Continuous monitoring of pH and Eh in bacterial plaque grown on a tooth in an artificial mouth. Apparatus which enables the simultaneous continuous monitoring of pH and Eh of bacterial dental plaque as it develops on a tooth surface in an artificial mouth is described.  Details of the electrodes used, monitoring equipment, and culture conditions are given.  Preliminary results are given showing the Eh and pH values of plaque produced in vitro to be in close agreement with readings reported for plaque in vivo.  The effect of the incorporation of 1 per cent sucrose in the medium on these parameters is reported and a distinct inverse relationship between pH and Eh recorded.


Dichlorofluoromethane inactivates Saccharomyces cerevisiae. Saccharomyces cerevisiae was incubated in aerosol cans containing YM broth and dichlorofluoromethane (f-21). The presence and number of viable cells were determined by inoculating (1% vol/vol) YM broth and by the plate count procedure (YM agar). Inactivation of the yeast was greater or more rapid when: (i) the thermodynamic activity (saturation value) of f-21 became greater through increasing the concentration of chemical from 0.5 to 1.5% (wt/wt) in a given volume (20, 40, or 80 ml) of broth, or by holding the concentration of chemical constant but increasing the volume of broth in the test vessel, (ii) the temperature of treatment was increased (7, 22, 37, and 47 C), (iii) samples with 1.5% (wt/wt) f-21 were agitated, (iv) young (8 h) rather than old (36 h or 10 days) cells were treated, and (v) cells were grown in YM broth without, rather than with, glucose. Adjusting the pH (6.3 to 4.0) of broth before treatment, pretreating the substrate with f-21, or distilling the chemical before use had no effect on viability of cells when treated with f-21. Yeast cells inactivated by f-21, chlorine, or heat were more resistant to disruption by sonic treatment than were viable cells.


Production and purification of the thermophilic bacteriophage TP-84. A new procedure for production and purification of the thermophilic bacteriophage TP-84 in high yields is described. Cultures of Bacillus stearothermophilus strain 10, enriched with nutrients to obtain heavy growth and to prevent sporulation and maintained at a pH of 6.5, were infected with the phage in a 100-liter fermentor.  Addition of magnesium chloride (0.01 M) and a temperature of 58-C were essential for maximal phage production.  Phage (5 times 1011 infective particles/ml) was precipitated with polyethylene glycol (molecular weight 6,000) in the presence of sodium chloride and was further purified by cesium chloride density centrifugation.


Adsorption of Vibrio parahaemolyticus onto chitin and copepods. Vibrio parahaemolyticus was observed to adsorb onto chitin particles and copepods.  The efficiency of adsorption was found to be dependent on pH and on the concentration of NaC1 and other ions found in seawater.  Highest efficiency was observed in water samples collected from Chesapeake Bay and lowest in water from the open sea.  V. parahaemolyticus was found to adborb onto chitin with the highest efficiency of the several bacterial strains tested.  Escherichia coli and Pseudomonas fluorescens did not adsorb onto chitin.  The adsorption effect is considered to be one of the major factors determining the distribution of this species and affecting the annual cycle of V. parahaemolyticus in the estuarine system.


Effect of L-histidine on the survival of a T-strain of mycoplasma. The addition of L-histidine to the growth medium prolongs the stationary phase and the survival of a T-strain of mycoplasma. Results of an experiment performed with (14)C-labeled urea demonstrate that the action of L-histidine is based on the retardation of the rise of pH.


Use of pH gradient plates for increasing the acid tolerance of salmonellae. Several strains of salmonellae survived higher concentrations of lactic acid after streaking on the surface of pH gradient plates. Most strains increased their acid tolerance by about 0.8 to 1.0 pH unit (9-to 10-fold), with Salmonella madelia showing the greatest differential, pH 5.2 in the wild strain and pH 4.2 after conditioning. The increased acid resistance was quickly lost after transferring to normal tryptic soy agar. Tests for survival in a liquid medium at pH values lower than those giving visible growth indicated that these pH values were bactericidal rather than bacteriostatic for both the wild and acid-conditioned strains.


Metabolism of glutamic acid in Aspergillus ochraceus during the biosynthesis of ochratoxin A. The uptake and utilization of glutamic acid in the biosynthesis of ochratoxin A by Aspergillus ochraceus were studied. Uniformly labeled L[14C]glutamic acid was incorporated into both the phenylalanine and isocoumarin moieties of ochratoxin A. Penicillic acid was also labeled. During the early stages of development, the amino acid was used mainly for the synthesis of ribonucleic acid and protein. A portion of glutamic acid was oxidized and was recovered as metabolic 14CO-2. The initial uptake  velocity of glutamic acid decreased with age and was pH and temperature dependent. No relationship was found between the initial uptake velocities and ochratoxin A biosynthesis.


Powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil. Agar media made with 0.4% colloidal chitin plus mineral salts and adjusted to pH 8.0 was superior to four other commonly used media for the isolation and enumeration of actinomycetes from water samples. More actinomycetes developed on chitin agar, and the development of bacteria and fungi was suppressed. Frozen and vacuum-dried chitin from aqueous colloidal suspensions was finely divided and gave results comparable to those obtained with media prepared from colloidal suspensions.


Effect of prednisolon on cell metabolism of candida. In vitro effects of 2 water-soluble prednisolon-derivatives on 10 Candida albicans and 10 Candida tropicals species were investigated. Tests were performed during resting and proliferation phases. Changes of cell metabolism were measured by O2-consumption by the Warburg technique. With increasing corticosteroid concentrations (10-10000 gammaml) a dose dependent increase of cell respiration was found with and without addition of nutritive substrates. Even high concentrations of corticosteroid did not cause an antimetabolic effect. The tests did not yield different results for the two Candida species examined.


The behaviour of collagen peptidase in the serum of different dermatoses. In 150 patients with different dermatoses and 15 controls the activity of collagen peptidase in serum was determined. No relation to specific skin diseases was found. Negative findings were also observed in the group of collagenoses: only 4 of 14 patients with progressive systemic sclerosis showed an increase of this enzyme activity. These negative results seem noteworthy because it thus is demonstrated that alterations of collagen structure are not necessaryly associated with alterations of collagen peptidase activity.


DDT and immunological responses. 3. Reduced anaphylaxis and mast cell population in rats fed DDT. Previous work showed that anaphylaxis, mast cell population, and tissue histamine content are reduced in guinea pigs given DDT injections. This study was intended to determine whether dietary intake of DDT causes similar effects. Rats immunized with diphtheria toxoid and fed diets containing DDT at 20 and 200 ppm levels for 31 days did not show effects on their serum antitoxin titers, but the numbers of metachromatically stained, histamine-containing mast cells in mesenteries were reduced: in the 20 ppm group by 46% and in the 200 ppm group by 61%. The severity of anaphylactic shock was also reduced in proportion to the DDT dietary levels, and, thus, the magnitude of mast cells. Apparently, daily dietary DDT intake above 2.2 mg DDT/kg of body weight alters the physiology of mast cells in the rat, and thus affects histamine-mediated reactions.


Different behaviors of erythrocyte delta-aminolevulinic acid dehydratase. A comparison study between lead workers and normals. Different characteristics of erythrocyte delta-aminolevulinic acid dehydratase (ALA-D1) between the workers with the history of occupational lead exposure and normals are described. In the blood of lead workers, when the hemolysates are heated at 60 C for five minutes, the activity of the erythrocytes ALA-D increases up to about 3.6-fold of an initial level, and as a result of heating the optimum in pH-activity curvee changes from pH 6.0 to pH 6.6, which is similar to the optimum pH of normal ALA-D. On the contrary, in normal blood, the optimum in the pH-activity curve is but little changed, even though the erythrocyte ALA-D activity is increased up to about 1.3-fold of the initial level by heating the hemolysates at 60 C for five minutes.


The fate of autologous fascia lata grafts in dogs. The fate of tricuspid fascia lata valves and intracardiac grafts was studied morphologically, histologically, and biochemically in dogs. Severe degenerative changes occurring by 21 days were largely correlated with graft thickening due to gross endothelialization. A primary event in graft alteration was an initial loss of the indigenous fibroblast population as detected both microscopically and by autoradiography after pulse labeling with 14C-Nethylmaleimide. Using different isotopic labeling procedures, both the in vivo and in vitro graft degradation patterns demonstrated that a second early eft degradation was correlated in vivo with endothelialization and collagenase secretion by invasive cells.


The hysterical personality. An attempt at validation with the MMPI. We attempted to validate the DSM-II diagnosis of hysterical personality and the depression often experienced by such patients by comparing mean Minnesota Multiphasic Personality inventory (MMP) scores of hysterical personality patients with those of paranoid schizophrenic patients and a group with mixed psychiatric diagnoses. Index patients had significantly higher scores than either control group and could be distinguished on individual scales from the paranoid schizophrenics. However, the mean MMPl profile of hysterical personalities was similar to that of depressed controls. Therefore, the MMPl alone could not differentiate these two groups nor could it confirm or refute the validity of the diagnostic concept of hysterical personality, but it did support the clinical observation that depression is a major risk for individuals given this diagnosis and that the experience of depression by these patients is genuine.


The offspring of schizophrenics. Fetal and neonatal deaths. This is the first report in a projected series on neurological development among the offspring of schizophernics. An increased incidence of fetal and neonatal deaths is noted among offspring of schizophrenics when compared to a closely matched control group, using data collected prospectively. In many cases, no apparent reason for death was determined; in some cases, major neurological malformations were found. It is necessary to consider three possible explantions for these findings--genotype of the child, adverse intrauterine environment, and medication toxicity.


Supervision of the initial interview. A study of two methods. Two methods of supervision of the initial evaluation of outpatients by psychiatric residents were compared. In condition 1, residents evaluated patients and then presented the case material to a supervisor in the traditional manner. In condition 2, the supervisor observed the interview directly. Supervisor and residents then independently completed ratings of psychopathology, motivation, insight, and prognosis of the patients. Significant differences in several variables between the two conditions were noted. Resident and supervisor ratings of patients were in clear agreement when both observed the interview.


Cardiorespiratory responses to dextran 40. Hemodynamic and oxygen transport changes in normal subjects and critically ill patients. Hemodynamic and oxygen transport were observed in 190 studies before and after administration of 500 ml dextran 40 in 12 normal, healthy subjects and 147 critically ill patients who were, or recently had been, in various degrees of shock. The major influence of dextran 40 was plasma expansion and hemodilution, which resulted in increased blood flow and blood volume. In general, the pressure-flow responses were greater in the ill patients than in the normal subjects. After dextran 40 infusion, oxygen transport increased in the critically ill patients, but not in the normal volunteers. Some of the effects of dextran 40 on oxygen transport in critically ill patients may be attributed to increased tissue perfusion from expansion of plasma volume, as well as to improved flow properties of blood in the microcirculation.


Survivor's and nonsurvivors' responses to dextran 40. Hemodynamic and oxygen transport changes in critically ill patients. Cardiorespiratory effects of dextran 40 administration were measured and compared retrospectively in 74 surviving and 73 nonsurviving critically ill patients. In the preinfusion control period, the survivors had higher mean arterial pressures and blood flow with lesser blood volume deficits. Dextran significantly improved pressure, blood flow, blood volume, oxygen transport, and derived calculations in both groups, but the average cardiorespiratory responses to dextran were somewhat greater in nonsurvivors than in survivors, About two thirds of the patients responded to dextran with significantly increased oxygen consumption (P smaller than .05), suggesting that this agent improved oxygen transport by its rheologic effects on the microcirculation. The cardiorespiratory effects of dextran were slightly greater in the patients who ultimately died; nevertheless, the salutary cardiorespiratory response did not reverse the clinical course in nonsurvivors, whose perfusion defect apparently had reached irreversible proportions prior to the administration of the drug.


Catecholamines in experimental brain ischemia. Local cerebral ischemia was produced in rats by internal carotid artery injection of 35 mu carbon microspheres, and brain norepinephrine (NE), dopamine, and cyclic adenosine 3, 5-monophosphate (cAMP) were measured in embolized and intact hemispheres at intervals up to four hours. Sham-operated animals were controls. There was an instantaneous increase of cAMP. Norepinephrine was reduced within two minutes after embolization and remained low for four hours. Dopamine increased by five minutes after embolization and returned to normal after four hours. Results were qualitatively similar, but less, in the nonembolized hemisphere. Accumulation of cAMP is thought to be due to a direct effect of ischemic hypoxia and may be the initiating factor in increased glycolysis that occurs in ischemia. Decrease in NE may be secondary to its generalized release from presynaptic terminals throughout the brain and could be a factor in cortical vasocontriction that follows embolization. Dopamine changes are a reflection of alterations in energy metabolism.


Amino acid composition of cerebrospinal fluid in actue neuroinfections in children. A survey of the cerebrospinal fluid (CSF) amino acids, glutamine, and glutamic and gamma-aminobutyric (GABA) acids was made in 168 children, aged 1 to 14 years, with various neurological infections. The glutamic acid and glutamine concentrations in the CSF of children with severe forms of acute serous and bacterial meningitis were about three to four times as great as in controls. The indices returned almost to normal during recovery. GABA is absent in normal CSF, but appeared in the CSF of patients with bacterial meningitis. Its determination may be used as an additional test to differentiate between serous and bacterial meningitis.


Postmortem changes in brain catecholamine enzymes. Postmortem changes in the activities of tyrosine hydroxylase, dopa decarboxylase, and dopamine-beta-hydroxylase were examined in various areas of rat brain. Tyrosine hydroxylase activity decreased in an exponential fashion with a half-time of two to four hours in caudate-putamen, substantia nigra, and locus ceruleus. Dopa decarboxylase activity remained within 20% of control values at five hours in these areas, but then decreased precipitously. Dopamine-beta-hydroxylase activity remained within 20% of control for at least 20 hours after death.


Microbiological sampling of the spacecraft atmosphere during a simulated skylab mission. A skylab Air Sampler (SAS) has been developed for the use during Skylab missions. The SAS was used in the Skylab Medical Experiments Altitude Test (SMEAT) to gather baseline data which could be directly compared to data obtained during actual Skylab missions. The results obtained in the SMEAT gave no evidence of consistent change in either concentration or types of microorganisms in the SMEAT atmosphere over the 56-d test. Microorganisms found included some potential pathogens but were largely normal human microflora. Few typical soil microorganisms were found. These findings are related to commonly anticipated effects of long-term spaceflights on environmental microflora and to other closed environment studies.


Solubility and net exchange of calcium, magnesium and phosphorus in digesta flowing along the gut of the sheep. 1. The changes in the solubility of calcium, magnesium and phosphorus in digesta flowing along the intestinal tract and the net movement across the intestinal wall of these elements were determined in six rams, each equipped with three T-shaped cannulas; cannulas were placed in a total of six different sites of the small intestine. Cr2O3 was used as a marker substance to measure the rate of flow of the digesta. 2. The concentrations of soluble Ca, Mg and P decreased as digesta moved along the intestine. TheMgreatest fall in soluble Ca occurred after the first 3 m of the intestine, while a significant decrease in soluble Mg was found only at 15 and 25 m from the pylorus. The concentration of soluble P in digesta decreased until the 7 m site and then remained stable. In the faeces, the level of soluble Mg was approximately 4 times higher than, and that of Ca equal to, the levels of Mg and Ca found in digesta flowing through the upper intestine. Unlike Ca and Mg, a very low concentration of soluble P was found in the faeces. 3. In the duodenum, 84, 78 and 62% of the total Ca, Mg and P respectively were soluble, whereas in the digesta flowing through the terminal ileum the corresponding values were 3-2, 7-2 and 19% for Ca, Mg and P respectively. 4. The forestomachs and the colon were found to be the main sites of  g net absorption; 1-12 mmol/h was apparently absorbed from the stomach and 1-05 mmol/h from the colon. The supper small intestine (1-3 m from the pylorus) appeared to be the major site of Ca and P absorption. 5. In the last 10 m of the small intestine, considerable amounts of minerals were secreted; 4-70, 0-96 and 1-85 mmol Ca, Mg and P/h respectively were added to the digesta flowing between 15 and 25 m from the pylorus. The effect of the increase in the pH of digesta along the small intestine on the solubility of these minerals is discussed.


Environment and conformation dependent sensitivity of the arsanilazotyrosine-248 carboxypeptidase A chromophore. Reaction of carboxypeptidase A crystals with diazotized arsanilic acid uniquely modifies Tyr-248 to form a monazo derivative, which-in solution-forms an intramolecular inner-sphere coordination complex in the active site zinc atom. tarsanilazocarboxypeptidase exhibits spectral properties that are closely similar to those of the model complex, tetrazolylazo-N-carbobenzoxytyrosine Zn2+, with a distinctive maximum at 510 nm. In addition, its circular dichroic spectrum reveals a negative extremum at this wavelength, also characteristic of this complex. Both spectra are exquisitely responsive to pth changes and serve to monitor formation and dissociation of the metal-azophenol complex. Two pKapp at 7.7 and 9.5 delineate the pH range over which the probe characteristics most effectively gauge conformational features of the active center of arsanilazcarboxypeptidase. Other environmental parameters, e.g., substrates and inhibitors, as well as crystallization of the enzyme also critically influence the formation and dissociation of the complex; the response of the probe suggests that they induce conformational movement of the azoTyr-248 residue away from the zinc atom. tthe now available chemical, functional, structural data bearing on the spatial relationships of Tyr-248 and Zn, both thought critical to catalysis, are evaluated, based on spectra of arsanilazo- and nitrocarboxypeptidase crystals and solutions as well as on detailed kinetic analyses of the native enzyme in both physical states and based on the X-ray structure analysis of the native enzyme and its Gly-L-Tyr complex. Collectively all of the data show that the conformation of carboxypeptidase in crystals differs from that in solution. Moreover, reexamination of the original X-ray maps reported in 1968 and thought to preclude a Tyr-248-Zn interaction now leads to the conclusion that in up to 25 per cent of the molecules in the crystals ttyr-248 interacts with the active site zinc atom (W.D. Lipscomb (1973), Proc. Nat. Acad. Sci U.S. 70, 3797). Thus, even in the crystals the enzyme exists in at least two different conformations. In one of these Tyr-248 is near while in the other it is far from the zinc atom. The spectral effects of Gly-L-Tyr and beta-phenylpropionate on solutions of arsanilazo- and of nitrocarboxypeptidase demonstrate that during the catalytic process Tyr-248 moves away from the zinc atom. This implies a mechanistic role for Tyr-248 different from that postulated on the basis of X-ray crystallographic analysis. Indeed, the proximity of ttyr-248 to the zinc atom, when altered by substrates and inhibitor, may reflect certain of the properties characteristic of the entatic, active site.


Proton magnetic resonance studies of carbonic anhydrase. I. Identification of histidine resonances. Nuclear magnetic resonance (nmr) spectra of human carbonic anhydrase B recorded in deuterium oxide reveal seven discrete single proton resonances between 7 and 9 ppm downfield from sodium 2,2-dimethyl-i-silapentane-5-sulfonate. Simplification of spectra by use of Fremy's salt, comparison of peak widths at intersections, and evaluation of the results of inhibition and modification experiments permit determination of the pH dependencies of these resonances. Five of these peaks change position with increasing pH; three move upfield by approximately 95 Hz and two move downfield by 10 and 23 Hz. The first three reflect residues with pK values of 7.23, 6.98, and 6 and can be assigned to the C-2 protons of histidines. The two remaining pH dependent resonances reflect groups with pK values of 8.2 and 8.24. Their line widths and T1 values are comparable to those of the first group, and they also appear to reflect C-H protons of histidines. Despite the structural and functional similarities of the B and C isozymes of human carbonic anhydrase, few of the low field resonances appear to be common to both. Six histidine C-2 protons are observed in the C enzyme and reflect groups with pK values of approximately 7.3, 6.5, 5.7, 6.6, 6.6, and 6.4. A seventh peak contains two protons and moves upfield with increasing pH without titrating. A final resonance to low field moves downfield with increasing pH and reflects a group with a pK between 6 and 7. Its behavior resembles that of peak 1 of the human B enzyme, and it also appears to be a histidine C-H proton. This peak may reflect a conserved residue in the two isozymes that plays an important role in enzymatic function, as discussed in the following paper.


Proton magnetic resonance studies of carbonic anhydrase. II. Group controlling catalytic activity. The seven resonances observed in the histidine region of the proton magnetic resonance (pmr) spectrum of human carbonic anhydrase B and reported in the preceding paper are studied in the presence of sulfonamide, azide, cyanide, and chloride inhibitors and in metal-free, cadmium substituted, cobalt substituted, and carboxymethylated forms of the enzyme. Results indicate that the two resonances that move-downfield with increasing pH and the two that do not move with pH reflect residues located at the active site. The first two resonances are assigned to the same titratable histidine whose pK value of 8.24 corresponds to that of the group controlling catalytic activity. Addition of anions or sulfonamides, removal of zinc, or substitution of cadmium for zinc at the active site, procedures known to abolish enzymatic activity, prevent titration of this residue. Partial inhibition of carbonic anhydrase by chloride slectively increases the pK value of the group controlling catalytic activity and of the histidine with pK equals 8.24. Experiments with metal-free and cadmium carbonic anhydrases and comparisons with model systems suggest that this histidine is bound to the metal ion at high pH; at low pH this complex appears to dissociate as protons compete with the metal for the imidazole group. It is proposed that ionization of the group controlling catalytic activity represents loss of the pyrrole proton of this neutral ligand when it binds to Zn(II), forming an imidazolate anion and juxtaposing a strong base and a powerful Lewis acid at the active site. When bound to zinc as an anion, this histidine can act as a general base catalyst in the hydration of carbon dioxide and be replaced as a metal ligand by an oxygen of the substrate in the course of the reaction. The histidine-metal complex is thought to exist in a strained configuration in the active enzyme so that its imidazole-metal bond is readily broken on addition of substrates or inhibitors. This model is consistent with the available data on the enzyme and is discussed in relation to alternative proposals.


Proton magnetic resonance studies of carbonic anhydrase. III. Binding of sulfonamides. Resonances of the histidine region of human carbonic anhydrase B have been studied by proton magnetic resonance spectroscopy in the presence of seven sulfonamide inhibitors. Results of difference spectroscopy and observation of the C-2 resonance of an additional titratable histidine in some of these spectra suggest a conformational change in the enzyme, while the large number of unaltered resonances indicates involvement of only a few residues. Inhibition of carbonic anhydrase by sulfonamides appears to involve: stabilization of an appropriately oriented initial complex by hydrophobic binding of the aromatic ring of the inhibitor to residues of the cavity forming the active site; ionization of the sulfonamido group, facilitated by its proximity to zinc; protonation and displacement of the high pH ligand to the metal controlling catalytic activity, thought here to be a histidine residue; and formation by the sulfonamido group of an ionic bond to zinc and a hydrogen bond to the hydroxyl group of serine or threonine. Diversity of spectra produced with various sulfonamides suggests that substituents on the ring and heteroatoms within the ring interact with additional groups at the active site. Increase in inhibitory potency appears to involve optimizing the number as well as the strength of these interactions. An upper limit for the dissociation rate of these complexes of 10 sec-1 was obtained.


Nicotinamide 3,N4-ethenocytosine dinucleotide, an analog of nicotinamide adenine dinucleotide. Synthesis and enzyme studies. A structural analog of NAD+, NICOTINAMIDE 3,N-4ethenocytosine dinucleotide (epsilonNCD+), has been synthesized, characterized, and compared in activity with the natural coenzyme in several enzyme systems. The Vmax and apparent Km values were determined for NAD+, epsilonNCD+, and epsilonNAD+ (nicotinamide 1, N6-ethenoadenine dinucleotide) with yeast alcohol, horse liver alcohol, pig heart malate, beef liver glutamate, and rabbit muscle lactate and glyceraldehyde-3-phosphate dehydrogenases. The Vmax for epsilonNCD+ was as great or greater than that obtained for NAD+ with three of the enzymes, 60-80 per cent with two others, and 14 percent with one. EpsilonNCD+ was found to be more active than epsilonNAD+ with all six dehydrogenases. EpsilonNCD+ served as a substrate for Neurospora crassa tnadase, but could not be phosphorylated with pigeon liver NAD+ kinase. NAD+ pyrophosphorylase from pig liver was unable to catalyze the formation of epsilonNCD+ from the triphosphate derivative of epsilon-cytidine and nicotinamide mononucleotide, but was able to slowly catalyze the pyrolytic cleavage of epsilonNCD+. The coenzyme activity of epsilonNCD+ with dehydrogenases can be discussed in terms of the close spatial homology of epsilonNCD+ and NAD+, which may allow similar accommodations within the enzyme binding regions.


Human pancreatic enzymes: purification and characterization of a nonelastolytic enzyme, protease E. resembling elastase. ?An enzyme with proteolytic activity has been isolated from activated extracts of human pancreatic tissue. The purification procedure included salt fractionation followed by ion-exchange chromatography on SE-TSephadex C-25 and on DEAE-Sephadex A-50. The homogeneity of this enzyme, designated protease te, was demonstrated by disc electrophoresis and by sedimentation equilibrium centrifugation stidues. The homogeneous enzyme shows the ability to hydrolyze many of the conventional synthetic substrates used for the identification of elastase activity; however, it demonstrates no significant elastolytic activity. A comparison of human protease E with porcine elastase reveals a high degree of similarity between the two proteases with respect to inhibition by active-site directed peptide chloromethyl ketones, stability, decreased susceptibility to naturally occurring proteinase inhibitors, and specificity for synthetic substrates as well as several other physical properties. The major difference between human protease E and porcine elastase, other than the lack of elastolytic activity by human protease E, seems to be in the ionic character and the amino acid composition of these two proteins. Porcine elastase is a cationic enzyme, while human protease E appears to be anionic in nature. These dissimilarities concerning elastolytic activity and ionic character appear to be directly related.


The solution conformation of nicotinamide mononucleotide: a quantitative application of the nuclear Overhauser effect. Torsion about the glycosidic linkage in nicotinamide mononucleotide has been investigated by quantitative application of the nuclear Overhauser effect. These measurements show that the syn (chi congruent to 20 degrees) and anti (chi congruent to 200 degrees) conformers of the title compound are isoenergetic, or nearly so, and interconverting rapidly. The syn/anti partition is not measurably affected by either changes in pH or temperature.


Partial characterization of the cytosol 3 alpha-hydroxysteroid: NAD(P)+oxidoreductase of rat ventral prostate. The cytosol 3alpha-hydroxysteroid dehydrogenase of rat ventral prostate has been partially purified. The rates of both the oxidation and reduction by crude and partially purified enzymes have been measured with a variety of radioactive substrates, and the effects of several inhibitor steroids have been assessed. Four conclusions have been drawn from the study. First, no detectable 3beta-androstanediol was formed from dihydrotestosterone, and the oxidation of 3beta-androstanediol was undetectable. Second, the cytosol enzyme exhibits a distinct and unique substrate specificity in that steroids with keto or hydroxyl substitution on the 11th carbon of the steroid cannot serve as substrates or as inhibitors of the enzyme. Third, either 5alpha or 5 beta reduction of delta4,3-keto steroids must take place before the steroids can serve as substrates of the enzyme. Fourth, many delta4,3-keto steroids that cannot act as substrates for the enzyme inhibit the enzyme competitively and may well serve as physiological regulators of the reaction in intact cell.


Membrane properties of Thermoplasma acidophila. Plasma membranes were isolated from Thermoplasma acidophila, a mycoplasma-like organism which grows optimally at pH 2 and 59 degrees. Cells in concentrated suspensions were lysed by titrating to pH 9.3. The membranes were purified by washing at pH 10 and centrifuging in a discontinuous sucrose gradient. Membrane purity was assessed by electron microscopy, determination of deoxyribonucleic acid content, polyacrylamide gel electrophoretic behavior. Gel patterns and amino acid composition of cells and membranes were found to differ significantly. The lipid contained small amounts of fatty acid esters and larger amounts of branched long-chain alkyl ethers.


Mutants of Escherichia coli K12 unable to grow on non-fermentable carbon substrates. Among a number of mutants unable to utilize non-fermentable carbon substrates, scoring for membrane ATPase and for ATP-driven transhydrogenase activity permitted to distinguish two phenotypes: (A) mutants lacking ATPase and ATP-driven transhydrogenase; (B) one mutant with an ATPase which behaved according to several criteria as released into solution instead of being membrane bound, a.o it exhibited no ATP-driven transhydrogenase activity. All A and B mutants exhibited a common nutritional pattern. The ATPase-deficient group, when scored for ATPase-binding sites on its membrane particles revealed three different subgroups: (1) mutants having free ATPase-binding sites, (2) mutants with ATPase-binding sites made available by the procedure which releases ATPase from wild-type membrane, and (3) mutants with no detectable ATPase-binding sites. Membranes of the mutant B with unbound ATPase also exhibited a deficiency in ATPase-binding sites, but its soluble ATPase was also found unable to bind to ATPase-binding sites of wild type membranes. The double alteration, namely abnormal or inactive ATPase and absence of ATPase-binding sites on the membrane is compatible with a single mutational defect.


Variations in the pathways of malate oxidation and phosphorylation in different species of Mycobacteria. Mycobacterium tuberculosis H37Rv, the slow-growing human pathogenic strain of tubercle bacilli and Mycobacterium smegmatis and Mycobacterium phlei, the fast-growing saprophytes, have shown variations regarding the type of dehydrogenase that initiates malate oxidation in the respiratory chain. M. tuberculosis H37Rv is characterized by having a malate oxidase system (designated MALNAD pathway) in which malate oxidation is mediated by the NAD+-dependent malate dehydrogenase (EC 1.1.1.37) but not by FAD-dependent malate-vitamin K reductase. M. smegmatis possesses a different malate oxidase system (designated MALFAD pathway) in which malate oxidation is exclusively carried out by the FAD-dependent malate-vitamin K reductase because NAD+-dependent malate dehydrogenase is absent in this organism. M. phlei has a mixed system of malate oxidase (designated MALNAD+FAD pathways) in which both the NAD+-and FAD-dependent dehydrogenases take part. In all the three systems, the rest of the electron transport chain is common.


Control of proton translocation induced by ATPase activity in chloroplasts. 1. Proton uptake was induced by ATP in the dark following light triggering of ATPase activity in chloroplasts. The accumulated protons were released when ATPase activity was inhibited by the energy transfer inhibitor DIO-9. 2. Approximately two protons were taken up for each ATP hydrolyzed at pH 8. A drop in H+/ATP ratio was caused by uncouplers such as NH4Cl and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These uncouplers caused an increase in the rate of ATP hydrolysis without a corresponding increase in proton uptake. 3. The energy transfer inhibitor dicyclocarbodiimide inhibited both ATPase activity and the rate of proton uptake without changing the H+/ATP ratio. 4. The antibiotic valinomycin caused an increase in the rate of both proton uptake and ATP hydrolysis without altering the ratio of H+/ATP. The H+/ATP ratio varied with changes in the external pH. The results were discussed in view of the chemiosmotic theory of oxidative and photosynthetic phosphorylation.


The mechanism of reduction of cytochrome c as studied by pulse radiolysis. 1. The reaction of hydrated electrons with ferricytochrome c was studied using the pulse-radiolysis technique. 2. In 3.3 mM phosphate-buffer (pH 7.2), 100 mM methanol and at a concentration of cytochrome c of less than 20 muM the reduction kinetics of ferricytochrome c by hydrated electrons is a bimolecular process with a rate constant of 4.5-10-10 M-1-S-1 (21 degrees C). 3. At a concentration of cytochrome c of more than 20 muM the apparent order of the reaction of hydrated electrons with ferricytochrome c measured at 650 nm decreases due to the occurrence of a rate-determining first-order process with an estimated rate constant of 5-10-6s-1 (pH 7.2, 21 degrees C). 4. At high concentration of cytochrome c the reaction-time courses measured at 580 and 695 nm appear to be biphasic. A rapid initial phase (75% and 30% of total absorbance change at 580 and 695 nm, respectively), corresponding to the reduction reaction, is followed by a first-order change in absorbance with a rate constant of 1.3-10-5 S-1 (pH 7.2, 21 degrees C). 5. The results are interpreted in a scheme in which first a transient complex between cytochrome c and the hydrated electron is formed, after which the heme iron is reduced and followed by relaxation of the protein from its oxidized to its reduced conformation. 6. It is calculated that one of each three encounters of the hydrated electron and ferricytochrome c results in a reduction of the heme iron. This high reaction probability is discussed in terms of charge and solvent interactions. 7. A reduction mechanism for cytochrome c is favored in which the reduction equivalent from the hydrated electron is transmitted through a specific pathway from the surface of the molecule to the heme iron.


Electric birefringence of myosin subfragments. Electric birefringence measurements have been made on aqueous solutions of myosin subfragments, heavy meromyosin, subfragments 1 and 2 (S-1 and S-2). All of these showed positive electric birefringence. Heavy meromyosin and S-2 showed a large intrinsic Kerr constant. From the analysis of the build up and decay process of the birefringence, the contribution of the slow induced dipole moment was concluded in heavy meromyosin and S-2, although the existence of the permanent dipole moment was not completely excluded. The decay process of the birefringence of heavy meromyosin was found to consist of two components; the fast one of which had a relaxation time of the same order as that of S-1. This is probably due to the presence of a flexible hinge in heavy meromyosin.


Heavy metal-nucleotide interactions. III. The participation of amino groups in the binding of methylmercury (II) to cytidine and adenosine 5'-phosphate in aqueous solution: studies by Raman difference spectrophotometry. Raman difference spectrophotometry has been used to study the interaction of CH3Hg(II) with cytidine and Ado-5'-P at high pH. In contrast to the binding reactions which occur at lower pH or in non-aqueous solvents such as dimethyl sulfoxide, a proton is transferred from the amino group; and the complexes are CH3HgCydH-1 and CH3HgAdoH-1-5'-P. The spectra are significantly different from those of the cationic complexes. The integrated intensities of ligand modes which shift upon metalation can be used to measure the concentration of unreacted ligand and consequently the extent of the reaction. Equilibrium constants for the reactions CH3HgOH + L yields CH3HgLH-1 + H2O were estimated to be log KCyd equals 0.63 plus or minus 0.05 and log KAdo-5'-P equals 0.85 plus or minus 0.05, in fair agreement with values determined under very different conditions by ultraviolet spectrophotometry. The vibrational spectrum of the ligand in CH3HgCydH-1 is virtually the same as that of UrdH-1- which is isoelectronic. The spectrum of the ligand in CH3HgAdoH-1-5'-P is more similar to the isoelectronic base InoH-1-than to Ado-5'-P, although the resemblance is not so close as in the CydH-1---UrdH-1-case. The structures of these complexes are discussed on the basis of their vibrational spectra and similarities in the spectra of related compounds. It is concluded that the CH3Hg(II) binds to the amino nitrogen at high pH with both cytidine and Ado-5'-P. In neutral solution with excess CH3Hg(II), metalation occurs on the amino groups, on the ring, and also on the ribose.


Cytoplasmic origin of the so-called nuclear neutral histone protease. 1. We have investigated the origin of proteolytic activity which causes degradation of histones in chromatin isolated from Xenopus liver and the rat liver at neutral pH. Polyacrylamide disc gel electrophoresis was used for detection of proteolytic products of histones. 2. No proteolytic degradation of histones occurs in chromatin isolated from Xenopus erythrocytes and rat liver according to our procedure even after prolonged incubation at pH 8.0 and pH 5.0. However with chromatin isolated from Xenopus liver a high level of histone degradation is observed under similar conditions. 3. Mixing isolated nuclei from Xenopus erythrocytes with a crude cytoplasmic fraction from Xenopus liver causes histone proteolysis in isolated chromatin at pH 8.0. In similar experiments with corresponding fractions from rat liver histone proteolysis can be introduced only after repeated freezing and thawing of the cytoplasmic fraction. 4. A purified lysosomal preparation from rat liver causes a similar type of histone degradation upon incubation with chromatin from Xenopus erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal fraction from rat liver is inhibited by sodium bisulphite. 6. We conclude that the neutral proteolytic activity which causes degradation of histones in isolated chromatin is due to a contamination with neutral protease(s) originating from cytoplasmic organelles.


Hydrogen-ion titration curve of ovomucoid. A systematic study of the H+ titration curve of purified ovomucoid was made at three temperatures (15, 25 and 35 degrees C) and three ionic strengths (0.05, 0.15 and 1.0). In all, 49 protons were dissociated reversibly in the pH range, 2.0-12.0. From the analysis of the results up to pH 12.0, the numbers of different dissociable groups per 28 300 g protein, together with their intrinsic pK values in parentheses were found tp be' 27 sode-chain carboxyl (pKint=4.0), four imidazole (pKint=6.5), one alpha-amino (pKint=7.5), 12 epsilon-amino (pKint=9.6), one guanidino (pKint=11.8) and one alpha-carboxyl group with abnormally low pK. The total number of basic nitrogens per mole of the protein was 22 so that four guanidino groups remained untitrated up to pH 12.0. Spectrophotometric titration showed that three out of five phenolic groups were titrated reversibly up to pH 11.9 with an intrinsic pK of 10.25; the remaining two groups became accessible only on protein denaturation. Viscosity results suggested absence of conformational change in the pH range 2.0-11.2. This explains the constancy of the pK values of carboxyl groups in the pH range 2.0-5.0. The empirical value of the electrostatic interaction factor, w, was 0.04, both in the carboxyl and phenolic regions.


Thyroid microsomal membrane proteins. Effects of solubilization on molecular size. The molecular size of microsomal membrane proteins from frozen porcine thyroids before and after solubilization by proteolytic and non-proteolytic techniques has been investigated by means of polyacrylamide-gel electrophoresis in the presence of 1% sodium dodecylsulfate. When thyroid microsomal membrane proteins are solubilized by non-proteolytic methods such as high pH, n-butanol, or deoxycholate, no major change in the electrophoretic pattern compared to untreated microsomes has been observed, thereby suggesting that these non-proteolytic methods are capable of extracting membrane proteins from thyroid microsomes without altering their molecular size. However, treatment of microsomes with protein-solubilizing levels of trypsin (1-5 mug trypsin per mg thyroid protein) results in degradation of all major proteins with a molecular weight greater than 30 000. The high-molecular-weight proteins are particularly susceptible to attack by trypsin. Thus, these experiments indicate that the use of trypsin to solubilize thyroid microsomal membrane proteins, particularly thyroid peroxidase, will result in fragmented proteins and should be avoided if intact membrane proteins are desired.


Isoelectric-focusing behavior of acid hydrolases in rat kidney lysosomes. Effects of the pH gradient, autolysis and neuraminidase. Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an  isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd


Changes in electronegativity of lysosomal hydrolases during intracellular transport. An isoelectric-focusing study in subcellular fractions of rat kidney. Isoelectric focusing was used to investigate the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase, beta-galactosidase and beta-N-acetylhexosaminidase in the following, previously characterized subcellular fractions from rat kidney: a special rough microsomal fraction, enriched up to 9-fold over the homogenate in acid hydrolases; a smooth microsomal fraction; a Golgi membrane fraction enriched about 2.5-fold in acid hydrolases and 10- to 20-fold in several glycosyl transferases; and a lysosomal fraction enriched up to 25-fold in acid hydrolases. The electro-focusing behavior of the hydrolases in these fractions was markedly sensitive to the autolytic changes that occur under acidic conditions, even at 4 degrees C. Autolysis was minimized by extracting fractions in an alkaline medium (0.2% Triton X-100, 0.1 M sodium glycinate buffer, pH 10, 0.1 % p-nitrophenyloxamic acid) and adding p-nitrophenyloxamic acid (0.1 %), AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND cathepsin D, to the pH gradient. The enzymes in the lysosomal fraction displayed a characteristic bimodal or trimodal distribution. Arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in an acidic form with an isoelectric point of 4.4, and a basic form with an isoelectric point of 6.2, 6.7 and 8.0, respectively. Acid phosphatase and beta-galactosidase occurred in an acidic, intermediate and basic form with isoelectric points of about 4. 1, 5.6 and 7.4, respectively. In the special rough microsomal fraction these enzymes were mostly in a basic form with isoelectric points between 7.5 and 9; these were 1-2 units higher than the corresponding basic forms in the lysosomal fraction. Treatment of extracts of the rough microsomal fraction with bacterial neuraminidase raised the isoelectric points of all five hydrolases by 1-2.5 units, indicating the presence of some N-acetylneuraminic acid residues in these basic glycoenzymes. The hydrolases in the Golgi fraction were largely in an acidic form with isoelectric points similar to or lower than those of the corresponding acidic components in the lysosomal fraction. The hydrolases in the smooth microsomal fraction showed isoelectric-focusing patterns intermediate between those in the rough microsomal and the Golgi fractions. These findings support the following scheme for the synthesis, transport and packaging of the lysosomal enzymes. Each hydrolase is synthesized in a restricted portion of the r


Purification, structure and properties of the respiratory nitrate reductase of Klebsiella aerogenes. 1. The respiratory nitrate reductase of Klebsiella aerogenes was solubilized from the bacterial membranes by deoxycholate and purified further by means of gel chromatography in the presence of deoxycholate, and anion-exchange chromatography. 2. Dependent on the isolation procedure two different homogeneous forms of the enzyme, having different subunit compositions, can be obtained. These forms are designated nitrate reductase I and nitrate reductase II. Both enzyme preparations are isolated as tetramers having sedimentation constants (s20,w) of 22.1 S and 21.7 S for nitrate reductase I and II, respectively. The nitrate reductase I tetramer has a molecular weight of about 106. 3. In the presence of deoxycholate both enzyme preparations dissociate reversibly into their respective monomeric forms. The monomeric form of nitrate reductase I has a molecular weight of about 260 000 and a sedimentation constant of 9.8 S. For nitrate reductase II these values are 180 000 and 8.5 S, respectively. 4. Nitrate reductase I consists of three different subunits, having molecular weights of 117 000; 57 000 and 52 000, which are present in a 1:1:2 molar ratio, respectively. Nitrate reductase II contains only the subunits with a molecular weight of 117 000 and 57 000 in a equimolar ratio. 5. Treatment at pH 9.5 in the presence of deoxycholate and 0.05 M NaCl or ageing removes the 52 000 Mr subunit from nitrate reductase I. This smallest subunit, in contrast to the other subunits, is a basic protein. 6. The 52 000 Mr subunit has no catalytic function in the intramolecular electron transfer from reduced benzylviologen to nitrate. However, it appears to have a structural function since nitrate reductase II, which lacks this subunit, is much more labile than nitrate reductase I. Inactivation of nitrate reductase II can be prevented by the presence of deoxycholate. 7. The spectrum of the enzyme resembles that of iron-sulfur proteins. No cytochromes or contaminating enzyme activities are present in the purified enzyme. Only reduced benzylviologen was found to be capable of acting as an electron donor. 8. p-Chlormercuribenzoate enhances the enzymatic activity at concentrations of 0.1 mM and lower. At higher p-chlormercuribenzoate concentrations the enzymatic activity is inhibited non-competitively with either nitrate or benzylviologen as a substrate. The inhibition is not counteracted by cysteine.


EPR studies of protein state transitions in Chromatium ferricytochrome c'. Recent magnetic studies have shown that in the pH range 1 to 11 the bacterial heme protein ferricytochrome c' can undergo reversible transitions between various pure high-spin and quantum mechanically mixed (intermediate and high)-spin protein states. The EPR data presented here extend the recent work to high alkaline pH and show that above pH 11.6 reversible transitions occur between various pure high-spin and pure low-spin protein states. The new data and the previous magnetic studies of the protein are discussed in the context of the entatic nature of the iron-porphyrin complex in ferricytochrome c'. EPR data are also provided concerning an anomalous, but reversible, transition to a protein state with reduced heme iron, triggered by freezing and thawing at physiological pH.


Study of a haploid yeast strain with an unusually high rDNA content. II. Fractionation of the DNA in preparative Ag+-Cs2SO4 density gradients. Yeast DNA has been fractionated in preparative Ag+-C-S2SO4 density gradients. After complexing with silver ions at a pH of about 7, the rDNA appears in a defined heavy satellite component in the gradient. For our particular strain, the satellite represents about 15% of the total nuclear DNA and has been  identified as the gamma-DNA. In alkaline CsCl density gradients, the satellite DNA forms two bands, of which the light component hybridizes with ribosomal RNA.


Effect of biotin on ribonucleic acid synthesis. A single injection of biotin to biotin-deficient rats produces a two-fold increase in the incorporation, both in vivo and in vitro of precursors into nucleic acids as early as 2 h after the biotin treatment. The specific activity of the precursor pool is not affected by biotin. Analysis of the polysome profile at various times following biotin treatment and a kinetic study of the effect of excess poly(U) on the incorporation of phenylalanine by cell-free amino acid incorporation experiments indicate a marked decrease in messenger-free ribosomes in rat liver after biotin administration.


Observations on the mechanical precipitation of oxy Hb S and other mutants. Oxyhemoglobin S exhibits greater mechanical instability than oxyhemoglobin A. The rate of precipitation of Hb S when agitated by vortexing depends upon the geometry of the tube, the volume of the hemoglobin solution, and the concentration of hemoglobin. The rate of precipitation is inversely related to concentration. Precipitation is inhibited by temperatures near 4 degrees C and alkylureas whose protective capacity is approximately proportional to the carbon chain length of the alkyl group. Blocking the beta93 -SH group with parahydroxymercuribenzoate has only a small enhancing effect on the precipitation rate. Other mutants such as Hb Gun Hill, Leiden, (both heat unstable), and C-HARLEM are also unstable. In the case of C-HARLEM, the precipitation rate is greater than that for Hb S. The heat-unstable mutants are not as well protected by cold temperatures or alkyl ureas. D2O has only a minor stabilizing effect on hemoglobin S, but NaCl and related salts markedly enhance precipitation at concentrations of 0.5 M. It is concluded that mechanical instability of oxyhemoglobins is a multifactorial process involving surface denaturation, pH, ionic strength, hydrophobic interactions, protein conformation, and primary protein structure. This phenomenon will require more extensive investigation.


Breakdown of prothrombin and proconvertin in rats with experimental extrahepatic cholestasis. Changes in the half-life of prothrombin and proconvertin were studied in rats with experimental extraphepatic cholestasis induced by ligation of the common bile duct. By the end of the second week of cholestasis the prothrombin activity was reduced almost by half and the proconvertin activity by more than two-thirds compared with the control. Meanwhile the half-life of prothrombin after cholestatsis for 14 days was increased by 1.f h and that of proconvertin by 4.7 h compared with the control. The slower breakdown of prothrombin and proconvertin during cholestasis is regarded as a compensatory reation.


Effect of o,p'-dichlorodiphenyldichloroethane on glutathione reductase activity and content of SH groups in the dog adrenals. The effect of o, p'-dichlorodiphenyldichloroethane (DDD), a compound inducing atrophy of the adrenal cortex and blocking steroid production, on glutathione reductase activity was studied. As a result of feeding dogs with DDD in a dose of 50 mg/kg body weight for 14 days activation of glutathione reducatase took place in the homogenate, cytoplasmic fraction, and disintergrated mitochondria of the adrenals. On the additon of DDD in vitro the activity of the enzyme was unchanged. The content of SH groups expressed per 100 mg tissue was reduced in the homogenate and in all subcellular fractions. If expressed per 100 mg protein in the level of SH groups was lowered only in the microsomes and the disintergrated mitochondria.


Method of investigation of deviations from the normal duration of the cardiac cycle. An electronic systems for determining the frequency of deviations from the normal duration of the cardiac cycle is described.


Usefulness of 99mTechnetium pertechnetate in periarteritis nodosa. Radioisotopes have a wide range of application in the clinical investigation of many organs. Muscle diseases are an exception in this expanding field. We report here a case of periateritis nodosa in which unusual scintigraphic images were obtained with 99mTc-pertechnetate. The kinetics of this isotope and the scintigraphic changes were followed during treatment and found to be useful clinical tools.


The uptake of GABA by clonal nerve glia. A number of clonal nerve and glial cell lines are able to accumulate exogenous GABA. The ability of the cells to concentrate GABA is dependent upon external calcium, while the uptake process is sodium dependent and temperature sensititive. GABA uptake by the cell lines is specific with respect to analogue inhibition, but this specificity is different from that observed in brain slices. It is, however, similar to that of glia in the rat sensory ganglia. Although clonal muscle cells can concentrate GABA to a limited extent, a number of other differentiated cells cannot.


Cerebral metabolism in hypoxic hypoxia. I. Pattern of activation of glycolysis: a re-evaluation. In order to evaluate the pattern of activation of glycolysis in cerebral cortex during hypoxic hypoxia, lightly anesthetized rats were subjected to a lowering of arterial Po2 to about 25 mm Hg and brains were frozen in situ for metabolite analyses either 1, 2, 5, 15 or 30 min following the induction of hypoxia. The lactate and pyruvate concentrations increased progressively during the 30 min period of hypoxia. At 1 and 2 min there were decreases in G-6-P and F-6-P, and increases in FDP, DHAP and 3-PG, indicating activation of phosphofructokinase. At 5 min this pattern of changes was less pronounced and at 15 min it was absent in spite of the fact that the lactate and pyruvate concentrations were further increased. At 30 min F-6-P and F-6-P had further increased but the levels of DHAP, FDP and 3-PG were normal. Evidently, phosphofructokinase activation can only be detected in the early stages of hypoxia, i.e. when the maximal increase in glycolytic flux occurs and before there has been a corresponding activation of other rate-limiting enzymatic steps. Signs of activation of phosphofructokinase were observed in the absence of changes in tissue concentrations of ATP or AMP, with minimal elevation of NH4plus, and in spite of increased (or unchanged) levels of citrate. However, since there were small but significant increases in ADP at 1 and 2 min, and pH-independent decreases in phosphocreatine, the results indicate that hypoxia is accompanied by an initial imbalance between production and utilization of ATP. The metabolic consequences of this imbalance (decrease in phosphocreatine, increases in ADP and P1) may be at least partly responsible for activation of phosphofructokinase.


Effects of putative neurotransmitters on neuronal activity in monkey auditory cortex. The effects of the putative neurotransmitters norepinephrine (NE), gamma-aminobutyric acid (BAGA), and acetylcholine (ACh) were tested on auditory cortex neurons which were activated acoustically by species-specific vocalizations in awake squirrel monkeys. Five-barrel glass electrodes were used to record the activity single neurons in the superior temporal gyrus and to apply NE, GABA, or ACh microiontophoretically. Poststimulus time histograms and raster displays of neuronal responses to the vocalizations were computed before, during, and after iontophoresis. Dose-dependent inhibition of spontaneous and vocalization-evoked discharge rates was seen with NE and GABA. Generally, excitation was observed with ACh. A given dose of NE or GABA reduced spontaneous activity by a greater proportion than it reduced activity evoked by the vocalizations. During excitatory responses, segments with lower discharge rates were reduced proportionately more than segments with higher discharge rates. Usually, response 'pattern' was not altered by iontophoresis of any of the substances. However, in some cases the differential inhibition of slow activity produced by NE or GABA did result in a 'patern' change. The demonstration that small amounts of locally applied NE and GABA substantially alter the specific neuronal activation produced by vocalizations provides additional evidence that these agents may function as neurotransmitters in this neocortical area and offers clues about their functional significance.


Selective uptake of (3H)beta-alanine by glia: association with glial uptake system for GABA. On the basis of inhibitor studies it has been suggested that the high-affinity GABA uptake system in glial cells differs in its chemical specificity from that present in nerve terminals. In this paper it has been demonstrated that the GABA analogue, beta-alanine, is almost as good a substrate as GABA itself in the glial cell uptake system whilst not being transported at all into nerve terminals. Not only has [3H]beta-alanine been shown autoradiographically to be exclusively accumulated in glial sites in toeh sensory ganglia and slices of cerebral cortex, but in addition a virtually identical high-affinity uptake process for [3H] beta-alanine was demonstrated at both these sites. Furthermore, non-radioactive GABA was shown to be an equipotent inhibitor of [3H]beta-alanine uptake in sensory ganglia as well as in both small slices and synaptosomes prepared from cerebral cortical tissue, Finally, the competitive inhibition kinetics of GABA on [3H]beta-alanine uptake in cortical slices furnishes proof that intact glial sites of GABA uptake were present in such slices in addition to the more prominent and well documented uptake system in nerve terminals. The possible imporatnce of such glial fragments in brain slice and crude synaptosomal preparations is discussed.


The cerebral ventricles as the avenue for the dipsogenic action of intracranial angiotensin. Using low doses of angiotensin II (1-128 ng), a mapping study was carried out to redefine the region within the brain from which the dipsogenic response can be elicited. The most sensitive sites were either close to the anterior cerebral ventricles or were at the tips of cannulae that traversed a ventricular space en route to the tissue site. Conversely, insensitive sites were remote from the ventricles and were not reached by cannulae with a ventricular trajectory. Therefore, a thorough assessment of the role of the ventricular system in the angiotensin thirst phenomenon was demanded. Further studies revealed that direct intraventricular injections were as effective as those made into any tissue site tested. Also, regardless of the similarity in the sites of their termination, cannulae which passed through a ventricular space en route to that site yielded highly sensitive preparations whereas those which did not were insensitive to the hormone. Autoradiographic and radioassay studies showed that tritiated angiotensin II injected into anterior diencephalic tissue through cannulae which traversed the ventricles was rapidly distributed throughout the ventricular system. Thus, efflux of the injected material up the cannula shaft and its entry into the ventricular system is essential for a dipsogenic response to low doses of the hormone. These results support the hypothesis of a periventricular receptor site for angiotensin. It is suggested that systemically generated angiotensin and angiotensin endogenous to the brain may use the ventricular route as a means of access to the sensitive periventricular site.


The soluble adenosine triphosphatase of Thiobacillus ferrooxidans. Adenosine triphosphatase (ATPase) from Thiobacillus ferrooxidans was purified 55-fold. Polyacrylamide gel electrophoresis of the most purified fraction showed only one major band; histochemical analysis showed that the ATPase activity was associated with this band. The pH optimum is 9-10. The enzyme hydrolyzed ATP stoichiometrically to ADP and inorganic phosphate, the Km for this substrate being 7.75 times 10-3 M. GTP and ITP are alternate substrates, the Km values for these being 6.71 times 10-3 M and 3.12 times 10-3 M, respectively. ADP is slightly hydrolyzed. Magnesium, manganese, and calcium can serve as cofactors; Km values for these are 2.0 times 10-3 M, 9.4 times 10-4 M, and 8.0 times 10-4 M, respectively. The enzyme activity was not activated by either sodium or potassium, but a combination of the two ions were inhibitory. Azide and p-hydroxymercuribenzoate strongly inhibited the enzyme activity, whereas cyanide, dinitrophenol, and N,N'-dicyclohexylcarbodiimide (DCCD) were without effect. The enzyme was cold labile at 0 degrees-C, but was more stable at 18-24 degrees-C.


Loss of primary alkylsulfatase and secondary alkylsulfatases (S-1 and S-2) from Pseudomonas C12B: effect of culture conditions, cell-washing procedures, and osmotic shock. Secondary alkylsulfatases (S-1 and S-2) were detected in Pseudomonas C12B cultured in minimal media lacking alkylsulfatases. The synthesis of these enzymes was not repressed by SO4-2- and L-cysteine or derepressed by L-methionine. Growth on 4% sodium citrate caused a 98% loss in the secondary alkylsulfatase activity of cells and 9% of this activity was detected in the culture medium. Citrate (20 mM) inhibited the activity of cell extracts (18%) but the inhibition was reversible by dialysis. The primary alkylsulfatase content of cells was not diminished by growth on citrate. The MgCl2 concentration of the medium also influenced the cellular levels of secondary alkylsulfatase. Bacteria grown on MgCl2 (0.05 mM - 40 mM) exhibited progressively increasing activity while the converse distribution was observed for activity present in the medium after growth at each MgCl2 concentration. Both primary and secondary alkylsulfatases were released when cells were either subjected to osmotic shock or treated for cell wall removal. Cells washed with 0.085 M sodium citrate-10 mM Tris-20%sucrose released roughly 87% of both enzymes and MgCl2 (0.04 M) inhibited the release of primary alkylsulfatase by 11% and secondary alkylsulfatase by 50%. It is suggested that citrate chelates divalent cations necessary for the attachment of secondary alkylsulfatase at the cell periphery.


Yeast-like growth of Mucor alternans (van Tieghem) in tissue-culture medium 199. Mucor alternans(van Tieghem) was found to be a diphasic species in that it grew exclusively in the yeast-like budding phase under anaerobic conditions in the complex medium yeast extract-peptone-glucose broth and in tissue-culture medium 199. In the latter medium this growth form occurred also at 37C, at an initial pH of 7.2, and at glucose concentrations of 0.1 and 1.0%. The authors suggest that because of its synthetic nature the tissue-culture medium could be used with advantage in the study of nutritional requirements of dimorphic mucors.


Stable isotope fractionation by Clostridium pasteurianum. 1. 34S/32S: inverse isotope effects during SO4-2- and SO3-2- reduction. During growth on minimal salts--sucrose media supplemented with various concentrations (10-4-10-2 M) of sodium sulfate, Clostridium pasteurianum grew at a normal rate and only evolved sulfide in late stages of growth on 10-2 M SO4-2-. The evolved sulfide was slightly enriched in 34S as compared to the medium sulfur. On the other hand, sulfide was evolved during growth on all concentrations of sulfite tested. Large normal and inverse isotopic effects were observed in the evolved sulfide during SO3-2- reductions. In contrast, the intracellular sulfur showed much smaller fractionations. The complexity of the isotopic patterns suggests that a dissimilatory sulfite reductase system may be induced by high concentrations of sulfite.


Occurence of lactose-negative mutants in chemostat cultures of lactic streptococci. Batch and chemostat cultures of Streptococcus cremoris HP and Streptococcus lactis 829 were examined for lactose-hegative (lac-)mutants on indicator agar. In batch cultures, S. cremoris HP gave less than 1% of the total count as lac- colonies while S. lactis 829 consistently contained about 15% of the total as lac- colonies. In chemostat cultures of S. cremoris HP in 2% skim milk containing casamino acids and yeast extract (0.1% each), the percentage of lac- colonies increased markedly when the temperature of growth was 18 degrees C but not when the temperature of growth was 25 degrees C. The percentage of lac- colonies in chemostat cultures in the skim milk medium at 25 degrees C was about the same as that in batch cultures. On the other hand, when chemostat cultures of S. lactis 829 in the skim milk medium were grown at several temperatures between 18 and 33 degrees C, the percentage of lac- colonies was markedly lower than that found in batch cultures of this organism. Cultivation of S. cremoris HP in chemostats with yeast extract-glucose broth at low temperature (18 degrees C) resulted in a selection of cells giving lac- colonies and atypical (small) lac+ colonies. The results show that cultivation of S. cremoris HP and S. lactis 829 in chemostats sometimes gave rise to altered populations. Conditions causing a change in one organism did not necessarily cause a similar change in the other. The results indicate that the successful propagation of lactic streptococci in chemostats for use as starter cultures in the dairy industry will require the careful establishment of optimum conditions for every strain so as to minimize the possible selection of undesirable populations.


Membrane mutations and production of enterotoxin B and alpha hemolysin in Staphylococcus aureus. Staphylococcus aureus strain S-6, which produces enterotoxin type B (SEB), and strain 10-275, a high toxin-producing mutant derived from S-6, display pronounced differences in dye sensitivity, osmotic stability, and bacitracin sensitivity. Such characteristics are consistent with the concept that strain 10-275 is a membrane mutant of strain S-6. Some membrane mutants of S. aureus strain 14458 exhibit about two- to three-fold increases in SEB production whereas other membrane mutants show about twofold increases in alpha-hemolysin production. It is suggested that specific and independent membrane mutations control the secretory processes resulting in the extracellular elaboration of these exoproteins.


Thiobacillus acidophilus sp. nov.; isolation and some physiological characteristics. After a brief exposition to glucose, Thiobacillus acidophilus was isolated from a culture of iron-grown T. ferrooxidans. Physicochemical analysis of its DNA showed a G+C content of 62.9-63.2%. The new isolate grows best at 25-30 degrees C and at pH 3.0. Growth is possible between pH 1.5 and 6.0. Thiobacillus acidophilus is apparently strictly aerobic. Ammonium salts are the only suitable source of nitrogen. The bacterium is a facultative autotroph. In addition to elemental sulfur, it obtains energy from organic compounds such as D-glucose, D-galactose, D-fructose, D-mannitol, D-xylose, D-ribose, D-arabinose, L-arabinose, sucrose, sodium citrate, malic acid,dl-aspartic acid, and dl-glutamic acid. Thiobacillus acidophilus possesses the key enzymes of the tricarboxylic acid (TCA) cycle including NAD-and NADP-linked isocitric dehydrogenase and alpha-ketoglutarate dehydrogenase, and the key enzymes of the hexose monophosphate pathway (glucose-6-phosphate and 6-phosphogluconate dehydrogenase, and fructose 1,6-diphosphate aldolase). NADH oxidase has been found in particulate fraction of extracts. Rhodanese and thiosulfate oxidase have also been detected.


Optimum conditions for initiation of filamentation in Candida albicans. A medium that initially produced filaments from almost all of its Candida albicans blastospore inoculum contained 1% mycological peptone and 0.2% glucose, final pH 7.4-7.5. The medium was inoculated to 10-6 cells/ml and incubated at 40 degrees C. Reversion to secondary blastospores began at a mean of 2.4 h after inoculation. The patterns of utilization of growth nutrients during optimal mycelial growth showed no correlation with the events of filamentation.


Aerosol BCG treatment of carcinoma metastatic to the lung: a phase I study. BCG (TICE) was safely administered to 15 patients with metastatic cancer to the lungs in weekly doses of up to 3 X 107 organisms by the aerosol route. The aerosol route of administration is associated in approximately 33% of the doses with a toxicity syndrome of malaise, fever, and chills beginning 4 to 8 hours after treatment and ending within 24-36 hours. This syndrome is experienced by all patients and symptoms gradually subside with continuation of therapy. No hepatic or pulmonary toxicity was documented during the 221 treatment doses.


Treatment of an established graft-versus-host reaction in AKR mice by adoptive immunotherapy. A model system in AKR mice for the induction and cure of a clinically evident graft-versus-host disease is reported. Graft-versus-host disease is inititated by i.p. injections of ecyclophosphamide (250 mg/kg body weitht ) into female AKR mice, on Day 0. This is followed by i.v. injections of 45 x 10-6 normal spleen cells (NSC) from male C57BL/6J mice. Median survival time for these mice is 33.4 plus or minus 4.5 days. Following the administration of C57BL/6J NSC, AKR mice were rescued from graft-versus-host disease by the following treatment protocol: (a) Day 6, 35 x 10-6 DBA/2 NSC given i.v.; (b) Day 10, cyclophosphamide i.p. (150 mg/kg body weight); (c) Days 11 and 26, 35 x 10-6 AKR NSC given i.v. These experiments demonstrate that graft-versus-host reaction can be elminiated by coupling a graft-versus-host reaction with a graft-versus-graft reaction and restoring the host by immunocompetent syngeneic cells.


Graft versus leukemia without fatal graft-versus-host disease in AKR mice. BW 5147 leukemia in AKR mice has been successfully treated by adoptive immunotherapy using allogeneic spleen cells from C57BL/6J mice. Graft-versus-host reaction was prevented by treatment with spleen cells from a second allogeneic strain (CBA, H-2 identical with AKR), followed by cycloposphamide and syngeneic spleen cells. Successful treatment of leukemia without graft-versus-host reaction is dependent upon a close relationship at the H-2 locus between the second allogeneic donor and the host AKR mice, since cells from a non-H-2 identical donor (DBA/2) do not increase survival. The doses of cyclophosphamide and of C57BL/6J spleen cells are also parameters of critical importance in successful treatment.


Therapy in an intracerebral murine glioma model, using Bacillus Calmette-Guérin, neuraminidase-treated tumor cells, and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. The s.c.-propagated murine glioma, GL-26, was established in tissue culture. The tissue culture line, with a doubling time of 36 hr, was used as the common source for all tumor cells. Suspensions of the tumor cells were transplanted intracerebrally in mice to produce an anaplastic ependymoblastoma. In vitro 51-Cr cytotoxicity assays did not detect any cellular immunity against GL-26 tumor cells in animals bearing either s.c. or i.c. tumors, indicating that the tumor itself is not highly immunogenic. Howeveer,significant cellular cytotoxicity was elicited in non-tumor-bearing animals by immunization with Vibrio cholerae neuramini-animals by immunization with Vibrio cholerae neuraminidase and mitomycin C-treated tumor cells plus complete Freund's adjuvant. In vivo therapy studies revealed significant increases in survival of animals preimmunized with V. cholerae neuraminidase- and mitomycin C-treated cells plus complete Freund's adjuvant. 1(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea, when given i.p. on Day 3 or 12 after tumor challenge, also resulted in significant increases in survival. Furthermore, the effects of 1-(2-chloroethyl)- 3-cyclohexyl-1-nitrosourea and preimmunization were additive, with significanchloroethyl)-3-cyclohexyl-1-nitrosourea. In contrast to results reported for several extracranial tumor systems, immunotherapy, using either V.cholerae neuraminidase- and mitomycin-treated tumor cells, Bacillus Calmette-Guerin, or both, beginning 3 0r 4 days after tumor challenge, did not produce any significant increases in survival.


The effect of B-cell immunosuppression on age-related resistance of chickens to Marek's disease. Chickens were bursectomized by cyclophosphamide treatment at hatching. At 8 or 9 weeks of age, bursectomized and unbursectomized hatchmates, free from prior infection, were challenged with pathogenic Marek's disease virus. Oncogenicity of the virus inoculum was confirmed by inoculating 1-day-old susceptible chickens. At the time of virus challenge, blood cells from the cyclophosphamide-treated chickens were able to mount a vigorous graft-versus-host reaction in allogeneic embryos. This ability indicated that the thymus function was intact. There were no significant differences in Marek's disease response of bursectomized and unbursectomized chickens, in spite of a severe defect in the bursa-dependent functions in the bursectomized chickens. Some bursa-deficient chickens had non-proliferating, presumably regressing lesions in peripheral nerves. Because these lesions lacked plasma cells, it was concluded that the plasma cell may not play a functional  role in recovery from Marek's disease.


Induction of enzymes by glucagon, glucose repression, adenosine 3',5'-monophosphate concentration during carcinogenesis and in Morris 6918A hepatoma. We have studied glucagon induction of enzymes, adenosine 3', 5'-monophosphate concentrations, and glucose repression in Morris 9618A hepatoma and in the liver of rats fed, for periods of up to 5 weeks, a solid diet containing 2-acetylaminofluorene or 3'-methyl-4-dimethylaminoazobenzene. While the basal levels of the enzymes serine dehydratase and tyrosine aminotransferase were the same as those found in control rats, their response to glucagon was reduced in experimental animals with or without tumors. However, the basal or glucagon-stimulated levels of adenosine 3', 5'-monophosphate in the liver of rats given the carcinogens were not changed. In Morris 9618A hepatoma, these parameters were, likewise, comparable to those in control animals. When glucose was administered to carcinogen-treated or tumor-bearing rats that had received a single dose of glucagon, there was no suppression of the increase in activity of serine dehydratase and tyrosine aminotransferase observed after glucagon treatment alone. The loss of glucose repression was seen already at 2 to 3 weeks following initiation of the carcinogenic diets. As previous studies had established for normal liver, the hormone-induced high levels of adenosine 3',5'-monophosphate remained unchanged also in Morris 9618A hepatoma and in rats given carcinogen. These results indicate that alterations in enzyme induction during chemical carcinogenesis are not the consequence of changes in adenosine 3',5'-monophosphate levels caused by carcinogens. The early disappearance of the glucose effect, which persists in slow-growing hepatomas, may be an expression of interference by carcinogens with the translation apparatus of the hepatic cell.


Renaturation effects of cis and trans platinum II and IV compounds on calf thymus deoxyribonucleic acid. The effects of cis dichlorodiammine platinum [cis Pt(II)], trans dichlorodiammine platinum (trans Pt(II)], cis tetrachlorodiammine platinum [cis Pt(IV)], trans tetrachlorodiammine platinum [trans Pt(IV)], and ethylenediaminedichloride platinum [Pt(II)en] on the absorption spectra, and thermal hyper- and hypochromicity of calf thymus DNA were investigated. Platinum-induced renaturation was studied as one parameter of interstrand cross-linking. Based on a DNA cross-linking hypothesis, the tumor-inhibitory platinum compounds cis Pt(II), cis Pt(IV) and Pt(II)en would be expected to induce renaturation following thermal denaturation, whereas the ineffective drugs, trans Pt(II) and trans Pt(IV) would not. All five bind to DNA in such a way as to induce renaturation. However, cis Pt(IV) requires at least a 3- to 4-fold longer incubation time than is required by the other compounds to form the coordination bonds necessary for renaturation. Maximum renaturation with all compounds was observed at a molar Pt/base ratio of 0.05 except cis Pt(IV), with which it was 0.25. The rate of the formation of the platinum-coordinated cross-links by fresh cis Pt(II) suggests two reactions or types of reactions occur. The first is rapid and destabilizes the DNA helix, whereas the second is slow and responsible for renaturation following thermal denaturation. These results suggest that cis Pt(IV) may be activated cellularly and that cross-linking is not the primary mechanism of action of the tumor-inhibitory platinum compounds.


Interactions of heme with hepatic microsomal mono-oxygenase. Effect on benzpyrene hydroxylation. The addition of heme (1-10 muM) to liver microsomes from phenobarbital (PB)-treated and 3-methylcholanthrene (MC)-treated male rats increased the rate of benzpyrene (BP) hydroxylation by about 20-40%. On the other hand, protoporphyrin IX caused only inhibition of BP hydroxylation. There was no increase of enzymatic activity by heme when solubilized preparations of liver microsomes were used. This suggested the possibility that an apo-cytochrome P-450 was present in intact microsomes. Higher concentrations of heme inhibited BP hydroxylation by either intact or solubilized microsomes. The inhibition by heme with solubilized microsomal preparations was noncompetitive, "mixed-type". However, with intact microsomes, the lack of linearity, precluded the determination of the type of inhibition. To examine possible effects of heme on the binding of BP to microsomal cytochrome P-450, the spectrum elicited by the addition of BP to microsomes was obtained in the presence or absence of added heme. The addition of heme to liver microsomes produced a marked increase in the trough (419-420 nm) of the difference spectrum formed by the subsequent addition of BP. These findings would suggest that heme increased the binding of BP to microsomes. However, the possibility that BP merely displaces the bound heme of the microsomes to yield, as expected, a trough at 413-416 nm (the addition of heme to microsomes yields a peak of 413-416 nm, unpublished) cannot be ruled out. Nevertheless, independent of our understanding of the mechanism involved in the spectral interactions between heme and BP with liver microsomes it is clear that an effect at their binding site(s) must have been elicited by the presence of both compounds.


Prior receptor occupancy as a determinant of the pressor activity of infused angiotensin II in the rat. The pressor responsiveness to angiotensin II and norepinephrine was examined in rats before and during blockade of coverting enzyme activity with the nonapeptide SQ 20881. Responses to angiotensin II were impaired by sodium deprivation but enhanced by sodium loading or bilateral nephrectomy. During the period of converting enzyme blockade, a twofold increase in the angiotensin II pressor response was observed in the salt-restricted rats, whereas only a small change occurred in the salt-loaded rats. Infusion of the inhibitor produced a profound fall in the blood pressure of the salt-depleted rats with a relatively minor fall in the sodium-loaded rats. Norepinephrine pressor responses were slightly potentiated in the salt-restricted rats after administration of SQ 20881, but no change occurred in the salt-loaded or the nephrectomized rats. These observations support the view that the decreased angiotensin II pressor activity during salt deprivation is the result of a prior occupancy of receptor sites by endogenous hormone. Therefore, a change in the number or the affinity of receptors consequent to changes in sodium balance need not be postulated to explain the phenomenon.


Increased plasma catecholamine concentrations and vas deferens norepinephrine biosynthesis in men with elevated blood pressure. Systemic biochemical indexes of sympathetic nerve functionplasma catecholamine concentrations and serum dopamine-beta-hydroxylase activitywere correlated with regional indexesnorepinephrine concentration activities of norepinephrine biosynthetic enzymes in the vas deferensānd with blood pressures and pulse rates of 57 men at the time of vasectomy. Mean plasma catecholamine content was increased 66% and 60% in men with elevated systolic and elevated systolic and diastolic blood pressures, respectively. The norepinephrine concentration, tyrosine hydroxylase activity, and dopa decarboxylase activity in the vas deferens were increased in 12%, 26%, and 17% of the men with elevated blood pressure, respectively. Tyrosine hydroxylase and dopa decarboxylase in the men with elevated systolic blood pressure were increased 41% and 68%, respectively, compared with values in the men with normal blood pressure (P less than 0.05 and P less than 0.02). Individual plasma catecholamine content was related directly to both systolic and mean blood pressure (P less than 0.01) and to activities of vas deferens tyrosine hydroxylases (P less than 0.05) and dopa decarboxylase (P less than 0.01). There was a weak, but not significant, correlation of plasma catecholamine concentration with serum dopamine-beta-hydroxylase activity. Raised plasma catecholamine concentration in men with elevated blood pressure prior to vasectomy was associated with increased neurotransmitter biosynthesis in their vas deferens, which may indicate increased sympathetic nerve tonicity and perhaps be a factor contributing to the blood pressure elevation.


Dobutamine: development of a new catecholamine to selectively increase cardiac contractility. We systematically modified isoproterenol's chemical structure to reduce chronotropic, arrhythmogenic, and vascular side effects. Experiments on dogs showed that the resulting drug, dobutamine, had an inotropic efficacy as great as that of epinephrine due to a direct action on beta1 cardiac receptors. However, unlike epinephrine, dobutamine's effect on alpha and beta2 vascular receptors was slight. At equivalent inotropic doses, dobutamine had less than a fourth of the chronotropic effect of isoproterenol. Desmethylimipramine (DMI), which blocks the sympathetic nerve fiber uptake mechanism, had no effect on dobutamine's actions. In contrast, DMI antagonized dopamine's inotropic effect, and marked chronotropic and pressor responses occurred when we used doses of dopamine large enough to elicit a direct inotropic effect. Dobutamine increased the contractility of isolated cat papillary muscles more but the automaticity less than did isoproterenol. In ischemic dog hearts, dobutamine lacked significant arrhythmic activity, whereas dopamine, norepinephrine, and isoproterenol caused severe ectopic activity. In dogs with experimentally induced low cardiac contractility, low cardiac output, and hypotension, dobutamine produced dose-related increases in cardiac contractility and output, restored arterial blood pressure, and reduced total peripheral resistance slightly. In contrast, isoproterenol failed to restore blood pressure, had only a meager effect on cardiac contractility and output, cuased extreme tachycardia, and lowered peripheral resistance more than did dobutamine. Norepinephrine, which did not increase cardiac contractility or output as much as dobutamine, excessively elevated peripheral resistance and arterial blood pressure.


Malignant hypertension resulting from deoxycorticosterone acetate and salt excess: role of renin and sodium in vascular changes. The evolution of malignant hypertension was studied under metabolic balance conditions in 11 uninephrectomized rats given deoxycorticosterone acetate and 1% NaCl as drinking water. Changes in sodium and potassium balance were related to changes in blood pressure, plasma renin activity, hematocrit, and kidney histology. After 3-4 weeks of steadily positive sodium balance accompanied by continuously increasing blood pressure up to 185 plus or minus 19 (SE) mm Hg, periods of sodium loss accompanied by evidence of hemoconcentration were observed marking the onset of the malignant phase as defined by the development of fibrinoid necrosis in the kidney. Plasma renin activity remained markedly suppressed both at the fourth week (0.33 plus or minus 0.02 ng/ml hour-1) when the sodium balance was positive and the kidney biopsy negative and at the end of the experiment (0.35 plus or minus 0.36 ng/ml hour-1) when the sodium balance was negative and the kidney histology revealed malignant vasculitis. Infusion of the angiotensin II inhibitor 1-Sar-8-Ala-angiotensin II consistently failed to affect blood pressure, and the kidney tissue norepinephrine level was reduced (0.054 plus or minus 0.01 mug/g) compared with the control level (0.132 plus or minus 0.02 mug/g). We conclude that malignant vasculitis in this model is preceded by hypertension associated with sodium and water retention and is accompanied by negative sodium balance, decreases in body weight, falling blood pressure, and hemoconcentration without demonstrable participation of the renin-angiotensin system or the renal catecholamines.


A renin inhibitor from rabbit kidney: conversion of a large inactive renin to a smaller active enzyme. Renin in extracts of frozen rabbit kidney exists in two forms: active (molecular weight about 37,000) and inactive (molecular weight about 55,000) renin. The inactive form becomes active after exposure to pH 2.5 at 4 degrees C. If extracts are chromatographed on DEAE cellulose, the inactive renin dissociates into active renin plus a renin inhibitor (molecular weight about 13,000). The inhibitor recombines with active renin if the two are incubated together at 37 degrees C. The inhibitor is destroyed by acid treatment at pH 2.5 at 4 degrees C. We conclude that the activation of inactive renin is due to destruction of the inhibitor by acid. The inactive material may be a renin proenzyme or a storage form of active renin combined with inhibitor.


Augmentation of myocardial digoxin concentration in hemorrhagic shock. The effect of the shock state on myocardial digoxin uptake and plasma digoxin levels was examined in unanesthetized dogs following hemorrhage. Five minutes after intravenous administration of tritiated digoxin the myocardial digoxin content in animals with shock was greater than in normal animals in both left ventricle (LV) (165 plus or minus 15 (SD) ng/g vs 130 plus or minus 26 ng/g, P smaller than 0.02 and right ventricle (RV) (142 plus or minus 13 ng/g vs 111 plus or minus 22 ng/g. P smaller than 0.02) as was the plasma digoxin concentration (61.6 plus or minus 11.8 ng/ml vs 44.3 plus or minus 4.6 ng/ml, P smaller than 0.02). After one hour, in another group of dogs, the difference in myocardial concentration of digoxin between test and normal groups was even greater (LV: 213 plus or minus 26 ng/g vs 133 plus or minus 13 ng/g, P smaller than 0.001; RV: 171 plus or minus 9 ng/g vs 111 plus or minus 8 ng/g. P smaller than 0.001) despite lower plasma digoxin concentration in the test group (12.9 plus or minus 2.9 ng/ml vs 17.3 plus or minus 2.5 ng/ml, P smaller than 0.05). Diminished peripheral blood flow, peripheral digoxin delivery and uptake were probably responsible for the early difference in plasma digoxin levels. Resultant greater plasma concentrations of digoxin presented to the myocardium in the early phase, coupled with relative preservation of myocardial blood flow, may explain the greater myocardial uptake in animals with shock although myocardial mechanical factors may also be implicated. Augmented uptake of digoxin by the myocardium in canine hemorrhagic shock may be relevant to the altered susceptibility to glycoside action in clinical shock syndromes.


Ultramicro analysis for copper, cadmium, and zinc in human liver tissue by use of atomic absorption spectrophotometry and the heated graphite tube atomizer. We describe a method of analysis for copper, cadmium, and zinc in a 15-mg (wet weight) sample of human liver by atomic absorption spectrophotometry.  The sample is digested with nitric acid (1.0 mol/liter), evaporated, and dilute HNO3 (10 mmol/liter) added.  The reconstituted acid mixture is injected into the graphite tube atomizer for analysis of Cu and Cd and aspirated into the air--acetylene flame for measurement of Zn.  The absorbance for each metal is suppressed with increasing pH.  NaNO3, KNO3, KCl, and NaCl (e.g.) quench the Cd absorbance in acid solutions that contain no protein, but not in the presence of protein.  Metal ions added to the predigestion human liver sample at 10 percent and 100 percent of the intrinsic metal concentrations were, respectively, 93 percent and 90 percent accounted for analytically in the case of Cu, 98 percent and 102 percent for Zn, and 101 percent and 93 percent for Cd.  Analysis of a National Bureau of Standards' Bovine Liver Standard Reference Material yielded results corresponding to 99 percent (Cu), 112 percent (Zn), and 91 percent (Cd) of the mean expected concentrations of these metals.  The between-run coefficient of variation for the bovine liver material was 6 percent for Cu, 9 percent for Zn, and 10 percent for Cd.  For 16 histologically normal samples of human liver, the mean values were: Cu, 26; Zn, 293; and Cd, 6.0 nanograms of metal per milligram dry weight, in agreement with values published previously.  The method can be easily and reliably applied to small samples of liver obtained by closed-needle biopsy.


Determination of serum copper by atomic absorption, with use of the graphite cuvette. We have established and evaluated a flameless graphite cuvette method for copper in serum.  This atomic absorption method provides substantial improvement in sensitivity, adequate accuracy, and acceptable precision, and little sample preparation is required before the analysis.  Standard addition studies and measurements of National Bureau of Standards materials indicated that the proposed method is accurate, but that sample pH must be kept between 2 and 3 for high accuracy.  Cations and anions that frequently are present in protein-containing samples do not interfere significantly.  Sample cross contamination in the syringe must be carefully avoided.  Finally, when results for more than 100 patients' sera by this method were compared to those obtained by flame atomic absorption for the same samples, no substantial bias or inaccuracies could be attributed to this new micro-scale method for serum copper.  Hence, this method is ideally suited for use on pediatric patients.


The contribution of the kidney to the removal of a lactic acid load under normal and acidotic conditions in the conscious rat. 1. The rate of removal from the circulation of an intravenous lactic acid load has been studied in conscious rats, previously subjected either to bilateral nephrectomy or to a sham operation. 2. In rats with normal blood pH, the apparent contribution of the kidneys to removal of the lactic acid load is 30%; less than 12% of the renal contribution is attributable to urinary excretion. 3. In bilaterally nephrectomized rats made acidotic by administration of ammonium chloride, the rate of removal of a half-neutralized lacrtic acid load is progressively decreased with increasing severity of acidosis. No such effect is seen in sham-operated animals. 4. An increase in the ability of the kidney to remove lactate during acidosis compensates for approximately half of the simultaneous fall in the capacity of the remainder of the body for lactate assimilation. 5. Basal blood lactate concentrations fall in the presence of metabolic acidosis.


The assessment of acid-base disturbance in man by the use of carbon dioxide titration curves. 1. Carbon dioxide titration curves were determined in vivo in dog and man at various degrees of acute non-respiratory acidaemia and alkalaemia. 2. The slope of the CO2 titration curve (delta log Pco2/delta pH) was found to increase with the severity of the acute non-respiratory alkalaemia the slope (delta log Pco2/delta pH) tended towards unity. 3. A simple scheme based on the CO2 titration curves determined in vivo has been proposed for the assessment of acute acid-base disturbances in man. 4. Carbon dioxide titration curves were also determined in vivo in patients with chronic respiratory and non-respiratory acidaemia and it was found that these curves were not significantly different from those obtained in states of acute acid-base disturbances. It is therefore suggested that the scheme described in this paper is applicable to all acid-base disturbances.