Proton magnetic resonance study of conformational dynamics, coordianted internal motions, and chemical shifts of tocinamide.

All proton magnetic resonance lines of the NH-CHalpha-CH2beta fragments of five amino acid residues of the heterodetic peptide, tocinamide, have been analyzed, including reassigment of certain NH, CHalpha, and CH2beta resonances. The 1H NMR spectral parameters evaluated from this analysis include (a) all chemical shifts, (b) (3JNHCH), (3Jalpha beta), and (2Jbeta beta) values, and (c) temperature dependencies of these coupling constants and chemical shifts--the latter includes aliphatic and aromatic CH protons. The (3Jalpha beta) coupling constants yielded Calpha-Cbeta rotamer populations, Pi, for five residues and these were used to explore conformational dynamics and coordinated internal motions of tocinamide. The rotamer populations and their temperature dependencies established, (1) that all Calpha-Cbeta bonds exhibit extensive internal rotation; (2) that rotamer preferences exist for every residue; (3) that the 162 conformations possible for completely free rotation around every bond of the Calpha1-Cbeta1-S1-S6-Cbeta6-Calpha6 disulfide fragment can be reduced to two or three if the concept of coordinated internal motion is introduced. We reject the possibility of a frozen conformation for the Cys1-Cys6 fragment in the eclipsed, staggered, or nonclassical rotamer states and propose the following possibilities: (a) three classical rotamers populated in the ratio 60:25:15, (b) three nonclassical rotamers not differing by +/- 15 degrees from the classical values of 180 degrees, + 60 degrees, and -60 degrees, or (c) two nonclassical but noneclipsed rotamers with a third rotamer essentially zero, and (4) that the Calpha-Cbeta rotamer populations of the Tyr2-Cys1-S-S-Cys6 fragment are consistent with coordinated internal motions of the Calpha-Cbeta bonds of all three residues. The existence of temperature coefficients, deltadelta/deltaT, POF +/- 2 ppb/degrees C for aliphatic protons can be accounted for, in part, by postulating intrinsic rotamer chemical shifts which are temperature independent. Deltadelta/deltaT values should therefore be useful for investigating conformational dynamics. A further consequence of this interpretation is that greater care should be exercised in using deltadelta/deltaT values to assign hydrogen-bonded amide protons, especially when deltadelta/deltaT approximately -2 to -4 ppb/degrees C.


Polymerizability of rabbit skeletal tropomyosin: effects of enzymic and chemical modifications.

Polymerizability of tropomyosin was unaffected by the removal of the three terminal residues 282, 283, and 284 using carboxypeptidase A. However, when residue 281 was removed, polymerizability was abolished. These results are consistent with a 9-residue molecular head-to-tail overlap in polymerized tropomyosin, in which residue 281 plays a space-filling role at the center of the overlap core. In acetylation studies, loss of polymerizability closely paralleled the extent of acetylation of lysine-7, and this residue was more susceptible to acetylation than any other. The effect of acetylation on polymerizability was probably caused not only by cleavage of salt-bridge between lysine 7 epsilon-NH2 and residue 284 alpha-COOH but also by distortion of the overlap core by the N-acetyl group. Specific modification of methionine in tropomyosin indicated that, in addition to residue 281, methionine-8 is also involved in formation of the overlap core. Modified nonpolymerizable tropomyosins could still bind to F-actin, indicating that the head-to-tail polymerization of tropomyosin is not a prerequisite for actin binding, although the regularity of tropomyosin molecules along the actin helix is presumably disrupted.


Isolation and characterization of bovine factor XII (Hageman factor).

Factor XII was purified approximately 14 000-fold from bovine plasma by ammonium sulfate fractionation followed by heparin-agarose, DEAE-Sephadex, CM-cellulose, arginine-agarose, and benzamidine-agarose column chromatography. By this method, about 15 mg of protein was purified from 15 L of plasma with an overall yield of 18%. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal analysis. Bovine factor XII is a glycoprotein with a mol wt of 74 000 as determined by sedimentation equilibrium centrifugation. It contains 13.5% carbohydrate including 3.4% hexose, 4.7% N-acetylhexosamine, and 5.4% N-acetylneuraminic acid. Factor XII is a single polypeptide chain with an NH2-terminal sequence of Thr-Pro-Pro-Trp-Lys-Gly-Pro-?-Lys-His. This sequence is homologous to the reactive-site regions of a number of protease inhibitors. The amino acid sequence of a carboxyl-terminal fragments prepared by cyanogen bromide digestion was found to be Leu-Cys-Ala-Gly-Phe-Leu-Glu-Gly-Gly-Thr-Asp-Ala-Cys-Gln-Gly-Asp-SER-Gly-Gly-Pro-Leu-Val-Cys-Glu-Asp-Glu. This sequence is homologous with the active site of a number of plasma serine proteases including thrombin, factor IXa, factor Xa, and plasmin. These data indicate that bovine factor XII is a precursor to a serine enzyme with an inhibitor sequence and a catalytic site located in the same single polypeptide chain.


Isolation and characterization of bovine factor XI (plasma thromboplastin antecedent).

Factor XI (plasma thromboplastic antecedent) has been purified approximately 28 000-fold from bovine plasma with an overall yield of about 30%. The isolation procedure involves barium sulfate adsorption of contaminants, ammonium sulfate precipitation, and chromatography on heparin-agarose, CM-Sephadex, and DEAE-Sephadex. The final product was homogeneous when examined by polyacrylamide gel electrophoresis and immunoelectrophoresis. A minimal mol wt of 124 000 was determined by sedimentation equilibrium. Factor XI is composed of two similar or identical polypeptide chain (mol wt of approximately 55 000), and these two chains are held together by a disulfide bond(s). Factor XI is a glycoprotein which contains approximately 11% carbohydrate including 5.4% heose, 4.7% N-acetylhexosamine, and 1.0% N-acetylneuraminic acid. Other properties of this coagulation factor including its amino acid composition and inhibition by antibodies prepared in rabbits are also reported.


Evidence for intermediate formation in the mechanism of potato starch -hosphorylase from exchange of the ester and phosphoryl oxygens of alpha-D-glucopyranose 1-phosphate.

We have examined under a variety of conditions the ability of potato starch phosphorylase to cause exchange of the ester and phosphoryl oxygens of alpha-D-glucopyranose 1-phosphate (Glc-1-P). In the presence of phosphorylase and strach, under conditions where 40-50% of the glc-1-P is consumed in starch elongation, little if any exchange occurs that cannot be accounted for by accompanying starch phosphorolysis. Nor are the oxygens scrambled in the same or longer times by enzyme only when no release of inorganic phosphate occurs. But when D-maltotriose is used as a primer, or during primerless synthesis, and in the presence of phosphorylase and alpha- or beta-cyclodextrin, a large degree of scrambling does occur. We conclude that under these latter conditions a glucosyl cation or covalent glycosyl-enzyme intermediate in formed. If this same intermediate is formed in the absence of starch or its analogue, then the phosphate counterion is not free to rotate; if the intermediate is formed with starch, then again one must assume that the rotation of phosphate ion is hindered, or that formation of the intermediate is rate determining.


Human hypoxanthine phosphoribosyltransferase. Purification and properties.

Hypoxanthine phosphoribosyltransferase (IMP:pryophosphate phosphoribosyltransferase, EC 2.4.2.8) from human erythrocytes has been purified 13 000-fold to apparent homogeneity. The native enzyme has a sedimentation coefficient of 5.9 S, determined by analytical ultracentrifugation, and a molecular weight of 81 000-83 000, determined by sedimentation equilibrium centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a subunit molecular weight of 26 000, suggesting that the enzyme is a trimer. Isoelectric focusing resolves three peaks of enzyme activity at pH 5.6, 5.7 and 5.9. The amino acid composition of hypoxanthine phosphoribosyltrasferase is 17 Lys, 5 His, 12 Arg, 0 Trp, 31 Asx, 12 Thr, 14 Ser, 16 Glx, 14 Pro, 19 Gly, 12 Ala, 5 Cys, 18 Val, 5 Met, 11 Ile, 20 Leu, 10 Tyr, and 9 Phe. The enzyme appears to have a blocked N terminus.


Characterization of myotoxin a from the venom of prairie rattlesnake (Crotalus viridis viridis).

A previously unknown polypeptide myotoxin, designated myotoxin a, was isolated for the first time from prairie rattlesnake (Crotalus viridis viridis) venom. Electrophoretic homogeneity of myotoxin a was shown in beta-alanine disc gel polyacrylamide gel electrophoresis and in isoelectric focusing gel electrophoresis. Molecular weight and isoelectric point estimates of 4100 and 9.6 were obtained by gel filtration and isoelectric focusing gel electrophoresis, respectively. Amino acid composition showed a total of 39 amino acid residues, with 10 lysine residues and two disulfide bridges. When the two disulfide brides were reduced and alkylated, the myotoxic activity was abolished, indicating that the disulfide bridges of myotoxin a are essential for its biological activity. The loss of the biological activity is probably due to a marked change in secondary structure. The circular dichroic spectrum indicates that the chemically modified, inactive myotoxin exhibits typical random-coil conformation.


Myoglobin as an oxygen indicator for measuring the oxygen binding characteristics of a modified myoglobin derivative containing covalently bound mesoheme.

By measuring the visible spectrum of a mixture of myoglobin and a modified derivative containing mesoheme in place of the normal protoheme, it is possible to evaluate the relative amounts of the oxidized, reduced, and oxygenated forms of each type of myoglobin. If the oxygen affinity of one myoglobin derivative is known, the oxygen affinity of the other can be determined from measurements at various oxygen partial pressures. In the absence of excess reducing agent, the rate of autoxidation can also be evaluated during the same experiment. The method described is suitable at very low oxygen partial pressures, where most previous methods are inaccurate, and it is very convenient to use, since no time-consuming calibration procedures are required. Using protoheme myoglobin as an oxygen indicator, the oxygen pressure at half saturation (P 1/2) of mesoheme myoglobin was shown to be 11% higher than the P 1/2 of a modified myoglobin derivative containing covalently bound mesoheme. The autoxidation rate of the covalent derivative is faster than that of the noncovalent derivative, but it is less dependent on oxygen pressure.


Incorporation into lipid bilayer membranes of a photo--sensitive pigment from the honeybee compound eye.

An increase of electrical conductance up to a factor 10(2)--5-10(2) was obtained by adding, in the dark, the honeybee photopigment to a positively charged lipid bilayer. The increase in conductance was made slower by illuminating the system during the incorporation of the protein into the membrane and it was negligible when the photopigment was bleached before the incorporation. The interaction of the photopigment with the membrane is tentatively interpreted in terms of formation of channels.


Vesicle penicillinase of Bacillus licheniformis 749/C. Apparent identity with the plasma membrane enzyme.

A substantial fraction of the total membrane penicillinase of Bacillus licheniformis 749/C is attached to the vesicles released during conversion of the cells to protoplasts. This enzyme was purified since there was indirect evidence that it differed from the enzyme that remained with the protoplast. The purified vesicle penicillinase has the same molecular weight and general properties as the plasma membrane (protoplast) enzyme and, similarly, contains a covalently linked phosphatidylserine residue. Treatment of the two enzymes with trypsin produced phosphatidylserine-containing peptides which could not be distinguished by gel or paper electrophoresis. The two membrane penicillinases are very similar, if not identical.


Glycosidases of the guinea pig brush border membrane.

The separation by polyacrylamide gel electrophoresis and subsequent enzymatic analysis of the components of the guinea pig intestinal brush border membrane revealed the presence of three enzyme complexes: maltase-glucoamylase, maltase-sucrase-glucoamylase and maltase-sucrase. Additional bands possessing lactase, trehalase and alkaline phosphatase activity were identified but no phlorizin hydrolase or palatinase was detectable. After exposure to strong dissociating conditions the bands possessing enzymatic activity were either absent or greatly reduced in intensity.


Interactions of histones and histone peptides with DNA Thermal denaturation and solubility studies.

The interactions of DNA with the five histone components (H1, H2B, H2A, H3 and H4) and with a number of histone fragments (N-H1 (1--72), C-H1 (73--216), N-H2B (l--59), C-H2B, (63--125), N-H2A (1-39), C-H2A (58--129), N-H4 (1--84) and C-H4 (85--102) have been studied by using the techniques of thermal denaturation and solubility behaviour. Complexes in 10(-3) M phosphate buffer, 2 - 10(-5) M Na(2)-EDTA, pH 7.0 were prepared by the direct mixing method. For lysine-rich histones (H1 and H2B) it has been found that the main characteristics which governs the interaction with DNA are located in the very lysine-rich part of the molecules, i.e. in the C-H1 and N-H2B segments. These regions are also responsible for a cooperative distribution of the histone along the DNA molecules in the artificial complexes. It appears from our studies that the tertiary structure of the moderately, arginine-rich histone (H2A) is an essential feature for its interaction with DNA. The two arginine-rich histones (H3 and H4) complexed with DNA behave in a similar way, both in thermal denaturation and in DNA precipitation. In the case of C-H4, a marked shift of the melting profile has been observed which is correlated with the presence in the peptide of the hydrophilic cluster Lys-Arg-Gln-Gly-Arg-Thr. Our results suggest that large segments rich in lysine and basic clustering within histones give rise to different modes of electrostatic interaction with DNA.


Interaction of acridines and tetrahydroacridines with DNA at low DNA/dye ratios.

This study examines certain aspects of the interaction of acridines with DNA. A comparative study of the methods available for the determination of the association constants (Kap) for compounds which interact with DNA has been pursued. A new equation which permits the spectrophotomeric determination of Kap has been derived. This equation can be applied to compounds which upon interaction with a polymer exhibit discrete absorption changes. Application of this equation to substituted acridines and tetrahydroacridines yields some preliminary information on the effect of ring substituents on the interaction of acridines with DNA. Low levels of DNA/dye ratios have been used in the studies reported herein.


DNA polymerases in adenovirus type 5-infected and uninfected KB cells. Induction of an alpha-type DNA polymerase in adenovirus type 5-infected and in fast growing cells.

DNA polymerase activities in uninfected KB cells or KB cells infected with adenovirus type 5 (Ad5) were compared by chromatography on DNA-cellulose and DEAE-cellulose and by isoelectric focusing. On DNA cellulose three components were found both in infected and in uninfected cells. The major component eluted at 0.15 M NaC1 and contained DNA polymerase alpha. Two minor components were found, one which did not bind to DNA-cellulose and one which bound strongly. This latter component contained DNA polymerase beta as characterized by DEAE-cellulose chromatography and sedimentation studies. No difference in properties between uninfected or Ad5-infected KB cells was found for the beta-polymerase. DEAE-cellulose chromatography of DNA polymerase alpha revealed the presence of two activities eluting at 0.11 and 0.13 M NaC1 designates as alphaI and alphaII, respectively. In Ad5-infected cells alphaII was the major component. In uninfected, stationary cells alphaI was the major component and alphaII was only detectable as a shoulder in the elution profile. However, fast growing, uninfected cells gave a similar pattern as Ad5-infected cells. These results indicate that the observed change of the DNA polymerase pattern after infection with Ad5 is related to the level of DNA synthesis and not to the induction of a viral enzyme.


Studies on almond emulsin beta-D-glucosidase. II. Kinetic evidence for independent glucosidase and galactosidase sites.

A purified beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) isozyme isolated from almond emulsin was found to catalyze hydrolysis of beta-D-glucopyranosides and beta-D-galactopyranosides but not the corresponding alpha-D-derivatives. Hydrolysis of the corresponding beta-D-thioglycopyranosides at rates 10(3)--10(4) times lower than those for the hydrolysis of the beta-D-glycopyranosides was also noted. The enzyme does not exhibit any transferolytic activity using D-glucose or D-galactose as acceptors. D-glucose, p-nitrothiophenyl-beta-D-glucopyranoside, 5-deoxy-5-thio-D-glucose and D-glucono-delta-lactone are shown to exert mainly competitive inhibition on beta-D-galactopyranoside hydrolysis. D-galactose, p-nitrothiophenyl-beta-D-galactopyranside and methylthio-beta-D-galactopyranoside are shown to inhibit the glucopyranoside hydrolysis mainly non-competitively and to exert competitive inhibition of galactopyranoside hydrolysis. The inhibition caused by the antibiotic Nojirimycin (5-amino-5-deoxy-D-glucose) is shown to be more complex. Analysis of the kinetic data indicates that the catalytic site of the enzyme responsible for the beta-D-glucosidase activity is kinetically distinct from the beta-D-galactosidase site.


Purification and characterization of adenosine nucleosidase from barley leaves.

Adenosine nucleosidase (adenosine ribohydrolase, EC 3.2.2.7) has been purified to a nearly homogeneous state from barley leaves. The enzyme is soluble in concentrated salt solution while it aggregates and precipitates at low ionic strength, factors which enabled a simple purification procedure to be carried out. A molecular weight of 66 000 +/- 3000 was estimated for the native enzyme by gel filtration. In sodium dodecyl sulphate polyacrylamide gel electrophoresis of the most purified fraction a single major band of polypeptide chains, with molecular weight of 33 000, was observed. Thus, the native enzyme seems to be dimer of alpha2 type. The pH optima are 4.7 and 5.4 for citrate and (N-morpholino)ethanesulphonic acid buffers, respectively. Adenine and adenosine protect the enzyme against heat inactivation. The enzyme is resistant to -SH reagents, dithiothreitol inhibits it. The Km for adenosine varied from 0.8 to 2.3 micronM depending on temperature and buffer system. The Km for deoxyadenosine was 120 micronM. Besides adenosine, of several nucleosides tested only adenosine N1-oxide, deoxyadenosine and purine riboside acted as substrates. Adenine as well as its derivatives, including plant hormones (cytokinins), have an inhibitory effect on the enzyme. The Ki values of some modified nucleosides and free bases were determined. The physiological role of adenosine nucleosidase in plants is discussed.


Conformational changes associated with the reversible cold inactivation of ribulose-1,5-bisphosphate carboxylase-oxygenase.

Crystalline ribulose-1,5-bisphosphate carboxylase-oxygenase (3-phospho-D-glycerate carboxy-lyase (dimerising), EC 4.1.1.39) isolated from tobacco (Nicotiana tabacum L.) leaf homogenates is partially inactivated by cold treatment and fully reactivated by simple heating in the absence of sulfhydryl reagents and effectors. Since the reversible cold inactivation of this bifunctional enzyme does not involve a gross change in the association state of subunits, a subtle conformational change induced by low temperatures was implicated (Chollet, R. and Anderson, L.L. (1976) Arch. Biochem. Biophys. 176, 344-351). Chemical modification of the cold-inactivated and heat-reactivated enzymes by 5,5'-dithiobis-(2-nitrobenzoate) and p-mercuribenzoate at 25 degrees C revealed no difference in the number of free -SH groups per mol protein. However, the reactivity of the sulfhydryl residues on the inactivated protein was considerably greater than that of the reactivated enzyme. Pretreatment of the two proteins with sodium dodecyl sulfate completely abolished the difference in -SH reactivity, indicating its dependence on protein conformation. Both the cold-inactivated and heat-reactivated enzymes enhanced the fluorescence intensity of 8-anilino-1-naphthalenesulfonate (ANS) and caused a blue shift of the emission maximum from 510 to 472 nm. When the inactivated enzyme was reactivated by heating, the increase in catalytic activity was closely paralleled by a concomitant decrease in the fluorescence intensity of the ANS - protein complex at 25 degrees C. Fluorescence titration experiments revealed that the decrease in fluorescence intensity accompanying heat reactivation of the inactivated enzyme was due to a reduction in the number of hydrophobic sites available for ANS binding rather than to a change in the dissociation constant of the ANS - protein complex. These results indicate that the reversible cold inactivation of ribulose-1,5-bisphosphate carboxylase-oxygenase is associated with a reversible change in the conformation of the protein. This cold-induced conformational change resluts in a greater exposure of sulfhydryl groups and hydrophobic regions to the external environment and is closely paralleled by changes in the catalytic activity of the protein. By analogy to other oligomeric enzymes also subject to reversible cold inactivation, perhaps low temperatures induce a partial dissociation of the octameric structure of the hydrophobic catalytic subunits, but complete dissociation is arrested in some unknown manner by the small hydrophilic subunits.


Kinetic properties of alpha-glucan phosphorylase from Indocibium guttattam.

The kinetic properties of alpha-glucan phosphorylase b (alpha-D-1,4-glucan : orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) purified from a deep-sea fish Indocibium guttattam were studied in the direction of glycogen synthesis. There was no homotropic site-site interaction between glucose 1-phosphate sites and between glycogen sites. However, heterotropic interaction was observed between substrate and activator sites. The kinetic data obtained for the fish enzyme were consistent with the rapid equilibrium random mechanism reported for alpha-glucan phosphorylase from other sources. All the dissociation constants were 2-3 times higher for the fish enzyme than for the rabbit enzyme. Although the fish enzyme exhibited a greater affinity for AMP at 30 degrees C as compared to the rabbit enzyme, these sites were characterized by a lack of homotropic cooperativity. Heterotropic cooperativity was observed between AMP and glucose 1-phosphate sites.


Studies on almond emulsin beta-D-glucosidase. I. Isolation and characterization of a bifunctional isozyme.

A beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) isozyme has been isolated from almond emulsin. The isolated enzyme is a glycoprotein and migrates as a single band on Sephadex G-200 filtration, CM 52 ion exchange chromatography, polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focussing. The glucosidase and galactosidase activities traverse together during Sephadex G-200 gel filtration. Polyacrylamide gels stained specifically for the 2 enzymes reveal that the two activities comigrate. The molecular weight of the isozyme has been found to be 135 180 +/- 770, and that of its protomers to be 65 150 +/- 650.


Distribution and metabolism of polyacetylenes in safflower.

Safflower seedlings, tissues and subcellular fractions were used to investigate the distribution and biosynthesis of polyacetylenes. The polyacetylenes rapidly increased during germination, but were not detected in mature seeds. The polyacetylenes in the seedlings accumulated mainly in cotyledons rather than in hypocotyls and roots. Polyacetylenic hydrocarbons were distributed in the microsomes obtained from leaves and roots of the mature plants, while polyacetylenic epoxide was found in chloroplasts. A 1000 X g pellet from the immature seeds showed the highest activity for the incorporation of labeled acetate into the hydrocarbons. The epoxide was synthesized by chloroplasts from the cotyledons. These results, together with the data of polyacetylene distribution and tissue culture, suggest that chloropoasts may be essential for the formation and metabolism of the epoxide.


The role of chain elongation systems in the supplying of fatty acids to the adipocyte membrane lipids.

A study of the fatty acid composition of the subcellular membranes and of the storage vacuole lipids of rat adipocytes indicated that the proportions of palmitic, stearic, oleic, linoleic and arachidonic acids of the total membrane lipids are essentially the same, while the storage fat is characterized by a higher proportion of oleate (31.5%), a lower proportion of stearate (3.7%) and by the absence of arachidonate. Adipocyte homogenate was incubated under conditions allowing for de novo synthesis, activation, elongation and esterification of fatty acids and the different subcellular fractions were then separated. Part of the newly synthesized fatty acids were found to be incorporated into the adipocyte membranes. The percentage of total fatty acid activity in the carboxyl was found to be 2.7, 2.4, 2.2 and 1.3 times greater, respectively, than the theoretical de novo synthesis value in plasma membranes, microsomes, mitochondria and supernatant. This suggests a functional relationship between elongation of fatty acids and their incorporation into the adipocyte subcellular membranes. The results of experiments in which fatty acid activation cofactors were omitted from the medium indicate that the membranous fatty acid elongation system, unlike fatty acid synthetase, is devoid of deacylase activity.


Unsaturated fatty acyl-CoA inhibition of cholesterol synthesis in vitro.

1. The influence of saturated and unsaturated fatty acids and fatty acyl coenzyme A thioesters on cholesterol synthesis in vitro has been studied in a rat liver post-mitochondrial supernatant system. 100 micronM free fatty acids do not influence in vitro cholesterol synthesis. Various fatty acyl-CoA thioesters at 10--100 microntm inhibit [14C]acetate incorporation into digitonin-precipitable sterols, the more unsaturated derivatives causing the greatest inhibition. 10 micronM arachidonoyl-CoA inhibits [14C]acetate incorporation into sterols 17% and 50 micronM inhibits 55%. [14C]Acetyl-CoA incorporation into sterols is similarly inhibited but [14C]mevalonate incorporation is not inhibited. Thus, the inhibition may be on the rate-controlling step of cholesterol synthesis, the conversion of beta-hydroxy-beta-methylglutaryl-CoA to mevalonate. Unsaturated fatty acyl-CoA thioesters may be important in regulating cholesterol synthesis. 2. Studies were undertaken to determine if the previously observed inhibition of cholesterol synthesis by thyroxine in vitro may relate to the thyroxine stimulation of fatty acid desaturation. 50 micronM thyroxine causes a preferential incorporation of [14C]acetate into unsaturated fatty acids while inhibiting acetate incorporation into sterols. However, a sufficient increase in unsaturated fatty acyl-CoA thioesters to account for the thyroxine inhibition of cholesterol synthesis was not demonstrated.


Lipoprotein lipase and uptake of triacylglycerol, cholesterol and phosphatidylcholine from chylomicrons by mammary and adipose tissue of lactating rats in vivo.

The relationship between lipoprotein lipase activity and uptake of triacylglycerol, cholesterol and phosphatidylcholine from chylomicrons was studied in mammary gland and adipose tissue of rats lactating 6--7 days. 60% of triacylglycerol [14C]oleic acid, 13% of [3H]cholesterol and 8% of [32P]phosphatidylcholine in chylomicrons injected intravenously were taken up within 11 min by mammary gland whereas negligible amounts were taken up by adipose tissue. Non-suckling for 44 h decreased markedly uptake of all lipids by mammary gland and retarded clearance of chylomicrons from blood, while it increased significantly uptake of triacylglycerol fatty acids and cholesterol by adipose tissue. Non-suckling also decreased lipoprotein lipase activity in mammary gland from 7.7 to 0.4 units/g, while it increased activity in adipose tissue from 0.1 to 2.7 units/g. These findings indicate that lipoprotein lipase is involved in uptake of chylomicron triacyglycerol and cholesterol by mammary gland and adipose tissue, and also in uptake of chylomicron phosphatidylcholine by mammary gland. They also show that reciprocal changes in lipoprotein lipase activity in mammary gland and adipose tissue, as occur during lactation, result in diversion of chylomicron lipids from one tissue to the other.


Studies on fatty acid omega-oxidation. Antiketogenic effect and gluconeogenicity of dicarboxylic acids.

1. Administration of dicarboxylic acids to starving rats decreased the concentration of ketone bodies in the blood. 2. Incorporation of 14C into blood glucose was greater from dicarboxylic acids than from monocarboxylic acids. 3. These results suggest that omega-oxidation may be important for production of succinyl-CoA from fatty acids. 4. In starving or diabetic rats about 15% of palmitic acid were subjected to omega-oxidation and then beta-oxidation.


Thermotropic transitions in fluidity of bovine adrenocortical microsomal membrane and substrate-cytochrome P-450 binding reaction.

Temperature dependence of the substrate-cytochrome P-450 binding reaction is investigated in the adrenocortical microsomes using 17-hydroxyprogesterone as the substrate. The apparent substrate dissociation constant ("Ks") increases with increase in temperature. Van't Hoff's plot of "Ks" shows breaks at 21 and 31 degrees C. These temperatures correlate well with the lipid phase transition temperatures as determined by the technique of fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene as the probe. The results indicate that the affinity of the substrate depends upon the temperature. In addition the results suggest that changes in the physical state of the lipids is a factor responsible for the temperature dependency of the reaction.


Comment on the carbon-hydrogen stretching region of vibrational Raman spectra of phospholipids.

The broad, environmentally sensitive 2935-2910 cm-1 feature in the vibrational Raman spectra of phospholipids originates primarily from methylene symmetric carbon-hydrogen (C-H) stretching modes. For ordered acyl chains exhibiting local C2h symmetry at low temperatures, the infrared active methylene C-H asymmetric stretching modes become increasingly apparent at higher temperatures in the Raman spectrum as intramolecular chain disorder (trans-gauche isomerization) leads to a loss of chain symmetry. The 2935 cm-1 shoulder, assigned to the acyl chain methyl group C-H symmetric stretching vibration, is difficult to distinguish as a separate spectral transition in the chain disordered state. An ordering of lipid fluidity for several phospholipids is presented in terms of the intensities of features in the 3000-2800 cm-1 region with particular emphasis upon the 2935-2910 cm-1 contour.


Mechanism of cholesterol side-chain cleavage. II. The enzymic hydroperoxide-glycol rearrangement of the epimeric 20-hydroperxycholesterols in 18O-enriched water.

Incubation of 20 alpha-hydroperoxycholesterol (I) and its 20 beta-isomer, 20 beta-hydroperoxy-20 isocholesterol (II) with adrenocortical mitochondrial preparations in the absence of molecular oxygen, in normal and 18O-enriched water, gave 20 alpha, 22R-dihydroxycholesterol (III) from I and 20 beta,21-dihydroxy-20-iso-cholesterol (IV) from II. Mass spectral analysis of the persilylated glycol products III and IV showed no uptake of 18O, indicating that the oxygen atoms of the C20-, C22- and C21-hydroxyl groups originated from the 20-hydroperoxy atomic oxygen complex is the intermediate in the enzymic oxidative reactions of cholesterol side-chain cleavage.


Catabolism of 6-ketoprostaglandin F1alpha by the rat kidney cortex.

Homogenates of the rat kidney cortex converted 5,8,9,11,12,14,15-hepta-tritiated 6-ketoprostaglandin F 1alpha into one major product identified by gas chromatography-mass spectrometry of the methoxime-methyl ester trimethylsilyl ether derivative as 6,15-diketo-9,11-dihydroxyprost-13-enoic acid. The sequence of derivatisation i.e. methoximation prior to methylation, was crucial as methylation of 15-keto catabolites of the E, F and 6-keto-F series affords degradation products. The corresponding 15-keto-13,14-dihydro catabolite was formed in much smaller quantities. Time course studies indicated that 6-keto-prostaglandin F1alpha was catabolised at a slower rate (about 2-5 fold) than prostaglandin F1alpha. The catabolic activity was blocked by NADH.


Thermally induced conformational transitions of Bence-Jones protein IVA and its proteolytic fragments.

The thermally induced conformational transitions of the kappa-type Bence-Jones protein IVA and its proteolytic fragments (variable and constant halves) were studied by differential adiabatic scanning microcalorimetry, circular dichroism and thermal differential spectroscopy. The striking feature of the results is the good agreement between the experimental heat of thermal denaturation of intact Bence-Jones protein and the heat calculated from the individual variable and constant halves. The results suggested that the variable and constant halves have independent secondary and tertiary structures. It is likely that in the intact light chain, the variable and constant domains have weak non-covalent interactions between themselves. It was shown that at pH values from 7.4 to 2.0 the variable halves exist in the dimeric form. Evidence was obtained that two relatively independent regions of strong non-covalent interactions stabilize the dimers of variable and constant domains.


Multiple hemoglobin alpha-chains in the sika deer (Cervus nippon).

Investigation of the hemoglobin alpha-chains of an Asiatic deer, the sika (Cervus nippon), was prompted by the heterogenity of alpha-chain gene loci in the Virginia white-tailed deer (Odocoileus virginianus). Although electrophoresis of hemoglobin chains from 10 sika revealed only a single alpha-chain band, peptide mapping demonstrated variations in the alpha-TPIII and alpha-TPIV peptides. Substitutions at positions 15, 20, and 22 produced a minimum of five alpha-chains; two possible additional chains could noy be proven because of inseparability of the whole alpha-chains. The most common chain contains Asp-15, Lys-20 and Pro-22 but in other chains glycine is present at position 15, Asx at position 20, and either serine or Asx at position 22. The probable explanation for the large number of alpha-chains is gene duplication which may have been produced by breedings between subspecies from different geographical areas. Comparison with the alpha-chain structure of the white-tailed deer suggests that the sika may have evolved from the lineage which produced the white tailed-deer after the alpha-chain genes of the latter species had duplicated. In addition, these data provide further examples of the unusual variability of this portion of the alpha-chain.


Isolation of prolactin from human amniotic fluid.

Amniotic fluid, obtained from women at term, was centrifuged and concentrated by ultrafiltration. The concentrated amniotic fluid was purified by gel filtration on Sephadex G-100 and ion-exchanged chromatography on DEAE-cellulose. The fractions obtained during purification procedures were assayed for prolactin and somatotropin activities by respective radioimmunoassays. The prolactin-rich fraction obtained from ion-exchange chromatography was further purified by isoelectric focusing. A yield of 0.21 mg of highly pruified prolactin containing 40 units/mg was obtained from 1 liter of the amniotic fluid. The prolactin was free of somatotropin and human placental lactogen.


The interaction of nonhistone chromosomal proteins HMG1 and HMG2 with subfractions of H1 histone immobilized on agarose.

Chromatographically isolated subfractions of calf thymus H1 histone have been covalently coupled to agarose beads and tested for their ability to form complexes with the non-histone proteins HMG1 and HMG2 (High Mobility Group proteins, Walker, J.M., Goodwin, G.H. and Johns, E.W. (1976) Eur. J. Biochem. 62, 461-469). When a mixture of HMG1 and HMG2 is passed through a column of H1 histone-agarose, the HMG2 does not bind. HMG1 does bind and can be eluted from the column with NaCl in the range of 0.05 M--0.15 M. The NaCl concentration required to elute HMG1 from each of the three H1 histone subfraction coulmns is different, suggesting that HMG1 has a different binding affinity for the three H1 histone subfractions.


The myoglobin of the fruit-bat (Rousettus aegyptiacus).

The myoglobin of the fruit-bat (Rousettus aegyptiacus) has been investigated. It has 153 amino acid residues. When the possible therian ancestral myoglobin is taken into consideration, the myoglobin of the fruit-bat resembles most closely that of the hedgehog.


Oxidative changes in human lens proteins during senile nuclear cataract formation.

1. Proteins from the cortex and nucleus of the human lens were studied to determine if any changes could be detected in their amino acids during senile cataract formation. 2. Senile nuclear cataract formation was found to be accompanied by a progressive oxidation of cysteine and methionine. The oxidation of methionine and changes in the distribution of the nuclear proteins did not appear to start until about 60% of the cysteine had been oxidized. 3. In the advanced nuclear cataractous lens, about 90% of the cysteine has been oxidized and 45% of the methionine is present as the sulphoxide in the nuclear proteins. The levels of other amino acids appeared to remain constant. 4. Similar, but smaller, changes were found in the cortical proteins in advanced nuclear cataractous lenses, suggesting that the oxidation spreads from the nucleus to the cortex. 5. These changes were discussed with regard to current views on cataract formation and it was concluded that they are probably the result of simple oxidation of the proteins with O2 or H2O2.


Relative affinities of bilirubin for serum albumins from different species.

Relative equilibrium constants ("affinity ratios") of complexes of bilirubin with a molar excess of charcoal-treated serum albumins from different species (human, bovine, rabbit and chicken) in aqueous solution, were estimated by circular dichroism measurements in the visible region at 26-27 degrees C, pH 7.4, and in the presence of 0.1 M NaCl. By variation of the mol ratios of the components of pairs of different bilirubin-serum albumin complexes showing circular dichroic bands of opposite sign, the apparent association constants of complexes of bilirubin with either human or chicken albumin were found to be greater by factors between 6 and 17 than those of bovine or rabbit albumins. The usefulness in the determination of affinity ratios is illustrated by the evaluation of single equilibrium constants of systems of high-ligand affinity from those of relatively lower affinity, the latter of which are more readily amendable to direct experimental measurement.


The amino-terminal tryptic peptide of bovine rhodopsin. A glycopeptide containing two sites of oligosaccharide attachment.

A glycopeptide (T1) composed of 16 amino acids has been isolated from a tryptic digest of bovine rhodopsin. Its sequence is Met-Asn(CHO)-Gly-Thr-Glu-Gly-Pro-Asn-Phe-Tyr-Val-Pro-Phe-Ser-Asn(CHO)-Lys. Both rhodopsin and peptide T1 are blocked at their amino terminals. When a method specific for isolating amino-terminal tryptic peptides from proteins is applied to rhodopsin, peptide T1 is demonstrated to be the amino-terminal peptide of rhodopsin. Peptide T1 contains two sites at which carbohydrate is attached, whereas rhodopsin was previously thought to contain only a single such site.


Polyalanylation of bovine somatotropin peptide 96-133.

Polyalanylation of bovine somatotropin peptide 96-133 has been investigated. Polyalanylated peptides contained an average of 3-50 additional alanines per mole of peptide. As indicated by circular dichroism, the parent peptide and the polyalanylated peptides were helical at pH 4.0. The biological activity per mole of peptide with 50 additional alanies was approximately the same as that of the parent peptide.


[Influence of the structure of lecithins on their interaction with cholesterol].

Interaction between cholesterine and phosphatidilcholines (PC) of three types: 1,2-disaturated (SS), 1,2-diunsaturated (UU) and 1-saturated-2-unsaturated (SU) was studied in bilayer vesicular membranes (BVM) by means of NMR-1H spectroscopy. BVM were obtained by sonification of aqueous (D2O) dispersions of individual (PC) and their mixtures. It is found that molecular special specificity of the interaction between PC AND cholesterine is preserved not only during phase division but also in the case when all the phospholipid components of the bilayer are in a liquid crystal state. At low content of cholesterine it favourably interacts with UU-PC, further on with the increase of concentration cholesterine starts to interact with SU- and SS-types of PC. It has been shown that such behaviour of cholesterine can not be explained by means of static models, i.e. by the differences in stability of its stechiometric complexes with CP molecules of different types. An attempt has been undertaken to interpret the selective character of PS-cholesterine interaction proceeding from the suggestion about the anisotropic character of lateral diffusion in the membranes, consisting of various structural types of PC.


[Modeling non-enzymatic influence of membranes on the kinetics of chemical reactions].

The reduction of nitroxide radicals by rho-phenylene diamine was studied in H2O, various organic solvents in liposome suspensions and oleic acid emulsion. Some new parameters, particularly the presence of acids, appeared to effect the rate of the process. The results obtained are consistent with the hypothesis that the electron transport in coupled membranes leads to the production of undissociated acids thus supplying protons for the ATP synthesis.


[Mathematical model of adaptation of the energy metabolism of a cell. Calculation of the influence of ATP on the activity and concentration of the initiator stage enzyme].

A simple kinetic model was constructed to study the adaptation of cell energy metabolism to a varying loading. In this model the initiatory step of energy metabolism, in which the initial substrate S is activated at the expense of ATP molecule energy, is catalyzed by an oligomeric enzyme E dissociable at high ATP concentration to monomers E1. It is assumed that the steady state level of monomers E1 in the cell is maintained by constitutive synthesis of E1 molecules, which balances their continuous hydrolysis by proteases. The properties of the kinetic model were studied using a mathematical model which is a system of nonlinear differential equations describing the change with time of the total enzyme E concentration and the concentration of ATP. The main isoclines of this system can intersect in one, two or three points. The mathematical analysis shows that the kinetic model considered exhibits adaptive properties. A sharp increase of the ATPase activity in the model initiates a transient process which leads to a rise in the total enzyme E concentration and in the efficiency of energy metabolism. As a result, the concentration of ATP drops only slightly. The establishment of a new level of the enzyme E concentration may proceed in the oscillatory fashion.


[Potential difference across the membrane of subcellular particles. IV. Study of the part of the respiratory chain from FAD to cytochrome c by the ion penetration technic].

Ability of mitochondria and submitochondrial particles (SMP) to generate a membrane potential on the addition of unpenetrating electron donors and acceptors and trimethylhydroquinone was used for specification of organization of the electron transfer chain. Ferri- and ferrocyanide added to mitochondria induce the membrane potential generation exchanging electrons only with cytochromes c or c1. Ferricyanide added to SMP induces the membrane potential taking electrons in three places: from NAD-H dehydrogenase, succinate dehydrogenase and in the presence of antimicyne A from cytochrome beta region. The difference of two sides of mitochondrial membrane is in agreement with the chemielectric hypothesis. According to this hypothesis protons from internal part of mitochondria are taken up to input H+-channel after electrons. That is why electrons come near the inner surface of the membrane and can be transferred from respiratory chain to water soluble acceptors. In output channels protons move under the action of intramembrane electrostatic field and in the direction of rising polarization of the medium. Protons are released to output channels while electrons are transferred to next carrier within the hydrophobic part of the membrane far from external surface of the membrane and cannot be transferred to water soluble acceptors.


[Potential difference across the membrane of subcellular particles. II. Proton channels in oxidative photophosphorylation].

A photo-electrochemical hypothesis of cyclic photophosphorylation in chromatophores suggests that electrons, thrown out by light from the reaction center, came back via the electron carrier chain within the hydrophobic part of the membrane taking up H+-ions through the entrance H+-channels. H+-ions and electrons are transferred to quinones or semiquinones which are transformed into semiquinones or hydroquinones accordingly. They release H+-ions into output channels, while the electrons are transferred to the next electron carrier. At non-cyclic photophosphorylation of the chloroplast the second photosystem takes electrons from water via primary donor and releases the H+-ions into the proton channels. After receiving an electron the oxidase takes up H+ through the H+-channels and gives the electron and H+ to O2. A direct method of the experimental check up of the electrochemical hypothesis is described.


[Model of the dynamics of the body's water metabolism under normal and pathologic conditions].

A theoretical and experimental study of metabolic processes is presented. Dynamics of water exchange in the living organism under normal and pathological conditions is described. The investigation performed states that the coefficients of analytical equations and constant removal of tritium water from the organism depends on the form and degree of pathology, i.e. pn the change of the constants of water passage from one water reservoir into another.


[Basic evidence for the active transport of carbohydrates in the membrane vesicles of Acholeplasma laidlawii cells].

A. laidlawii membrane vesicles are able to accumulate C14-glucose as well as maltose and fructose against the concentration gradient in the absence of exogeneous entergetic sources. Sugar transport is inhibited by anaerobiosis and by the electron transfer inhibitors such as rotenone and amytal, and by proton conductors such as carbonylcyanide-m-chlorophenylhydrazone. Arsenate and dicyclohexylcarbodimide (inhibitor of membrane-bound ATPase) do not inhibit the sugar transport. It is concluded that sugar transport in the membrane vesicles can be driven by the high-energy state of the membrane or the membrane potential.


[Mathematical model of an immune response. II. Stochastic aspects].

In the mathematical model describing the development of infection and its suppression with antibodies worked out of the course of the delayed immune response a problem of complete destruction of antigen is considered. A method of calculating the probabilities of antigen destruction is advanced. The optimal cure tactics is discussed. It is shown that the highest probability of the destruction of antigene is achieved if the serum is injected in the moment of antibodies peak and when the cure with antibiotics is started at the antigen maximum.


[Blood flow in a renal artery with a deformed vessel wall].

Blood flow in a vessel of the kidney arterium type with a deformed vascular wall in the form of stenosis or atherosclerotic patches is described. Rate fields and pressure distribution in the vessel are described. Conclusions are made concerning the behaviour of blood forming elements. Tension of the vascular wall depending on its deformation and blood current parameters are evaluated. The calculation results are in a good agreement with the experimental data.


[Analysis of the electrophysiologic characteristics of the trabecular membrane in the atria of frogs by recording ionic currents during potential fixation. II. Accomodation. Repeated responses. Anode-breaking excitation].

Measurements were carried out by the method of double succrose gap. Accommodation was practically absent in all the preparations studied: the minimal gradient was not observed, the mean value of accommodation was 0.02 rheobasa/s, saturation current/H practically coincided with the current of rheobase IO(IH/IO = 1.0 +/- 0.1). Repeated responses were initiated in 1/3 of samples. The current of initiating the repeated responses IRR was insignificantly higher than the rheobase one (IRR/IO = 1.2 +/- 0.1). In the samples where the replated responses were absent the effect of uneven depolarization was observed. At depolarization current which was insignificantly higher than the threshold one (IO), the potential did not return to the resting potential and was settled at the level of -20 mV. Anode disconnecting excitation was absent in all the samples. The hyperpolarization level varied from -90 to -160 mV. The data obtained well agree with the results of the analysis of the mathematical model of frog auricle trabecula membrane constructed on the basis of the data on the potential fixation.


[Identification of "slow" cells in insects eyes].

Responses of "slow" cells of the compound eye of grasshopper to equal-energetic flashes of monochromatic light of different wavelengths were recorded by intracellular microelectrode. Majority of cells had the curve of spectral efficiency of 540 nm. This curve coincided practically with the curve of spectral efficiency measured with ERG. Colour adaptation significantly changed the curve of spectral efficiency of the "slow" cells. Intracellular staining of the "slow" cells of grasshopper, cricket and locusta by procion yellow M-4 or by sulphide of cobalt have shown that the "slow" cells are the cells of crystaline cone.


[Work of the human visual system. VII. Opposite color difference and anticolor. 4th series of experiments].

The "opposite color differences" were considered in the case of decreasing of color difference, when k ln a2 a1 was changed to k ln a2+a3 a1+a3 and in the case of disappearance of color difference, when k ln a2 a1 was changed to k in a3 a3. It is shown that the changes indicated evoke the appearance of correlated opposite color differences; k is the coefficient of proportionality with a dimension of sensation; a1, a2 and a3 are the actions of light.


[Models of the neuronal network of the lateral geniculate body].

Mathematical identification of neuron structures forming different types of receptive fields (RF) of the outer geniculate is carried out on the basis of the results of neurophysical and morphological studies. Net models of overlapping RF of different types are studied. It has been shown that the difference in time characteristics of different RF responses is mainly determined by the constants of excitation and inhibition while the difference in the character of RF response dependence on the stimulus parameters--by different types of dependence of the time of delay, on stimulus parameters and different form of spatial weight functions. It has been analysed what functional difference of neuron channels composed of various types of RF is conditioned by the differences in the parameters of neuron structures which form various RF.


[Use of linear proportional chambers for small-angle synchrotron radiation scattering in solutions of biopolymers].

The results of applying linear position sensitive proportional counters to diffuse small-angle synchrotron radiation scattering to biopolymer solutions are presented. Synchrotron radiation of the VEPP-3 storage ring (Institute of Nuclear Physics, Novosibirsk) was used. On the basis of small-angle scattering curve obtained from protein pepsinogen it has been shown that the exposure is about 100 times less than with conventional X-ray technique.


[Model of a binaural hearing system. II. Monaural processing of information concerning short sounds].

The first part of the model of the auditory system determining the direction on the source of short (shorter than 10 msec) sound signals is presented. The processes in perceptors, the neurons of spiral ganglion, anterior ventral cochlear nucleus and medial superior olivary nucleus are simulated. According to the model the information about a sound signal has undergone several stages of processing and finally a resultant pulse is formed. The moment when this pulse appears on the input of the comparing neuron (in the medial olive) is determined by the two parameters of the sound: its intensity and the moment when a sound arrives to one of the ears.


Substituent effects on the redox reactions of tetraphenylporphyrins.

The effect of substituents on pi radical reactions of para-substituted tetraphenylporphyrins was investigated by cyclic voltammetry in methylene chloride. In all cases electron donating substituents produced a more difficult reduction and an easier oxidation. Plots of E1/2 vs. a yielded Hammett linear free energy relationships for cation radical and dication formation and anion radical and dianion formation. An average reaction constant of p=0.07+/-0.01 V was obtained. This was true for tetraphenylporphyrins containing the central metals VO, Mn, Fe, Co, Ni, Cu, and Zn, as well as the free base H2 (p-X)TPP. The value of p appears not to be directly affected by the central metal oxidation state or the overall charge on the complex.


[Study of the mitochondrial Ca2+-transport system on lipid bilayer membranes (LBM)].

Electrical properties of BLM formed from lipid components of rat liver mitochondria were studied. The lipids were obtained after preliminary 40 second incubation of mitochondria in the medium containing succinic acid. BLM electroconductivity in the medium with calcium chloride (8--20 mM) is 10--100 times higher than the initial electroconductivity in tris-HC1 at pH 7.5, and in the presence of potassium chloride and magnesium chloride it differes little from the initial value. The transmembrane potential difference per 10-fold concentration gradient of Ca2+, Mg2+, and K+ is approximately 27 mV, approximately 15 mV and approximately 22 mV correspondingly. By means of biionic potential method the following selectivety line for cations is formed: Ca2+ greater than Sr2 greater than or equal to Mg2+ greater than K+. La3+ ions inhibit the transport of Ca2+ through the BLM studied.


Sequence structure relationships in metalloproteins-I. A predictive model of hemerythrin.

Utilization of simple algorithmic schemes coupled with chemical data and the known conformational requirements of metal ions has resulted in the prediction of the basic tertiary structure of a metalloprotein, the hemerythrin subunit.


Definitive evidence for the existence of the "sitting-atop" porphyrin complexes in nonaqueous solutions.

Definitive evidence for the intermediate in metalloporphyrin formation produced in the interaction of meso-tetraphenylporphine and [Rh(CO)2Cl]2, the "sitting-atop" complex (SAT), is given in this article. The second-order kinetics of SAT complex formation, the small rate constant for the formation reaction, and the equilibrium study indicate a one-to-one complex between the meso-tetraphenylporphine and [Rh(CO)2Cl]2. The analytical data, infrared spectrum, and especially the proton magnetic resonance lead to a formulati-n of the "sitting-atop" complex shown in Fig. 8.


Multiple roles of metal ions in the reaction catalyzed by yeast inorganic pyrophosphatase.

Yeast inorganic pyrophosphatase has three roles for metal ions in its reaction: activator, substrate and structural. Out of a wide variety of metal ions tested, only Mg2+, Zn2+, mn2+ and Co2+ can fulfill both the activator and substrate roles. Several other metal ions inhibit the Mg2+-stimulated activity; the strong inhibition by Ca2+ (and probably Cd2+) is due to interference with both activator and substrate roles, while the weaker inhibition by Sr2+ (and possibly Cu2+ and Ni2+) is due to interference with only the substrate role. Rare earth ions strongly stimulate nonenzymic PPi hydrolysis but do not activate the enzyme. Despite its ability to fulfill both the activator and substrate roles. Zn2+ causes inactivation of the enzyme, probably by interference with the "structural" Mg2+. The results suggest that the three roles for metal ions are independent (an individual metal ion can satisfy only one at a time) and that the metal ion specificity for the three roles declines in the order: structural greater than substrate greater than activator.


Interaction of DNA with anti-cancer drugs: copper-thiosemicarbazide system.

The interaction of copper-thiosemicarbazide complexes with DNA was investigated using ultraviolet and infrared spectroscopy. Evidence for the interaction of the complexes with nucleic acid bases and with the phosphate group is presented.


[Model of temperature distribution on the body surface of homeotherms].

A mathematical model is considered which describes a change of temperature on the body surface under the effect of the heat source located near the surface. Solutions of heat conductance equations for semi-infinite isotropic and homogenous medium are used as a model. A point source with capacity Q is placed into the medium at the depth zota at the moment t=0. The heat sources are focuses of heat increased production--tumours. Temperature distribution of the skin obtained for a dynamic and static case permits to determine the time of transition processes, efficiency of the application of dynamic methods of studies etc.


A study of the redox potentials and electron transfer rates of several naturally occurring and synthetic iron porphyrins in DMF.

The reduction potentials and formal electron transfer rate constants of several iron porphyrins were measured in DMF by the technique of cyclic voltammetry. The ease of each reduction was found to be a function of porphyrin ring basicity and was dependent on the type of electrode reaction. Greatest effects of ring basicity were observed for pi anion radical formation whil smaller effects were obtained for reactions involving the central metal. Electron transfer rate constants for the ferric-ferrous reaction also varied with ring basicity and paralled trends in the redox potentials, that is, the most basic porphyrin yielded the most difficult reduction, both kinetically and thermo-dynamically, while the least basic prophyrin yielded the most facile kinetic and thermodynamic reduction. Based on this data a mechanism of ferric-ferrous electron transfer involving transfer via the pi ring system is proposed.


[Effect of light negative aeroions on the human body].

Information is given about the effect of light negative aeroions on human organism. This phenomenon is explained by the effect of the change of ion charge. The latter causes the ionization of gas mixture components and their active perception by the organism. Besides during the formation of negative ions at the expense of electron seizure on outer levels ions in electron-excited state can be formed. Such ions have higher chemical activity which contributes to their effect on human organism.


[Properties of mitochondrial monoamine oxidase isolated from rabbit brain and liver].

The effect of pO2 on the monoamine oxidase activity of mitochondria from rabbit brain and liver was investigated using the substrates tyramine, dopamine, tryptamine and serotonin. The effect of the second substrate (oxygen) was dependent upon the concentration of the first substrate (the amine). At amine concentrations below 50 micronM, the reaction rate as measured by a radiometric assay, was not affected by variations in the pO2. It was found that both phenazine methosulfate (PMS) and chlorpromazine (CPZ) are reversible inhibitors of monoamine oxidase, the former was a potent inhibitor (Ki=3X10(-6) M) and the latter relatively weak (Ki=5X10(-4) M). Inhibition by both compounds was non-competitive with respect to the amine substrate. Imipramine was a weak inhibitor of purified MAO from beef kidney and of the MAO activity of mitochrondria from brain and liver. Using tyramine or dopamine as the substrate (0.5-1.0 mM), inhibition ranging from 6-30% was observed at 5X10(-4) M imipramine. With tryptamine or serotonin (0.5-1.0 mM) as the substrate in the presence of 5X10(-4) M imipramine the drug seemed to have no net effect on MAO activity since the average value in the presence of imipramine for a number of experiences was the same as the average for control experiments. For p-iodo-phenyl-3-p-nitrophenyl tetrazolium chloride, a Ki of 43X10(-6) M was found using dopamine as the substrate and oxygen as the gas phase.


[Effect of immobilized noradrenaline on human plasma arginine esterase activated by factor XII].

An attempt to study direct hormonal regulation of the blood coagulating system by catecholamines, using the affinity chromatography method, was made. It was found that human blood plasma is adsorbed and the enzyme hydrolyzing N-benzoyl-L-arginine, is activated on the column. It was found that human blood plasma after the contact with norepinephrine loses its ability to increase the BAEE-esterase activity, when tanic acid, which activates factor XII (Hageman factor), was added.


[Presence of an active fibrinogen--heparin complex in the total blood plasma fraction of plasma fibrinogen degradation products].

It is found by means of different methods (the administration of 35S-heparin, the determination of fibrinolytic activity of blood plasma fractions containing fibrinogen degradation products and fibrinogen--heparin complex, spectral analysis of fibrinogen--heparin complex etc.), that thrombin injection into animals results in the formation of fibrinogen--heparin complex, which is mainly discovered in the fraction of fibrinogen degradation products.


[Macromolecular organization and biochemical characteristics of the genome of phage FI-1].

The molecular weight of phage FI-1 DNA is determined by the methods of sedimentation and kinetics of reassociation, as well as from buoyant density values of virion components and specific partial volume of DNA (85X10(6) daltons). The spectral analyses showed that the distribution of guanine-cytosine pairs along the whole length of the molecule is Gaussian. The DNA content in the particle of FI-1 makes up to 41%, which is 7% less as compared to the morphologically related phage T4. The protein component analysis of phage FI-1 demonstrated that the genome of this virus is capable to encode at least 20 different proteins. Possible reasons for differences in the molecular weights of the genomes of T4 and FI-1 phages are discussed.


[Allosteric properties of muscle creatine kinase].

The dependence of the reaction rate on substrate concentrations at pH 8.0--7.5 does not submit the Michaelis-Menten kinetics. The dependence of v on Mg-ATP is described with a curve having an intermediate plateau. The dependence of v on the creatine concentration is expressed by a curve, which is not hyperbolic. In this case the index of the substrate concentration, (q), is variable, and it increases with the increase of creatine concentration from 1 to 2 (at the presence of effectors, PEP and ADP,--from 1 to 3.5). The specific creatine kinase activity increases 3--4-fold with protein dilution, but this effect is not observed in the presence of inhibitors to FDP and PEP. Creatine kinase is desensibilized with respect to FDP and PEP after a prolonged storage. The data obtained and the presence of an effector set indicate, that muscle creatine kinase is a regulated allosteric enzyme.


[Change in the concentration of total lipids, phospholipids and neutral lipids in rat liver mitochondria and microsomes during chemical carcinogenesis].

Sharp increase in the content of total lipids and, especially, neutral lipids, and considerable decrease in the content of phospholipids were observed in liver microsomes and mitochondrias from liver of tumour-bearing rats in the process of the growth of sarcoma, induced by a single injection of 3,4-benzpyrene. The injection of anthracene, a non-cancerogenous hydrocarbon, practically did not affect the lipid composition of rat liver subcellular particles. Thus, the disturbances in normal functioning of subcellular particles under cancerogenesis are due to considerable change in the chemical composition of biomembranes. The data obtained confirm our hypothesis on lipid mobilization under tumour growth.


[Products of bovine prothrombin citraconylation and their activation by factor Xa].

Products of bovine prothrombin acylation by citraconic anhydride, modified to 20--90% have been obtained. Disc-electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate has shown that the citraconylation of prothrombin is accompanied with spontaneous activation, resulting in formation of products with molecular weights identical to those of neoprothrombin C, neoprothrombin T, profragment and fragments A and C. Spontaneous activation upon the citraconylation is never completely recovered: the reaction products always contain a fraction, corresponding in molecular weight to prothrombin. Citraconylation products possess neither coagulating, nor esterase activity. Generation of esterase activity requires the presence of an enzyme--factor Xa, which splits the 323-324 peptide bond (Arg-Ile) in the molecules of prothrombin and intermediate products of its activation. The mechanism of activation of citraconylated prothrombin products by factor Xa does not differ from the mechanism of native prothrombin activation by the enzyme. The esterase activity, which is generated after the incubation with factor Xa, is due to the building of citraconylthrombin and partly of the native thrombin; the latter may be formed at the low degree of prothrombin modification.


[Lysosomal neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase of regenerating liver tissue].

Neuraminidase (EC 3.2.1.18) beta-galactosidase (EC 3.2.1.23) and beta-N-acetylglucosaminidase (EC 3.2.1.30) were studied in normal and regenerating rat liver. All these glycosidases were shown to be predominantly localized in lysosome rich fraction. The activity of lysosomal neuraminidase in regenerating rat liver increased 24 hours after partial hepatectomy, whereas those of beta-galactosidase and beta-N-acetylglucosaminidase decreased. The presence of inhibitor of neurominidase in regenerating liver homogenate was shown. Actinomycin D and cycloheximide were shown to have no significant effect on lysosomal neuraminidase in regenerating rat liver.


[Photoreduction of bacteriophenophytin b in the primary light reaction of Rhodopseudomonas viridis chromatophores].

Photoconversions of the reaction center pigments in chromatophores of nonsulfur purple bacteria Rhodopseudomonas viridis have been studied as a function of redox potential of medium (Eh). It has been shown that at a decrease in the Eh values from +400 mV to--100 divided by--600 mV a photo-induced accumulation of P980+ (oxidized primary electron donor in R. viridis) is replaced by the photoaccumulation of a reduced pigment complex P800 (bleaching of bacteriopheophytin b absorption bands at 545 and 800 nm, a development of broad bands at 680 and 430 nm and a blue shift of the bacteriochlorophyll band at 830 nm). The P800 photoreduction is observed under illumination by light with lambda greater than 900 nm between +20 divided by--196 degrees C at pH 3,5--12,5 and is accompanied by oxidation of the cytochrome and an increase in fluorescence yield of bacteriochlorophyll. It is suggested that P800 accepts an electron from the P980 in the primary photoreaction, which preceeds ubiquinone reduction. A midpoint redox potential (Em) is found to be of --620(+/- 20) mV for the P800/P800- and +515 (+/-20) mV for the P980/+P980. At a decrease in the Eh value down to -400 mV luminescence has been detected with T1/2 8 nsec, an activation energy of 0,065 +/- 0,02 ev and quantum yield being close to the fluorescence yield. It is assumed that this luminescence is a result of charge recombination in the biradical P980+ -- P800-.


[Possible mechanism of action of oxythiamine on nucleic acid metabolism].

Hydoxythiamine injections (400 mg/kg for 72 hours) resulted in the inhibition of transketolase activity and of 14C-orotic acid incorporation into RNA in rat liver tissue. At the same time, the decrease of RNA and phosphoribosylpyrophosphate levels has been observed, which suggests a regulation effect (via pentospohosphates) of hydroxythiamine on nucleotide and nucleic acid metabolism.


[Isolation of purified aspergillopepsin A].

Two methods of isolation of pure aspergillopepsin A have been developed. The first method is based on sequential chromatography of a crude preparation on gramicidin C-sepharose 4B and ECTEOLA-cellulose (35% yield, 100-fold purification). The second method consists in sequential chromatography of evaporated extract of Asp. awamori surface culture on Acrylex P-10, aminosilochrome and ECTEOLA-cellulose (40% yield, 430-fold purification). The methods discussed may be used to isolate pure aspergillopepsin A preparations in order to establish the primary structure of the enzyme.


Whole and disaturated lung phosphatidylcholine in cortisol-treated, intrauterine growth-retarded and twin control lambs at different gestational ages.

Lambs 116--124 days gestation infused in utero for 75 h with cortisol showed, when compared to twin controls, more mature lung histology and pressure-volume relationships. 32P orthophosphate incorporation into whole lung phosphatidylcholine (PC) was increased in the four cortisol-treated lambs at 116--117 days but not at 121, 123, and 124 days gestation. 14C palmitate incorporation into PC or disaturated phosphatidylcholine (DSPC) was not enhanced at 116--117 days gestation. At 121 days in a cortisol-treated and at 128 days in a growth-retarded lamb fetus not treated with cortisol, a larger quantity of DSPC  was present although the incorporation of 14C palmitate into DSPC per milligram DNA was the same. This indicated that the synthesis of DSPC had been initiated in the cortisol-treated and growth-retarded animals prior to the controls and at the time of sacrifice both were incorporating 14C palmitate at a similar rate suggesting similar rates of synthesis.


Site of horseadish peroxidase transport by the small intestine of the neonatal rat.

The absorption of horseradish peroxidase was estimated in 10-day-old rats by measuring plasma peroxidase levels following intraduodenal admininstration. Peak levels occurred 45 min after injection. Plasma peroxidase levels, 45 min after injection into five ligated segments along the small intestine, were significantly lower in rats with distal segments than in rats with proximal segments. This indicates that the proximal small intestine, including the duodenum, may play a significant role in nonantibody macromolecular transport as well as in immunoglobulin absorption.


Early postnatal development of the intestine in progeny of protein-deprived rats.

Development of the small intestine was studied in newborn, and 4-, 8-, and 12-day-old young of rats fed diets, during gestation, containing 24 or 4% casein as the source of protein. Newborn prenatally protein-deprived rats had shorter, narrower intestines, reduced numbers of villi per unit length, shorter villi, and reduced numbers of absorptive cells and crypt cells. These differences had disappeared by the age of 12 days in adequately fed pups that had survived to that age.


Plasma renin activity in sheep pregnancy after fetal or maternal nephrectomy.

The effect of bilateral fetal or maternal nephrectomy on basal and diuretic-stimulated plasma renin activity (Pra) was examined in 9 chronically catheterized Dorset sheep. After fetal nephrectomy, there was an initial rapid decrease in fetal PRA with t1/2 of 42-84 min, followed by a slower decrement with 1/2 350-720 min. There was no change in maternal PRA during this period. Similarly, after maternal nephrectomy, the decrease in maternal PRA had two exponential components and there was no associated change in fetal PRA. These data, and the maternal and fetal PRA responses to intravenous furosemide, have demonstrated that renin does not cross the sheep placenta and that fetal PRA is independent of the maternal PRA level.


Role of respiration in effecting transfusion at cesarean section.

The transfer of blood from placenta to baby (placental transfusion) was examined in 29 full-term babies delivered by elective Cesarean Section. Events surrounding the delivery were carefully monitored using a stopwatch. Residual placental blood volume (RPBV) and change in hematocrit (Hct) were measured. In 13 infants held above the level of the placenta, the mean RPBV was significantly greater, and the change in Hct significantly lower, than in 16 infants held below the level of the placenta. Increasing duration of respiration resulted in increasing amounts of placental transfusion, even in those babies held just above the placenta. These results indicate that there are separate effects of gravity and respiration which facilitate a placental transfusion at Cesarean Section.


Multiple forms of thymidine kinase during human development.

The developmental progression of thymidine kinase from the electrophoretically slow-migrating 'fetal' forms to the fast-migrating 'adult' form was examined in human serum and fetal liver. In fetal liver, only the fetal forms of the enzyme were present at 17 weeks' gestation. A transitional period followed in that both enzyme forms were identified and by 24 weeks only the adult form was detected in fetal liver. This same enzyme changeover pattern--fetal to transitional to adult--occurred at a later time in human serum as it took place between 30 and 40 weeks' gestation.


Phototherapy in Gunn rats. A study to assess the photobiologically most effective radiant energy and dose/response relationships.

In order to get a more realistic spectral efficiency curve and to evaluate dose/response relationships in phototherapy, homozygous weanling Gunn rats -- nondepilated, with fur -- were illuminated under standardized conditions with 8 different fluorescent tubes. Some of the tubes were operated with different electric power. Clear spectal differences in the extent and the rapidity of the bilirubin decay could be ascertained. Furthermore, the sharpness of the bilirubin decrease depended on the baseline concentration. For the calculations the animals were therefore divided into 3 groups with starting levels of larger than or equal to 8 mg%, 6.5--7.9 mg% and less than 6.5 mg%. Correlating the spectral power distribution of the lamps with the bilirubin decomposition found in the experiment, the spectral response function s(lambda)bili, rel was calculated by an integral method. A comparison of our results with data from the literature shows that so far near UV radiation was evaluated too high. A new radiometer for digital measuring the effective irradiance Ebili was developed. On a logarithmic scale a comparatively sharp dose/response relationship could be demonstrated in dependence on the measured effective radiant exposure. Serum bilirubin decrease is directly proportional to log Ebili. A dose of about 2.5 mW - h/cm2 is necessary to achieve a constant serum bilirubin decrease at all. Good results were obtained at doses of about 35 mW - h/cm2 with the most efficient being at 160 mW - h/cm2. Highly effective doses can be applied with different types of lamps. However, there are great differences in the time of illumination required. 24 h are necessary with daylight tubes (Osram L 20 W/19) to apply 20 mW - h/cm2, whereas the same dose is already attained after 4 h with BAM blue tubes (Philips). The accuracy of the radiometer was finally controlled by screening Westinghouse special blue and Osram standard blue tubes with black tapes, so that the effective irradiance (Ebili) corresponded to that of Osram daylight tubes. The bilirubin decrease observed after this manipulation was nearly identical (not exceeding 1 SD of the daylight values). Since bilirubin decrease was sharper in the first few hours of illumination, continuous phototherapy was compared in some tests with 3 intermittent schedules: (a) 4 h light, 4 h dark, 4 h light; (b) 5 h light, 2 h dark, 5 h light, (c) alternately 2 h light and 2 h dark. There was no indication that more bilirubin could be removed per hour with intermittent phototherapy than with continuous illumination.


Latency of multiple skin conductance responses in differential classical conditioning.

Latencies of multiple skin conductance responses were observed in 30 female undergraduate students who participated in a classical differential conditioning experiment employing a 10 sec interstimulus interval (ISI). Responses occurring in both the early and late portions of the ISI were examined. Pairing delayed the onset of early responses and hastened the onset of responses occurring later in the ISI. Additionally, the latency changes observed did not appear to be an artifact of the higher response frequencies typically associated with conditioning.


Preception (UCR diminution) in normal and neurotic subjects.

Three groups of subjects, viz. low trait anxious (LA), high trait anxious (HA) and neurotic patients (PAT) recieved a series of warned unpleasant auditory stimuli. The expectation that the warned stimuli would evoke smaller accelerative heart rate responses than the unwarned ones was not confirmed. On the contrary, the responses to the warned stimuli stimuli were larger than to the unwarned stimuli. Evidence was provided for the idea that homeostatic mechanisms were, at least in part, responsible for this result, though the possibility that even homeostatic heart rate changes can have psychologically relevant effects on central structures was not excluded. Neither the responses during the ISI nor the responses to the unpleasant stimuli (UCS) differed between groups although the response in the IS was in the predicted direction, i.e. a stronger initial acceleration in the LA group as compared to the HA and PAT groups. It was further suggested that the difficulty separating homeostatic effects from changes in sensitivity of the organism makes the operationalization of preception in terms of UCR amplitude of heart rate questionable.


Differential effects on test stress on the heart rates of extraverts and introverts.

A mental arithmetic task was administered to 39 subjects under conditions which imposed social stress. Using the relaxed state to provide a baseline, changes in heart rate and finger blood volume pulse were recorded. The subjects were divided to obtain groups with extreme scores on Eysenck's PEN scales, and the groups were compared on the two psychophysiological measures. Contrary to Eysenck's theory of Autonomic Lability as the neurological basis of N, the high N scoring group was not differentiated from the low N group by change in level of sympathetic activity as indicated by blood volume pulse, while, on the heart-rate measure, introverts showed a large increase and extraverts considerably less: there was no overlap between groups (p less than 0.001). It was concluded that extraverts exhibit greater parasympathetic activity relative to sympathetic arousal under this stress condition.


The effect of chronic anxiety level upon self control of heart rate.

A sample of 90 subjects were rated on the IPAT anxiety scale and 5 subjects (3 male and 2 female) per group were selected from each of the high and low anxiety categories. All subjects were naive participants in heart rate biofeedback sessions utilising analogue visual feedback. Following a heart rate baseline (no feedback) session, all subjects were required to raise their heart rate and lower their heart rate in two separate trials, a visual "target" criteria being provided. The data collected consisted of a resting baseline heart rate value, mean heart rate increase, and mean heart decrease values expressed as beats per minute. Results indicated a marked ability for highly anxious subjects to self-induce heart rate increases only and for low anxious subjects to decrease their heart rate only.


The contingent negative variation (CNV) and speech production: slow potentials and the area of Broca.

The lateral distribution of the CNV was investigated during conditions that required normal male and female subjects to vocalize simple English words in a CNV paradigm. In addition to a non-verbal or comparison condition (Tone-Clicks/Button Press), two word conditions presented stimulus words in either S1 or S2 positions (Word-Tone/Speak; Tone-Word/Speak) and a third condition required a one-word association to the stimulus words (Word-Tone/Speak Association). Monopolar scalp activity was collected from sites that included a location approximating Broca's speech area on the left hemisphere,  a homologous comparison site on the right hemisphere, frontal sites (F3, F4) and the vertex (Cz). Although several subjects showed some asymmetrical activity in some of the word conditions, amplitude measures based on CNV averages indicated no consistent lateralization effects over the left hemisphere prior to word vocalizaiton. Negative pretrial shifting was suspected in those conditions in which words appeared as S1 signals.


DNA polymerase activity and antibody against it in sera of patients with systemic lupus erythematosus.

In the sera of one half of patients with sytemic lupus erythematosus (SLE) the endogenous and exogenous DNA polymerase activities were observed. The simultaneous presence of DNA polymerase activity and the antibodies to denatured DNA in the half of polymerase positive SLE sera was proved. The polymerase activity was not associated with virus-like particles. The endogenous polymerase activity probably used RNA, the exogenous one prefered DNA as a template. The determination of template requirement was complicated by the complexity of SLE sera. The product of polymerase activities were DNA-RNA hybrid and DNA. In some sera of the SLE patients without polymerase activity the antibodies against the polymerase were found. The occurrence of DNA polymerase activity and possible viral origin of SLE was discussed.


Elastogenesis and elastinolytic activity in human breast cancer.

Polymeric elastin was isolated and chemically characterized from 34 human breast cancers. There exists a good correlation between the histological and biochemical determinations of elastin; the breast cancer elastin resembles the other elastins isolated from ligamentum nuchae or aorta. Meanwhile it differs by its lower proline content and by its degree of crosslinking as determined by the ratio (Des + IDes/4)Lys. An elastinolytic activity (elastase) was found in human breast cancer extracts. This activity increased with the elastin content of the tumors.


Hepatic uptake and biliary excretion of a bile salt analog : glycodihydrofusidate.

Glyco-24, 25-dihydrofusidate is efficiently excreted in the bile by the liver. This compound partially inhibits hepatobiliary transport of bile salts and bilirubin. In order to determine if bile salts and glycodihydrofusidate share common pathways at the hepatocyte level, we studied the effect of sodium dehydrocholate on the blood clearance of glycodihydrofusidate. Two groups of rats were perfused, controls with 0.15M NaCl, and test animals with 10 micronmol + min-(1) + kg(1) of dehydrocholate; both groups received 1 micronCi of 14C-glycodihydrofusidate intravenously. Carotid blood was removed every minute and the disappearance of radioactivity from the blood was monitored. The results are consisgent with a theoretical model of two compartments. The experimental curves were fit to the sum of exponentials.


Secretion of labelled proteins by the isolated dog pancreas in the response to secretin and cholecystokinin.

We compared the effects of secretin (10 clinical units/hur.) and cholecystokinin-pancreiozymin (CCK-PZ 10 Ivy units/hr.) from G.I.H. Laboratory in Stockholm, Sweden, on the secretion of radioactively labelled proteins in the pancreatic juice. This was carried out on isolated dog pancreas perfused "ex-vivo" with whole heparinized oxygenated blood. CCK-PZ and secretin were infused together and the results compared with a second series of experiments where secretin alone was administered, at the same dose. Both hormones caused a discharge of labelled proteins, which was linear over 55 minutes. Secretin increased the flow rate of the pancreatic juice and to a lesser extent the enzymatic protein output. CCK-PZ stimulated the mass flow of secretory proteins.


Matrix biology and pathology, a new branch of biomedical sciences.

The macromolecules of the intercellular matrix (MM) : collagen, elastin, proteoglycans and structural glycoproteins) are present in all tissues in variable amount and proportion. Some tissues, particularly rich in MM are designated as "connective tissues". Matrix macromolecules assure the integration of cells in tissues and of tissues in organs and in the whole organism. Differentiation, morphogenesis, maturation and aging are characterized by the variation of the raltiave rates of synthesis of individual MM-s. Several post-transcriptional and post-translational steps play an important role in biosynthesis of MM-S offering a multitude of possibilities for genetic and/or aquired anomalies. Recent progress in the descriptive and dynamic biochemistry of MM-s sheds new light on these anomalies which condition a whole class of diseases (the diseases of the intercellular matrix or matrix pathology). The molecular and cellular mechanisms of several of these diseases start to be understood. As the great majority of the important disease of occidental societies (such as vascular and articular diseases, diabetes, and the pathology of aging) belong to this category, intensive research in matrix biology and pathology as well as of its teaching in the medical curriculum should be considered as first priorities.


Correlation between calcitonin and carcinoembryonic antigen levels in medullary carcinoma of the thyroid.

We have detected high values of carcinoembryonic antigen in the blood of patients suffering from medullary carcinoma of the thyroid (MCT). Levels of the antigen and calcitonin, the hormone which is specifically secreted by these tumours, are positively correlated. Stimulation of calcitonin secretion has no effect on carcinoembryonic antigen levels. Though the antigen assay is non specific for MCT, it may improve the diagnosis and follow-up of MCT-patients.


Xanthinuria : study of a large kindred with familial urolithiasis and gout.

We have carried out biochemical and clinical studies on a large family in which xanthinuria, xanthine lithiasis, uric acid lithiasis and/or gout were discovered. The analysis of its pedigree has shown that : a) the mode of transmission of xanthinuria is autosomal recessive; b) the occurence of xanthine urolithiasis is likely to be due to the association of a second genetic disorder.


The roles on intraluminal oxygen and glucose in the protection of the rat intestinal mucosa from the effects of ischaemia.

Rat ileal loops were subjected to 15 minutes ischaemia by clamping both mesenteric and collateral vessels, after which the functional capacity of their mucosae was assessed according to their ability to accumulate phenylalanine "in vitro". Groups of intestines were rinsed before being subjected to ischaemia. Independent and additive protective effects were observed when the rinsing buffer was oxygenated and/or when it contained glucose. Indeed, an oxygenated, glucose-containing buffer afforded full protection to the mucosa, whereas a nitrogenated, glucose-free buffer had no action.


The activation of prothrombin complex concentrates by calcium in vitro.

The activation of prothrombin concentrate mixtures incubated "in vitro" was investigated. "Activation" was assessed by the correcting effect of suct mixtures on the APTT of a test plasma containing a factor VIII inhibitor and also by the generation of free thrombin. Thrombin itself was found to have insignificant activating effect. Prothrombin concentrates incubated with near-physiological levels of calcium appeared to correct the abnormal APTT to an increasing degree as the calcium concentration was increased. Free thrombin was generated with calcium cencentrations greater than those required for APTT correction. The activation process could be stopped at any stage by adding citrate which removed ionized calcium from the incubation mixture.


Inhibition of human platelet mitochondrial protein synthesis by chloramphenicol.

Human platelet mitochondrial protein synthesis, assayed by "in vitro" 3H-leucine incorporation into the protein moiety of isolated mitochondria, was shown to be inhibited by a therapeutic concentration of chloramphenicol (CAP). This finding is in accordance with the ultrastructural damage caused to these organelles by the antibiotic, and with the inhibition of mitochondrial protein synthesis found in other kinds of mammalian cells.


Production of thymic cells by mouse spleen and bone marrow.

Karyotype difference between the AKR/TIALD strain (T1) that bears 2  metacentric markers, and the AKR strain (no marker) was used to follow the thymic repopulation of lethally irradiated (AKR X T1) F1 hybrids restored by AKR bone marrow (BM) or spleen cells. Eleven days following radiation exposure, 40-50% of the thymic cells were BM-derived in the mice restored with BM cells whereas spleen-derived cells remained below 10% in those restored with spleen cells. The thymic repopulation by spleen-derived elements was enhanced either by injecting a larger munber of spleen cells or by adding thymic cells to the spleen inoculum; however in both cases the appearance of the spleen-derived karyotypes still required a delay of about 11 days. The thymic cells could either recruit thymic precursor cells or trigger their multiplication. On the opposite, it has not been possible to demonstrate a favorable effect of the injected thymic cells on the repopulation of the thymus by BM-derived elements.


Plasma free fatty acids and cholesterol in propranolol-treated rabbits.

The effects of propranolol on circulating free fatty acids (FFA) and cholesterol were studied in rabbits. The first injection (5 mg/kg) of the beta-blocking agent strongly stimulates FFA mobilization. The effect lasts less than 3 hours in fasting animals and more than 6 hours in fed animals. Propranolol also causes a drop in plasma cholesterol in fed rabbits. After prolonged treatment (5 mg/day/10 days), basal FFA levels were higher than in controls while plasma cholesterol levels of fed animals were lower than in normal rabbits.


Sex chromatin in tumors of the female breast (the problem reviewed).

The authors looked for the percentage od sex chromatin in benign (73 cases) and malignant (131 cases) female mammary tumors. The sex chromatin was present in at least 30% of nuclei in benign tumors. On  the other hand, in malignant tumors, the sex chromatin was present in 4 to 30% of nuclei in 42 cases and over 30% of nuclei in 76 cases. We observed one or two sex chromatin in the same nuclei in 12 malignant tumors. No correlation was observed between the percentage of sex chromatin and other prognostic factors : TNM (tumor, nodes, and metastasis), histological grading, nodal involvement, hormonal status. The authors also observed the survival after radical treatment. Therefore, this may raise the question of the value of the test.


On some mathematical techniques for the analysis of visual spectral sensitivities.

Starting from known spectral properties of visual photopigments and photoreceptors, a mathematical construction of neural response functions to visual stimuli is obtained. Included in this is a somewhat general derivation of the univariance principle. Temporal dependence of response on stimulus is included in the formulation. Special attention is given to the case of flash stimuli and their resulting spectral sensitivities. This formulation is applied to certain physiological spectral sensitivity measurements that are at variance with known spectrophometric results. An analysis of the data in these cases suggests that they correspond to "pseudo-pigments" arising from the neural interaction of several photopigments. The method of analysis is constructive and identifies the gamma-max's of the interacting photopigments. These are found to be in good agreement with existing spectrophotometric measurements.


Measurement and modification of forces between lecithin bilayers.

We probe in two different ways the competing attractive and repulsive forces that create lamellar arrays of the phospholipid lecithin when in equilibrium with pure water. The first probe involves the addition of low molecular weight solutes, glucose and sucrose, to a system where the phospholipid is immersed in a large excess of water. Small solutes can enter the aqueous region between bilayers. Their effect is first to increase and then to decrease the separation between bilayers as sugar concentration increases. We interpret this waxing and waning of the lattice spacing in terms of the successive weakening and strengthening of the attractive van der Waals forces originally responsible for creation of a stable lattice. The second probe is an "osmotic stress method," in which very high molecular weight neutral polymer is added to the pure water phase but is unable to enter the multilayers. The polymer competes for water with the lamellar lattice, and thereby compresses it. From the resulting spacing (determined by X-ray diffraction) and the directly measured osmotic pressure, we find a force vs. distance curve for compressing the lattice (or, equivalently, the free energy of transfer to bulk water of water between bilayers. This method reveals a very strong, exponentially varying "hydration force" with a decay distance of about 2 A.


Cation concentrations in the hemolymph of Loligo pealei.

Hemolymph and protein-free hemolymph obtained from Loligo pealei were analyzed for cation concentration by the method of atomic absorption spectroscopy, for the following ions: Na+, K+, Ca++, and Mg++. No significant differences were seen in ion concentration between hemolymph and protein-free hemolymph. Of particular neurophysiological significance is that K+ ion concentration is much closer to that of seawater than previously reported.


Proton and deuteron relaxation of muscle water over wide ranges of resonance frequencies.

The spin-lattice relaxation time (T1) of water protons in mouse muscle was studied from 10(4) to 10(8) Hz at several temperatures, and the deuteron T1 of muscle water was studied from 2.0 X 10(3) to 1.54 X 10(7) Hz at several temperatures. Proton T1's of muscle and brain water with different D2O contents were measured at 25 degrees C and 35 MHz. From the results of variable frequency and temperature measurements and the data of isotope substitution, it is concluded that the major relaxation mechanism for the protons in muscle water is the intermolecular dipolar interaction between the protons of the macromolecules and the protons of the water molecules in the hydration layer. It is also suggested that the relaxation of deuterons can be accounted for a very small fraction of water molecules directly hydrogen-bonded to the macromolecules.


Human marrow cells capable of erythropoietic differentiation in vitro: definition of three erythroid colony responses.

A systematic study has been undertaken to analyze the spectrum of erythropoietic colonies obtained in cultures of human marrow cells plated in methyl cellulose. Colonies were identified as erythropoietic on the basis of the appearance in them of hemoglobin-containing erythroblasts. As found previously in mouse marrow cultures, three sequentially appearing types of colonies which differed in their ultimate cluster content could be readily distinguished. Small erythroid colonies containing 1-2 clusters reached a peak after 7-8 days; small bursts containing 3-8 clusters reached a peak after 10-12 days; and large bursts containing greater than 16 clusters reached a peak after 17-20 days. The previously reported enhancing effect of human leukocyte conditioned medium on burst formation seen in cultures of human nonadherent cells was found to be due largely to an effect on the formation of the largest, late appearing type of burst. By analogy with the mouse, the progenitors of such bursts would represent a primitive cell type which has a close relationship with pluripotent stem cells, as well as a second and independent close relationship to the progenitors of granulopoietic colonies.