiazepine esters. The method involves the extraction of pivoxazepam and 2'-chloropivoxazepam from blood and urine samples and the hydrolysis of both drugs with strong acid to the corresponding 2-amino-5-chlorobenzophenone (ACB) and 2-amino-5,2'-dichlorobenzophenone (ACDB). Recoveries of 97-98% have been obtained and the trifluoroacetic (TFA) derivatives of both benzophenones (ACB-TFA and ACDB-TFA) can be chromatographed on OV-17, in a short time, with good electron-capture detector (ECD) responses. The mass spectra of these derivatives provide abundant molecular ions suitable for mass spectrometric detection by multiple ion detection (MID),  with a considerable improvement in specificity compared with the ECD responses. The detection limits in blood specimens have been established at 5-10 ng/ml by ECD and 0.5-0.8 ng/ml by MID. The method has been applied to the study of blood levels attained after i.v. and oral doses in rats and in man.


Characterization of bacteroides melaninogenicus. Fifty-eight human isolates of Bacteroides melaninogenicus, 42 from a variety of clinical infections and the rest from normal flora, were studied for pigment production and ultraviolet light fluorescence and by forty biochemical and other tests, including end-product analysis by gas-liquid chromatography. In a number of instances, tests were repeated several times and the results were reproducible. Agar plate dilution susceptibility tests were also performed to 12 antimicrobial agents. These 58 strains could be reliably placed into three groups, corresponding to the three subspecies described, based on seven characteristics. These included acid production in peptone-yeast-glucose medium, production of n-butyric acid from peptone-yeast-glucose medium, esculin hydrolysis, starch hydrolysis, indole production, effect on milk, and lipase production. Production of hydrogen gas in peptone-yeast-fructose medium may be another distinguishing characteristic. In general there was not much difference in the susceptibility of the three groups to the various antimicrobial agents tested. Two strains had a minimal inhibitory concentration of penicillin G of 16 and 32 U/ml, respectively. Three strains did not produce a black pigment in spite of prolonged incubation on blood-containing media.


Recent rural health research.

Recent rural health research. Recent rural health research may be examined in two ways: needs and solutions. A definition of needs requires an evaluation of the social factors that affect the expectations and the behavior of both the provider and the consumer. Three types of solutions should be considered: the appropriate utilization of manpower, including the efforts to influence physician location and specialty distribution, new health practitioners, and team approaches; the new technology for transportation and communication; and the organization of new delivery systems. Two areas of rural health research that need more attention are program evaluation and financial planning.


Mouse ovarian alkaline phosphatase activities that respond to gonadotropins: histochemical and biochemical studies. Alkaline phosphatase activity was measured in whole ovarian homogenates from pre-pubertal mice of different ages, with and without prior injection of human chorionic gonadotropin. Alkaline phosphatase activity was also scored in the different cell types in sections of similar ovaries, using two distinct histochemical procedures. The results from those methods differed. Biochemical studies indicated the presence of three distinct alakaline phosphatase activities: I and Ib, both optimal at pH 10.4 and with similar substrate requirements and inhibitor sensitivities (phosphatase I being characteristic of unstimulated ovaries and Ib of ovaries stimulated with human luteinizing hormone or human chorionic gonadotropin), and phosphatase II, optimal at pH 9.4, with different substrate requirements and inhibitor sensitivities. The differences observed using the histochemical procedures can probably be accounted for by the effects of different incubation conditions on the activities of these three enzymes.


The occurrence of salmonellas, mycobacteria and pathogenic strains of Escherichia coli in pig slurry. Ninety-eight samples of pig slurry from 54 farms were examined for the presence of salmonellas, porcine enteropathogenic strains of haemolytic Escherichia coli and mycobacteria. Salmonellas were isolated from 12 farms (22%) and enteropathogenic E. coli from 13 farms (24%). Pathogenic mycobacteria were not isolated. Salmonellas were isolated from 7 of 16 farms (44%) stocked with 'minimal disease' pigs compared with only 5 of 38 farms (13%) stocked with conventionally reared pigs. Conversely enteropathogenic coliforms were isolated from 3 of 16 farms (19%) stocked with 'minimal disease' pigs compared with 10 of 38 farms (26%) stocked with conventionally reared pigs.


Detection of serum proteins in the electrophoretic patterns of total proteins of mycoplasma cells. The contamination of mycoplasma cell preparations by serum proteins originating from culture medium was studied. A. laidlawii and M. arthritidis cells were grown in the presence of [14C]-aminoacids, and the cells were washed with 0-9% NaC1 by threefold centrifugation. Total proteins of the washed cells were analysed by SDS gel electrophoresis. Coomassie-stained electrophoretic patterns were compared with autoradiographs of the same gels. The stained electrophoretic pattern of washed A. laidlawii grown without serum was identical with autoradiographs of the same cells grown without or with serum. That of washed A. laidlawii grown with serum differed from the corresponding autoradiography by the presence of extra protein bands I, II, III, and IV with molecular weights of over 160,000, 80,000-87,000, 55,000 and 25,000, respectively. The same extra bands were found in stained electrophoretic patterns of washed: (a) A. laidlawii cells grown without serum and mixed with serum in the stationary phase, (b) M. arthritidis cells, as compared with their autoradiographs, (c) serum precipitate. The bands III and IV may be due to the heavy and light chains of gamma-globulin, the band II might belong to transferrin or to some component of complement. Acidification of serum to pH 5 brought about 100-fold rise of amount of serum precipitate, the number of bands in the electrophoretic pattern of the precipitate being also increased. Stained electrophoretic patterns of cells purified by twofold centrifugation in step sucrose density gradient (1-20-1-27 g./cm.3 for A. laidlawii, and 1-15-1-25 for M. arthritidis) contained no extra bands and matched completely with their autoradiographs. It was concluded that contamination of washed mycoplasma cells by serum proteins is mainly due to co-precipitation of aggregated serum proteins together with cells during centrifugation rather than to adsorption of serum proteins on the cell surface.


Comparison of the inactivation of IgM and IgG complement fixation sites by acid and base. Rabbit IgM antibodies to denatured mammalian or T6 bacteriophage DNA or poly(A)-poly(U) irreversibly lost complement-(C) fixation reactivity on exposure to low pH and reneutralization, with a halving of the complement-fixation titer occurring after treatment at about pH 3. The titers of IgG antibodies to denatured phage DNA, to poly(A)-poly(U), or to hemocyanin were halved only after exposure to pH 2. Inactivation by acid was enhanced by low protein concentrations, incubation at higher temperatures, and by slow reneutralization; under all these conditions it was more extensive with IgM than with IgG. Inactivation of IgM C-fixation activity at pH 2.5 and room temperature was a first order reaction, with a half-time of about 20 min. Both classes retained antigen-binding activity after exposure to pH 2. In the alkaline range, full C-fixation reactivity was retained by both classes after reneutralization from pH 11.5, some loss occurred at pH 12, and total irreversible inactivation occurred by pH 12.5. In the latter case, antigen-binding activity was also lost. The C-fixation inactivation curves in the alkaline range were similar for IgG and IgM antibodies.


Genetic control of the antibody response to type III pneumococcal polysaccharide in mice. III. Analysis of genes governing the expression of regulatory T cell activity. Recombinant-inbred strains of mice, as well as the progenitor strains from which they were derived, were evaluated with respect to the capacity of B cells to respond to an optimally immunogenic dose of Type III pneumococcal polysaccharide (SSS-III) and the amount of suppressor and amplifier T cell activity present. None of these functional activities was found to be linked to genes within the major histocompatibility (H-2) or the IgCH allotype complex, and several autosomal genes appeared to govern the expression of each of these characteristics.


Bacterial interference by oropharynegeal and clinical isolates of anaerobic bacteria. Anaerobic isolates were tested for bacterial inhibitory activity. Of 144 isolates, 102 were from oropharynegeal washings, and 42 were from clinical specimens. Thirteen facultative bacterial species (seven members of the Enterobacteriaceae and six species of gram-positive cocci) were used as indicators of inhibition. Eleven anaerobic species were isolated from oral secretions. All isolates of Bacteroides melaninogenicus, the most commonly recovered species, consistently inhibited several species of indicator bacteria. Bacteroides fragilis, Bacteroides oralis, and Peptostreptococcus anaerobius had unprecictable inhibitory activity, whereas most of the other oral anaerobes were noninhibitory. The 42 clinical species were generally noninhibitory.


Inhibition of pituitary prolactin secretion by human placental lactogen in rats. Intact female rats given twice daily injections of 1 mg human placental lactogen (HPL) showed continued dioestrous vaginal smears and their ovarian corpora lutea were found to be hypertrophied and functiona. The serum prolactin level was significantly lower in these rats than in the controls at dioestrus as well as at pro-oestrus. Twice-daily injections of 0.5 or 2 mg HPL to ovariectomized rats decreased serum and pituitary levels of prolactin and increased hypothalamic activity of prolactin inhibiting hormone, although the effect was less at the lower dose. Human placental lactogen had no direct effect on pituitary prolactin secretion in vitro. These findings have demonstrated that HPL, like prolactin itself, inhibits prolactin secretion by actin