Showing papers in "Physiological chemistry and physics in 1975"

A potent new mesodermal growth factor from mouse submaxillary gland. A quantitative, comparative study with previously described submaxillary gland growth factors.

Abstract: A new growth factor with potent growth stimulating effects on connective tissue cells of the cornea has been isolated and partially purified from mouse submaxillary glands. A computerized image analysis system has been used to quantitate the growth responses of connective tissue fibroblasts to this new growth factor and to compare its activity with that of four previously described growth factors [nerve growth factor (NGF), epidermal growth factor (EGF), mesodermal growth factor (MGF), and thymocyte-transforming factor (TTF)] also obtained from mouse submaxillary glands. The new factor is as potent a growth stimulator as the previously described MGF. All five of the growth factors possessed esteropeptidase activity (trypsin-like) and general proteolytic activity. There was no correlation between the degree of growth stimulation of the connective tissue cells and either the esteropeptidase or general proteolytic activities of any of the growth factors.

Effects of several unusual sulfur-containing amino acids on rat liver cystathionine-gamma-lyase

Abstract: 1. The mode of inhibition of rat liver cystathionine-gamma-lyase [L-cystathionine cysteine-lyase (deaminating), EC 4.4.1.1] was studied by using several unusual sulfur-containing amino acids newly found in this laboratory. Some cysteine conjugates (CMC, Beta-CEC, HCETC and HCPC) inhibited noncompetitively both homoserine dehydratase and diaminopropionate ammonia-lyase activities, and competitively gamma-cystathionase activity. CMTC exhibited a mixed type inhibition on both homoserine dehydratase and gamma-cystathionase activities, and a noncompetitive inhibition on the diaminopropionate ammonia-lyase activity. Some homocysteine conjugates (CMHC, beta-CEHC and HCEHC) inhibited competitively both the activity of homoserine dehydratase and of gamma-cystathionase, and exhibited a mixed type inhibition on the diaminopropionate ammonia-lyase activity. beta-CEC, CMHC and beta-CEHC were also effective inhibitors to cysteine desulfhydrase activity. 2. Among the other amino acids tested, DL-homocysteine and D-cysteine, irrespective of their concentration, exhibited a mixed type inhibition on the homoserine dehydratase activity. However, they promoted gamma-cystathionase activity at their lower concentrations and inhibited at their higher concentrations, more so than cystathionine. DL-alpha-Aminobutyric acid was a weak competitive inhibitor of the homoserine dehydratase, gamma-cystathionase and diaminopropionate ammonia-lyase activities. DL-alpha-Aminopimeric acid has the same chain length as beta-CEC, CMHC and CMTC, but it showed a very weak inhibitory effect compared with the latter sulfur-containing compounds. L-Methionine, DL-methionine sulfoxide, L-ethionine, L-cysteic acid, L-aspartic acid, L-asparagine, L-glutamic acid, L-glutamine, D-alanine, beta-alanine, L-ornithine and L-lysine had little or no effect on any activities of the enzyme preparation. These results were discussed in relation to the catalytic center of cystathionine-gamma-lyase.

The effect of chlorpromazine on some properties DNA in solution.

Abstract: The effect of chlorpromazine [2-chloro-10-(3-dimethylaminopropyl)-phenothiazine] on calf thymus DNA has been investigated by spectrophotometric, equilibrium dialysis, thermal denaturation, sedimentation and viscosity methods. The absorption spectra of DNA undergo two important changes upon binding to chlorpromazine, namely, the displacement of peaks to longer wavelength (ranging from 5-8 nm) and a decrease in the optical density. The extent of binding of chlorpromazine to native calf thymus DNA, AS MEASURED BY SPECTROPHOTOMETRIC METHOD, IS DECREASED WITH INCREASING SODIUM CHLORIDE Concentration. A curvature in the Scatchard plot suggests two types of binding processes. Chlorpromazine decreases the optical density at higher temperatures without affecting the Tm of DNA. In its presence, the absorption spectra of purine deoxynucleosides (dA, dG) and of deoxynucleotides (dAMP, dGMP) are modified, i.e., the maxima are displaced to longer wavelength (ranging from 5-17 nm) and there is a general decrease in the optical density. No such effect is observed with pyrimidine deoxynucleosides (dC, dT) and deoxy-nucleotides (dCMP, dTMP). A combination of electrostatic binding of the amino group of chlorpromazine sidechain with the negative phosphate groups of the DNA and a partial insertion of either of its two phenyl rings between the nucleotide base pairs of the DNA plus the binding caused by mutual interaction between different chlorpromazine molecules at higher concentration is proposed as a probable mode of binding of chlorpromazine to DNA.

Trehalose and glycogen levels during the initial stages of growth of Candida albicans.

Abstract: Yeast phase cells of Candida albicans were extracted with cold 0.5 M trichloroacetic acid. The only quantitatively significant anthrone-positive material in these extracts was trehalose which was identified chromatographically and enzymatically. Glycogen in the residue was digested with amyloglucosidase to free glucose which was assayed by a glucose oxidase method. Washed cells from a stationary phase culture of C. albicans rapidly decreased their trehalose content during the early phases of growth on a fresh glucose-containing medium. Concurrent increases in total glycogen concentration were of a greater magnitude than could be accounted for by trehalose mobilization. Thus a specific role for trehalose during initial growth is indicated and is compared with other fungal systems. Trehalase activity was measured in situ and showed only minor changes during the time intervals of these experiments in contrast to other reports on S. cerevisiae. A large fraction of the enzyme in C. albicans behaved as external to the protoplasmic membrane. On one type of growth medium C. albicans exhibited (in addition to trehalose) variable amounts of a sugar alcohol in trichloroacetic acid extracts. This has tentatively been identified as arabitol.

NMR in cancer. VII. Sodium-23 magnetic resonance of normal and cancerous tissues.

Abstract: Sodium-23 magnetic resonance was performed on four types of cancers and six types of normal tissues of rats and mice. The spin-lattice relaxation time of the tumors was generally longer than that of the normal tissues, with the most marked difference occurring between rat liver (T1 = 6.5 msec) and Novikoff hepatoma (T1 =23.7 msec). Estimation of tissue sodium from the signal intensity of the resonance indicated that all four types of tumors contained more sodium than any of the normal tissues.

Isolation of two new sulfur-containing amino acids from the urine of a cystathioninuric patient.

Abstract: Two new sulfur-containing amino acids are isolated from the urine of a cystathioninuric patient. They are presumed to be cystathionine sulfoxide and perhydro-1,4-thiazepine-3,5-dicarboxylic acid.

Length-dependent optical diffraction pattern changes in frog sartorius muscle.

Abstract: The length dependence of the laser light diffraction pattern produced by R. pipiens whole sartorius muscle has been examined at rest and during tetanic contraction. The muscle diffraction pattern was scanned by a vidicon camera; camera output was digitized and processed by an on-line digital computer to allow a real line display of a section through the diffraction pattern. Analysis of the first order diffraction line profiles yielded values for line amplitude, intensity, center of gravity, line width and percent dispersion. The calculated dispersion provided a measure of the muscle's sarcomere length distribution. First order line amplitudes and intensities were observed to increase, then decrease, with progressive stretch from 1.0 to 1.3 reference length. The percent dispersion among sarcomeres in resting muscle averaged 11% and was approximately proportional (coefficient = .0341) to length. The amplitude and intensity of zero and first order diffraction lines decreased during tetanus, while the line widths and dispersions increased. First order line intesity during tetanus was maximum at about the same length as during rest, i.e., at approximately 2.5 mu sarcomere length. Sarcomere dispersion increased by about 6% during tetanus.

Simultaneous efflux of K+ and Na+ from frog sartorius muscle freed of extracellular fluids: evidence for rapidly exchanging Na+ from the cells.

Abstract: SUMMARY After removal of radioactivity trapped in the extracellular space and correcting for the contribution of connective tissue elements, the Kt -efflux curve of frog sartorius mus cles becomes a perfect straight line in a semilogarithmic plot. The simultaneously recorded Na+-efflux curve from the same muscles remains strongly curved, and can be resolved into a slow fraction (which conventionally has been regarded as representing the entire cell Na t ) and at least one fast fraction. The fast fraction of Naf could not have originated from a sarcoplasmic reticulum or any other extracellular space extensions; otherwise a similar fast fraction should exist for K'. The data agree with the interpretation that it is the fast fraction that is rate limiting by cell permeability and the slow fraction by desorption from intracellular adsorption sites. INTRODUCTIOh When Levi and Ussing' first studied labeled Na f efflux from frog sartorius muscles, they recognized two fractions of labeled ~a+ from the tissue, one fast and one slow. They considered the slow fraction with a half time exchange (t%) of about 30 minutes as representing ~a+ from the cell and the faster fraction as representing ~a+ from the extracellular space. Levi and Ussing's original assignment has become widely accepted. Indeed, reading recent literature on this and related subjects, one cannot escape the impression that its validity must have been long proven. The truth is different. Evidence exists (ref. 2, p. 293; ref. 3, p. 838; ref. 4; ref. 5, p. 19) which hes pointed to the need for further study on the subject. The results of such an undertaking are reported in this communication.

The mechanism for the exclusion of sugars from the water in a model of the liveing cell: the ion-exchange resin: pore size or water structure?

Abstract: SUMMARY The equilibrium distribution coefficients (p-value) of D-arabinose between the water in sulfonate ion-exchange resin and the external aqueous solution vary with the nature of the five alkali metal counterions studied. The strongest exclusion (lowest p -value) is found in the Li+ resin and the least exclusion (highest p -value) in the Cs+ resin. The p-value decreases with the increasing atomic weights for the alkali-metal ions: pCs+> p~b+>p~+>~~a+>~~ i+. The water contents of these resins, on the other hand, vary in the opposite direction, being highest for the LiC resin and lowest for the Cs+ resin. These data disprove the pore size theory but fully substantiate the predictions of the associationinduction hypothesis.

Inhibition of adenosine 3',5'-monophosphate phosphodiesterase by nicotinamide and its homologues in vitro.

Abstract: Inhibition of cAMP phosphodiesterase from rat liver by nicotinamide and its homologues was studied. Among the several compounds tested, such agents as nicotinamide, N2-ethylnicotinamide, N2-methylnicotinamide, N,N-diethylnicotinamide, 3-acetylpyridine, methylnicotinate, and ethylnicotinate showed potent inhibition of cAMP phosphodiesterase, with an over 90% inhibition by 5 mM ethylnicotinate when cAMP was used as substrate at a 0.48 x 10(-7) M concentration. A comparison of the inhibitory curves of theophylline, papaverine, and ethylnicotinate on enzyme activity showed them to be approximately coincident. Furthermore, the plots with abscissa of equal increments per concentration of ethylnicotinate in the presence of theophylline or papaverine coincided with that in the absence of these agents.

Change in electrophoretic mobility of human erythrocyte as the result of membrane shape change in vitro.

Abstract: Shape change of human erythrocyte due to the membrane externalization induced by incorporation of excess lysolecithin or by hypertonic treatment in vitro, was accompanied by a decrease in the electrophoretic mobility of the cell. No change in sialic acid content of the membrane, mostly responsible for the cell surface charge, was observed in any of these treatments. Therefore, the membrane externalization seems to cause a redistribution of the surface charge and the resulting alteration of the electrophoretic mobility of the cell. Heating at 48 degrees C for 1 hour induced both the shape change and change in the mobility, but at the same time it reduced the membrane sialic acid content.

A circular dichroism study of charged polypeptides interaction with salts.

Abstract: The effect of salts on the experimental circular dichroism spectra of polypeptides is presented using poly-L-lysine as the main model. Salt effects are analyzed into: (a) shielding at low (less than 0.5 M) concentrations of all salts; (b) binding to positively charged and some neutrally charged side-chains by certain anions (e.g., CCl3COO-, CF3C00-, ClO4-), with induction of helicity; (c) binding of these same anions, at high concentration, to the backbone leading toward random structure; (d) binding of high concentration of denaturing cations (La+3, Ca++, Li+) to the backbone, with La+3 and Ca++ leading to collapsed random structure (R) while Li+ tends to leave the polypeptide somewhat extended; (e) indirect interaction of salting-out salts (NaH2PO4, (NH4)2SO4, NH4F), at high concentration, leading toward complete alpha helicity, probably by competition with the polypeptide and the anion for available water. Effects of changing the temperature from 5 degrees to 50 degrees on the circular dishroism spectra of different polypeptide-salt solutions throughout the region from extended (LES) to alpha helical conformation are analyzed in terms of introduction of randomness (R) at high temperature. Applications to effects of salt on protein structures are considered.

Hydroxyamino compounds produced by the oxidation of diamines with diamine oxidase in the presence of alcohol dehydrogenase.

Abstract: When the diamines putrescine, cadaverine, cystamine and lanthionamine are oxidized by purified pig kidney diamine oxidase in the presence of NADH and either liver or yeast crystalline alcohol dehydrogenase, NADH is oxidized. Chromatographic evidence obtained in the case of putrescine and cystamine indicates the production of the respective hydroxy-amino compound. In the case of cystamine, the product of the reaction is mercapto-ethanol-cysteamine mixed disulfide which may represent a biological source for the production of mercaptoethanol used for other reactions.

Electrophoresis of acid soluble proteins from rat liver nuclei and polysomes on large two-dimensional polyacrylamide gels.

Abstract: Increased resolution of 0.4 N H(2)SO(4) soluble rat liver polysomal and nuclear proteins was achieved by initial electrophoresis on 15 percent polyacrylamide disc gels (25 times 0.5 cm) followed by electrophoresis on 16 percent polyacrylamide gel slabs (25 times 15 cm). With this method, it was found that the histones were resolved into 12 spots, the polysomal proteins A1 and A9 were each resolved into two components, and polysomal proteins A1, A1 and A1 with molecular weights of approximately 14,000 were separated from the other proteins by their rapid migration in the first dimension. Moreover, no histones were found in the acid extracts of the polysomes.

Biochemical effects of bleomycin A2 on Novikoff hepatoma ascites cells.

Abstract: Purified nucleolar DNA was markedly degraded at a concentration of 13 mug/ml by bleomycin A2; bleomycin concentrations 20-30 times greater were required to degrade nucleoplasmic DNA. Whole nuclear DNA was degraded to only a small extent at 13 mug/ml but was markedly degraded at higher bleomycin concentrations. Treatment of the various types of DNA with high concentrations of bleomycin A2 produced low molecular weight (approximately 6S) fragments that were no longer sensitive to degradation by bleomycin A2. Hybridization studies demonstrated a loss of ribosomal DNA sequences from nucleolar DNA treated with bleomycin A2 in vitro. Studies on RNA synthesis in Novikoff hepatoma ascites cells in vitro showed there was a decreased uptake of 32Pi into high molecular weight nuclear RNA in the presence of bleomycin A2. These results indicate that nucleolar function is inhibited by a direct effect of bleomycin A2 on nucleolar DNA.

The effects of cholera enterotoxin on intestinal tissue water as measured by nuclear magnetic resonance (NMR) spectroscopy.

Abstract: Cholera enterotoxin has been postulated to change the configuration of the intracellular protein-water system, altering the permeability of the cell to water. Using nuclear magnetic resonance (NMR) spectroscopy, this protein-water relationship can be examined. Small intestinal loops in the rat were injected with 0.5 ml of either Schwarz/Mann cholera enterotoxin (40 mug/cc saline solution) or normal saline. Full thickness segments of intestine from each loop were taken and percentage water (using a gravimetric procedure which includes a correction for fat) and NMR relaxation times were determined. The mean value +/- S.D. for tissue water was 79.49 +/- 2.65% in the controls and 84.52 +/- 2.01% in the cholera specimens (p less than .001). T1 (spin-lattice) relaxation times were 521.22 +/- 69.5 msec in the controls and 667.96 +/- 119.25 msec in cholera tissue (p less than .001). T2 (spin-spin) relaxation times were 62.34 +/- 9.59 msec in controls and 80.35 +/- 21.46 msec in cholera tissue (p less than .02). These findings are consistent with the theory that cholera enterotoxin acts to alter intracellular protein-water relationship.

Effect of alpha-tocopherol on the rate of photohemolysis of human erythrocytes.

Abstract: The results of this study demonstrate that alpha-tocopherol has a significant ability in vivo to prohibit the production of cholesterol hydroperoxide in the erythrocyte membrane, and also significantly reduce the degree of cell membrane deformation upon exposure of the blood to light and oxygen (photooxygenation conditions). This study suggests that alpha-tocopherol prohibits the production of cholesterol hydroperoxide in the membrane which if produced leads to a weakened membrane observed as a "budded" cell in the electron micrographs.

Characterization of nuclear and chromatin poly A-containing RNA.

Abstract: Poly A-containing RNA was isolated from Novikoff hepatoma cell chromatin on nitrocellulose membrane filters after the cells were labeled with 32P-orthophosphate for 6 hours. Under denaturing conditions, the poly A-containing chromatin RNA had a sedimentation coefficient of approximately 8-18S. The presence of poly A tracts in the chromatin RNA fractions indicate that poly A addition occurs almost immediately after transcription. Poly A-containing chromatin RNA and nuclear RNA were approximately 1/5 as active as cytoplasmic poly A-containing RNA in the wheat germ translational system. Since chromatin poly A-containing RNA is the precursor of cytoplasmic poly A-containing RNA, further processing of this RNA prior to its attachment to polysomes may account for its increase in translational capacity.

Intracellular resistance and conduction in bullfrog atrium.

Abstract: The present study was undertaken to study the mechanism of transmission across the junctions between cells of cardiac muscle. Two cases, one with high resistance junction and the second with low resistance junctions, were modeled mathematically and each model was analyzed to predict the ratio of the transverse to longitudinal threshold field strengths. This ratio was measured experimentally for atrial trabeculae from Rana catesbiana and agreed well with the value predicted by the model in which the resistance of the membranes at the junctions is low. It can be inferred, therefore, that the resistance at the junctions between cells of bullfrog atrial muscle is low enough to permit the flow of sufficient current from the pre- to postjunctional cell to cause excitation of the latter. One parameter required in the analysis was the space constant, lambda. This was measured with the trabecula in a volume conductor using a large planar external electrode to produce a change in the transmembrane potential and a microelectrode to measure the spatial decrement of the electrotonic potential. The measurements of electrotonic decrement in 16 trabeculae are pooled to provide an estimate of the value of the space constant in frog atrial trabeculae: lambda= 0.863 +/- 0.041 mm, s.d., n=96.

Correlation between in vivo and in vitro metabolic measurements. Maximum capacity for urea synthesis.

Abstract: Estimations of enzyme activity in vivo have been or can often only be done at unphysiological conditions. A main biochemical goal is to correlate in vivo and in vitro measurements. A possible approach to this problem is presented based on forcing metabolic activity in vivo to the maximum for a certain metabolic sequence. Since the urea synthesis system, including maximal rates of enzyme activities, is well known, we have compared in vitro maximum rates for the individual enzymes of urea synthesis with in vivo rates as judged by urea levels in blood of rats given large amounts of protein. The excellent agreement found between the calculated maximum activities from in vitro measurements to the time needed to metabolize a protein overload is presented and comments made on its significance and on the importance of maintaining protein intake at moderate levels, for the capacity of the urea system is limited. Since the intake of large quantities of protein increases the urea level in blood and in other tissues and since high urea levels are somewhat deleterious "per se" and particularly due to equilibrium with cyanate, ingestion of excessive amounts of protein is at best expensive and possibly hazardous.

Osmotic pressure variations used to elicit oscillations and changes in stiffness of muscle fibers.

Abstract: By observing diffraction spectra produced by passing a laser beam through stimulated muscle fibers, the magnitude of sarcomere length change and the relative rate of filament sliding were found to decrease in hypertonic solutions. The first order diffraction for both resting and stimulated muscle fibers faded and then returned to its original intensity as the bathing solution was changed from isotonic to hypertonic and then back to isotonic. This is explained as being due to a change in optical contrast between the A and I bands. 7 Hz oscillations were detected at 4 degrees C during stimulation as the fibers equilibrated in hypertonic solutions. The stiffness of resting muscle in the hypertonic solutions increased while the stiffness of stimulated muscle decreased until at a concentration of 3.10 R they were equal. All processes were reversible.

Urinary purine levels in suicide.

Abstract: A high-resolution liquid chromatographic system was utilized to analyze urine samples of twelve nonchemical suicide victims for the relative composition of ultraviolet-absorbing compounds. The analyses demonstrated a decrease in the output of uric acid, characteristic of severe depression, and an increase in the excretion of hypoxanthine. The excretion of 7-methylxanthine was found to be variable and xanthine was normal. Language: en

13C NMR or coenzyme A and its constituent moieties.

Abstract: Natural abundance 13C nuclear magnetic resonance spectra, in D2O, were obtained for the following: pantoyl lactone, beta-alanine, cysteamine hydrochloride, cystamine dihydrochloride, calcium pantothenate, beta-aletheine oxalate, pantetheine, pantethine, pantetheine 4'-phosphate, oxypantetheine 4'-phosphate, desulfopantetheine 4'-phosphate, N-acetyl-aminodesthiopantetheine 4'-phosphate, adenosine 2',5'-diphosphate, adenosine 3',5'-diphosphate, and coenzyme A. A complete assignment of the 13C chemical shifts in the NMR spectrum of CoA is reported. Comparison of spectra indicates that CoA most likely exists in an extended conformation.

Red blood cell ghosts: hollow membranes or solid bodies?

Abstract: Human red blood cell ghosts prepared by a widely used hypotonic lysis method are shown to be not hollow membranes but are solid bodies of distorted shapes.