The synergism of cardiotoxin and phospholipase A2 in hemolysis. The synergistic effect of exogenous cobra phospholipase A2 on the hemolysis brate of guinea pig erythrocytes by highly purified snake venom cardiotoxins was investigated. In the presence of phospholipase A2 the reaction was not only faster and had a lower activation energy but followed a sigmoidal instead of a linear time course. Similar results were obtained using porcine pancreatic phospholipase A2. Significantly, addition of even a trace of cobra phospholipase A2 (approx. 0.1%, w/w) was sufficient to bring about the full synergistic effect, emphasizing the stringent purity requirements for any meaningful investigation of cardiotoxin's own action. The possibility that the action of cardiotoxin on its own may involve the stimulation of an endogenous phospholipase is discussed in the light of the results obtained with exogenous cobra enzyme.


Kinetics of the absorption of amino acids by the rat intestine in vivo. The kinetics of L-phenylalanine and L-lysine absorption by the rat small intestine in vivo have been studied by perfusing intestinal segments and monitoring simultaneously the uptake of the substrate into the intestinal tissue and its disappearance from the perfusate. The rate of phenylalanine disappearance is a linear function of the substrate concentration. Its uptake into the tissue is rapid and obeys saturation kinetics, but is not concentrative. Both tissue uptake and disappearance rate can be inhibited by leucine or methionine, but are not influenced by hydrophilic neutral or dibasic amino acids. Lysine disappearance from the perfusate and its uptake into the tissue both display saturation kinetics. Lysine transport is quantitatively smaller than that of phenylalanine. Both uptake and disappearance are inhibited by arginine and leucine, but are unaffected by other neutral amino acids or sugars. To analyse the kinetic results, integrated equations were developed to express the final concentration in the perfusate in terms of the original concentration. The disappearance rate was considered as a mixed process (saturable and non-saturable in parallel) in a one-compartment system, and the uptake by the tissue was treated as a two-compartment system in which the amino acid entered the cells by a mixed process but left them by a pure non-saturable mechanism. The results concerning disappearance from the lumen are compatible with the one-compartment model. Phenylalanine absorption can be described by a major non-saturable component and a minor saturable one, while lysine absorption occurs almost entirely by a saturable process. The two-compartment model does not adequately describe the tissue uptake results.


The basic asymmetry of Na+-dependent glycine transport in Ehrlich cells. Influx and efflux of glycine have been examined as a function of external and internal Na+ concentrations, respectively, when : formula: (see text) = O. With : formula: (see text) = O it was found that at comparable external and cellular Na+ levels, the Km for efflux was larger by an order of magnitude than the value for influx and the V for efflux was several times greater than the V for influx. For both fluxes the major effect of Na+ was to decrease the Km value. The observations are consistent with the conclusion that the Na+-dependent transport system is asymmetric per se. Influx and efflux of glycine were increased in a near linear manner by increasing the Na+ concentration from 13 to 100 mM, the half-time for glycine equilibration being a funcion of the Na+ concentration in absence of an electrochemical potential difference for Na+. In Na+-free media ([Na+] less than 5 mM) equilibration of glycine between cells and medium was not achieved after 60 min at 25 degrees C. With : formula: (see text) = O, efflux (or uptake) of glycine was not affected by internal (or external) K+ between 20 and 120 mM suggesting that K+ plays no direct role in Na+-dependent transport of glycine in Ehrlich cells.


Effects of insulin on the uptake of D-galactose by isolated rat epididymal fat cells. In muscle, insulin stimulates uptake of D-galactose as well as D-glucose and certain other sugar isomers (Kono, T. and Colowick, S.P. (1961) Arch. Biochem. Biophys. 93, 514--519). In fat cells, the hormone also stimulates uptake of D-glucose and certain other monosaccharides. Nonetheless, the hormone does not increase the uptake, as determined by the utilization, of D-galactose by fat cells (Ball, E.G. and Cooper, O. (1960) J. Biol. Chem. 235, 584--588; Kuo, J.F. and Dill, I.K. (1969) Biochim. Biophys. Acta 177, 17--26). As pointed out by Ball and Cooper, this does not necessarily indicate that insulin has no effect on the membrane transport of D-galactose in fat cells. The possible effect of the hormone on transport may not be seen in the utilization data if the intracellular metabolism is considerably slower than the rate of transport and insensitive to insulin.


The susceptibility of cholesterol-depleted erythrocytes to saponin and sapogenin hemolysis. The assumption that complex formation between erythrocyte membrane cholesterol and saponins or sapogenins is the cause for their hemolytic activity, was tested by measuring the susceptibility of cholesterol-depleted erythrocytes towards these hemolysins. For some of the hemolysins cholesterol depletion caused inhibition of hemolysis, for others an augmentation. The results suggest that cholesterol does not serve as a specific binding site for these hemolysins.


Involvement of glycoconjugates in insulin-receptor interactions. Studies in liver plasma membranes of control and diabetic mice. The involvement of glycoconjugates in the insulin-receptor interactions in mouse liver is tested by digestions of membranes with various enzymes. Trypsin decreased the binding of [125I]insulin to liver membranes. After digestion with beta-galactosidase no ""high affinity'' receptor sites could be detected. The effects observed with plant lectins confirm the involvement of galactoconjugates in the insulin binding process. Sophora japonica and Ricinus communis lectins (with galactose specificity) and concanavalin A largely inhibit the binding process of insulin and those effects concern the ""high affinity'' receptor sites. Other lectins (wheat germ agglutinin, Dolichos) and enzymes (alpha-L-fucosidase, beta-N-acetyl-hexosaminidase and neuraminidase) are without effect on insulin binding. Comparative studies performed on diabetic mouse liver membrane (KK mice), previously characterized by decreased number of insulin receptors, are in good agreement with qualitatively similar receptor sites in both non-diabetic (control) and diabetic mice. Effects of enzymes and lectins yielded same results as compared to control membranes. Plasma membrane proteins and glycoproteins in both types of mouse are indistinguishable with respect to enzymic and chemical analysis. Sodium dodecyl sulphate acrylamide gel electrophoresis shows identical patterns. Moreover, the decrease in the number of insulin receptors is easily reversed with diet restriction. These data are consistent with the similarity of receptor sites in control and diabetic liver membrane.


Non-isothermal potential of phospholipid bilayer films. Influence of cholesterol and macrocyclic carrier effects. The effect of cholesterol on the ion selective behavior of phoshpolipid (phosphatidylcholine or phosphatidylethanolamine) bilayer films is studied through the measurement of the membrane non-isothermal potential. It is shown how the mixed phosphatidylcholine-cholesterol membrane can be either cation of anion permselective according to the film composition (cationic behavior is met in the 0--10% cholesterol composition range while anionic selectivity appears in the 20--50% range). On the contrary, mixed phosphatidylethanolamine-cholesterol membranes show the absence of ionic selectivity already met with pure phosphatidylethanolamine films. The presence of a cationic carrier as Dibenzo-18-crown-6 in the film transforms all the studied films (cationic, anionic and no selective bilayers) into ideally cationic selective membranes. These results are discussed on the basis of the current ideas on the charge distribution through the bilayer membranes. Moreover, the role of the permeating ions as potential determining species is stressed.


Cholesterol-lipid interactions in membranes. The saturation concentration of cholesterol in bilayers of various lipids. 1. The integration of cholesterol in a lipid bilayer can be visualized by changes in the fluorescence properties of the probe N-phenyl-1-naphthylamine (NPN). An increasing cholesterol content in the lipid phase corresponds to decreasing fluorescence intensity of NPN and a short wave shift of the emission spectrum. 2. Equilibrium constants for the partition of NPN between water and the various lipid phases are reported. An increasing cholesterol content in a bilayer decreases the solubility of NPN in the bilayer. 3. The saturation concentration of cholesterol in bilayers of various lipids prepared by ultrasonication is determined using the flourescence probe NPN. The maximal molar ratio of cholesterol : lipid is 2 : 1 for sphingomyelin or egg phosphatidylcholine and 1 : 1 for cerebroside, dipalmitoyl phosphatidylcholine, or dipalmitoyl phosphatidylethanolamine. 4. The comparison of the maximal molar ratio of cholesterol : lipid with the number of proton donor and proton acceptor sites in the lipid moiety is used for a discussion of the polar interactions of cholesterol within a lipid bilayer.


Turbidity changes of lipid vesicles near the phase transition temperature as an indication of fusion. Sonicated liposomes of dipalmitoyl phosphatidylcholine show sharp turbidity changes on heating at two distinct temperatures. A decrease in turbidity at the lower temperature (approx. 37degrees C) is thought to be associated with the phase transition of small vesicles and a decrease at about 44 degrees C with larger vesicles or multilayer. An increase of turbidity between 38 and 43 degrees C is attributed to the fusion of small vesicles. The turbidity changes were studied under various modes of vesicle preparation to confirm the interpretation of the turbidity data. Alternate interpretations are discussed.


Local perturbation of the double helical DNA structure near single breaks. The influences of DNA composition and solvent modification on the stability of the native DNA structure near a single break have been studied. The value of the magnification parameter h of the double helix breakdown probability in a haplotomic degradation process is used as an index of the perturbation range. Methodological and technical details are discussed and comments are made on previously reported data on the h value. From this study it may be concluded that the width of the perturbation zone next to a nick is influenced by solvent-induced changes in the general stability of the native DNA molecule, and by compositional-dependent local interactions. Our results point to entropic effects as the more important contributions to stability perturbation near a discontinuity in the native DNA molecule.


Analysis of plant genomes. III. Denaturation and reassociation properties of cryptic satellite DNAs in barley (Hordeum vulgare) and wheat (Triticum aestivum). A cryptic satellite fraction was isolated from barley and wheat by preparatory ultracentrifugation of total DNA in Ag+-Cs2SO4 density gradients and was characterized by studying its denaturation-reassociation properties. Wheat satellite DNA underwent thermal denaturation as a single component with a Tm of 81 degrees C while barley satellite DNA consisted of one major (Tm = 82.5 degrees C) and one minor (Tm = 91 degrees C) component. When the barley and wheat satellites were reassociated and then melted, the Tm values were found to be 6--7 degrees C lower than those of the corresponding native DNA preparations. Examination of the C0t curves of these two satellite DNAs revealed the presence of a major, fast reassociating and a minor, slow reassociating fraction. The fast reassociating DNA fraction of barley was found to have a complexity of 9.7 . 10(5) daltons while that of wheat satellite was 5.8 . 10(5) daltons. Since these satellites reassociated with about 4--5% base mismatching, as judged by their deltsTm (6--7 degrees C), they each appear to consist of rather similar base sequences.


Endonuclease activities from a permanently established mouse cell line that act upon DNA damaged by ultraviolet light, acid and osmium tetroxide. The activity of damage-dependent endonuclease in mouse plasmacytoma cells (line MPC-11) has been studied using damaged phi X 174 RFI DNA as substrate. The DNA was treated with ultraviolet light, acid, or osmium tetroxide to introduce different types of lesions. Ultraviolet light-damaged DNA was cleaved at approx. 1.1 sites per 35 thymine-containing dimers by the extract, which indicates no specificity towards this type of lesion. The acid-treated DNA, which contains apurinic sites, was enzymatically broken in every alkalilabile site and this strongly suggests the presence of an apurinic-specific endonuclease activity in the nuclear extract. The activity which acts on ultraviolet-irradiated DNA and that which acts on acid-treated DNA have different specificities as shown by their salt requirements and the extent to which they are stimulated by magnesium. While the ultraviolet-endonuclease activity was very little affected by reducing the KCl concentration, the apurinic-specific activity was almost completely abolished. Osmium tetroxide renders the DNA an excellent substrate for endonucleolytic activity in the mouse cell extract. The response to KCl and MgCl2 of the osmium tetroxide-specific endonuclease activity is qualitatively similar to that of the endonuclease activity, which acts on ultraviolet-irradiated DNA. Treatment of DNA with osmium tetroxide is known to produce 5,6-dihydroxydihydrothymine which is a minor photoproduct in DNA after irradiation, suggesting that the ultraviolet-specific endonuclease activity acts upon this lesion.


An exonuclease activity associated with DNA polymerase I of Micrococcus radiodurans. An exonuclease activity is associated with one of three DNA polymerase in Micrococcus radiodurans. The nuclease activity co-sedimented with its DNA polymerase I of this bacterium on glycerol gradient centrifugation. Both activities show the same optimum pH and heat-inactivation kinetics. This nuclease hydrolyzes preferentially double-stranded DNA in an exonucleolytic manner from both ends of the duplex DNA. The products of hydrolysis are mostly deoxyribonucleoside 5'-monophosphate and no nucleosides are released into the acid-soluble fraction. Di- or other oligonucleotides are also produced but their relative amounts are constant during the time of incubation. The exonuclease activity requires Mg2+ and is inhibited by high concentrations of KCl as is DNA polymerase I of M. radiodurans.


DNA-dependent RNA polymerases from Acanthamoeba castellanii. Multiple forms of the class III enzyme and levels of activity of the polymerase classes during encystment. Multiple forms of class III DNA-dependent RNA polymerase have been found in a number of higher eukaryotic cells types (Chambon, P. (1975) Annu. Rev. Biochem. 44, 613--638). Similar multiple forms are reported here from a lower eukaryote, the soil amoeba Acanthamoeba castellanii. The levels of activity of all three RNA polymerase classes in whole cell extracts of Acanthamoeba during cellular differentiation were examined. In contrast to our previous observation (Detke, S. and Paule, M.R. (1975) Biochim. Biophys. Acta 383, 67--77) that the amount of the alpha-amanitin-sensitive polymerases (II + III) increase in the nucleus during encystment we find that the amount of polymerases II and III when solubilized from whole cells remains constant. The level of activity of polymerase I, however, remains constant both in nuclei and in whole cell extracts. Thus, the shutdown of ribosomal RNA synthesis occurring during encystment is not due to a decrease in the number of polymerase I molecules in the cell.


Effect of L-ethionine on macromolecular synthesis in mitogen-stimulated lymphocytes. 2--4 mM L-ethionine completely inhibits DNA synthesis in phytohaemagglutinin- or concanavalin A-stimulated lymphocytes even though it does not prevent the morphological changes characteristic of blast formation. Evidence is presented which indicates that complete commitment to DNA synthesis as well as a substantial increase in the rates of RNA and protein synthesis can occur in the presence of ethionine. Ethionine, however, does inhibit methylation of tRNA and prevents mitogen-induced increase in the activity of histone-modifying enzymes. All of these effects of exposure to ethionine are completely reversible. Removal of ethionine after 24 h or more of exposure results in a rapid, synchronous wave of DNA synthesis, an increase in the rate of methylation of RNA and an increase in activity of histone-modifying enzymes.


S-adenosylhomocysteine analogues as inhibitors of specific tRNA methylation. Of 17 base- or amino acid-modified analogues of S-adenosylhomocysteine, six were found to produce at least 50% inhibition of the activity of an unfractionated tRNA methyltransferase extract at concentrations of 200 micron. The inhibitory effects of these six analogues on five purified rat liver tRNA methyltransferases were examined. The purified enzymes differed greatly in their sensitivity to the analogues. Ki values for the inhibitory analogues were determined for the three most highly purified methyltransferases. The kinetic analyses indicated that inhibition is competitive for nearly all enzyme/inhibitor combinations. The Ki values for good enzyme/inhibitor pairs were in the range of 0.11--2 micron. Each analogue appears to inhibit one methylation more strongly than others; e.g. the Ki values obtained for N6-methyl-S-adenosyl-L-homocysteine are approx. 0.4 micron for guanine-1 tRNA methyltransferase, 6 micron for adenine-1 tRNA methyltransferase and 100 micron for N2-guanine tRNA methyltransferase I. Structural features which are important for inhibitory activity are presence of a terminal amino group on the amino acid and the presence of adenosine rather than any other base. Ring nitrogens, a terminal carboxyl group and conformation at the asymmetric carbon appear to be important for some but not all of the tRNA methyltransferases examined.


Replication of the genes coding for 5 S RNA in synchronized HeLa cells. The duplication of the genes coding for 5-S RNA has been followed during the S phase of HeLa cell cultures synchronized by mitotic detechment. Replication was analyzed by exposing the cultures to bromodeoxyuridine (BrdUrd) for 3 h at successive intervals and analyzing the DNA product in CsCl density gradients. DNA containing the 5-S genes was detected in the gradients by molecular hybridization using purified 125I-labeled 5 S RNA. In addition, as an internal marker of S phase chronology, late DNA replication was followed by examining the incorporation of [3H]thymidine into repetitive DNA (C0t less than 0.01 fraction). The results indicated that greater than 85% of the cells in the culture were synchronously dividing and that greater than 80% of the 5-S genes replicate in the first half of S phase, whereas the rapidly reassociating fraction of the DNA replicates in the second half of S phase.


Estrogen withdrawal in chick oviduct. Characterization of RNA synthesized in isolated nuclei using a mercurated precursor. 1. As a prerequisite for analyzing the effect of estrogen on transcription in chick oviduct, we describe suitable methods for the synthesis (under conditions restricting reinitiation), and isolation of RNA transcripts from oviduct nuclei in vitro, utilizing mercurated UTP (Hg-UTP) as an RNA precursor and chromatography on sulphydryl-Sepharose (SH-Sepharose) to recover mercurated RNA (Hg-RNA). The techniques described include treatment of Hg-RNA with p-hydroxymercuribenzoate, to improve the efficiency of binding to SH-Sepharose, and elution of Hg-RNA from SH-Sepharose after treatment with 60% formamide at 90 degrees C, to eliminate contamination by aggregated nucleic acid. 2. RNA synthesized by endogenous form B RNA polymerase (using either UTP or Hg-UTP as precursor) was recovered in nuclear lysates in the form of 30--85-S heterogeneous RNA . protein complexes, and after removal of protein, was 10--12 S in size. 3. The nature of RNA transcripts synthesized in vitro was examined by hybridization. More than 90% of the RNA was complementary to "unique" DNA sequences, and 50--60% of the hybridized RNA could be competed with homologous, steady-state nuclear RNA, indicating a significant degree with homologous, steady-state nuclear RNA, indicating a significant degree of homology between in vitro transcripts and in vivo RNA. The level of homology was similar whether RNA synthesis was performed in low salt, or in high salt in the presence of heparin. Possible reasons for only partial competition in these experiments are discussed. 4. Withdrawal of estrogen from chicks leads to a 50% reduction in endogenous RNA polymerase activities in nuclei within 48 h. Similar levels of competition with Hg-RNA transcripts for "unique" DNA were obtained using oviduct nuclear RNAs isolated before or after estrogen withdrawal, and even with liver nuclear RNA. Thus, in oviduct, those sequences present in primary transcripts, and analyzed under our experimental conditions, are present in different hormonal states and also in other chick tissues.


Conformation of pyridazine nucleosides: the molecular structure of 4-hydroxy-1-(beta-D-ribofuranosyl)pyridazin-6-one (3-deaza-6-azauridine). The glycosyl torsiona angle in 4-hydroxy-1-(beta-D-ribofuranosyl)-pyridazin-6-one (or 3-deaza-6-azauridine) is in the "high-anti" region. This is similar to the torsional angles observed for 6-azapyrimidine and 8-azapurine nucleosides, but in marked contrast to those found in uridine, 3-deazauridine and other pyrimidine nucleosides.


Influence of a few coat protein subunits on the base-paired structure of the RNA species of alfalfa mosaic virus. Differentiated thermal melting profiles were made of all the three RNA species (RNAs 1, 2 and 3), constituting the genome of alfalfa mosaic virus and of the subgenomic coat protein messenger RNA (RNA 4) of this virus. Whereas all profiles showed multiphasicity the profile of RNA 4 was most clearly subdivided showing three clear transitions with tm values of 25.5, 35.5 and 53 degrees C. The first transition disappeared upon addition of 6 coat protein molecules per molecule of RNA 4. The effect of coat protein on the melting profiles of the genome RNAs was much less clear.


Studies on sheep kidney nuclease. II. Limited digestion of phiX174 single-strand DNA. Single strand DNA of bacteriophage phiX174 was digested by sheep kidney nuclease (nuclease SK) at 37 degrees C for 2-6 h and the digests were examined by 2.5% polyacrylamide gel electrophoresis. On this electrophoresis five bands were detected corresponding to the DNA fragments by scanning the gel at 260 nm. After these DNA fragments from the gel with the electrode buffer used for the electrophoresis, the molecular weights of fragments I, II, III, IV and V were determined by sucrose density gradient centrifugation. The molecular weights of these DNA fragments were 1.7 . 10(6), 1.2 . 10(6), 8.6 . 10(5), 4.5 . 10(5) and 2.8 . 10(5). From the melting curves and the base compositions of these fragments it was suggested that phiX174 single-strand DNA might have intramolecular double helical regions, which were hardly susceptible to attack by nuclease SK.


Age-dependent DNA labeling and deoxyribonucleotide synthesis in wheat seeds. Utilisation of ribonucleosides as precursors of DNA biosynthesis was studied in germinating wheat embryos because the reductive pathway leading to deoxyribonucleotides is very difficult to demonstrate in extracts of higher plants in vitro. [5-3H]Cytidine and [6-3H]uridine are incorporated into wheat DNA (RNA-free) via ribonucleotide reduction without intermediate scission of the glycosidic bond. This reaction is observed at 20-30 h after the onset of germination only in aged (2-4-year-old) seeds while the embryos isolated from fresh grains show very little cytidine incorporation; in contrast, thymidine incorporation into DNA between 10 and 18 h of germination is not age dependent. Fresh wheat contains a soluble, heat-stable inhibitor fraction, most probably a modified oligonucleotide, which efficiently prevents cytidine incorporation when added to old embryos together with the labeled nucleoside. This material also inhibits purified Escherichia coli ribonucleotide reductase and is thought to be part of the control system for ribonucleotide reduction in wheat; it may gradually decay during storage of the seeds. Dry wheat embryos do not contain deoxyribonucleoside triphosphates. Pool sizes of dATP and dTTP in germinating embryos were found to reach 1 pmol/microgram DNA at 10-15 h of germination (i.e. before ribonucleotide reduction) and were independent of the age of seeds. These data suggest that wheat contains other preformed dexoyribonucleoside derivatives which are phosphorylated at an early time and can initially sustain DNA synthesis. Induction of measurable ribonucleotide reductase activity in fresh winter wheat was for the first time accomplished by 15 days of vernalization of the seeds at +2 degrees C.


Characteristics of fatty acid oxidation in rat liver homogenates and the inhibitory effect of malonyl-CoA. Experiments were carried out to study the control of fatty acid oxidation and ketogenesis in rat liver homogenates. In contrast to findings with the perfused liver, rates of fatty acid oxidation were high and equal in liver homogenates from fed and fasted animals. No difference in apparent Km values for oleate, ATP, coenzyme A or carnitine could be detected in the two types of homogenate. Over the concentration range 20--40  micron, malonyl-CoA inhibited oleate oxidation by 50--75%. The fact that the inhibitory effect could be removed by pre-treatment with alkali or fatty acid synthetase indicated that the inhibitory molecule was malonyl-CoA rather than a contaminant. The effect was readily reversible and appeared to be competitive with oleyl-CoA. Malonyl-CoA also inhibited oleate oxidation in homogenates of heart and kidney cortex but this is unlikely to have physiological relevance since, in contrast to liver, neither tissue contains an active cytosolic pathway for the generation of malonyl-CoA and the synthesis of fatty acids.


Glycolipids of a halotolerant, moderately halophilic bacterium. A 247 000 x g particulate fraction from a moderately halophilic halotolerant bacterium incorporated [14C] glucose added as UDP[14C]glucose and 32P-labeled phosphatidylglycerol into glucosylphosphatidylglycerol. Exogenously added phosphatidylglycerol was available to the enzyme only when dispersed in a detergent, preferably Triton X-405, by sonication. The 14C- or 32P-labeled glucosylphosphatidylglycerol was degraded with phospholipase C. The water soluble product formed was isolated and identified by paper chromatography as glucosylglycerolphosphate. The system required Mg2+ or Ca2+ for activity. KCl and NaCl were inhibitory even when added at low concentrations.


The lipid composition of isolated rat spermatids and spermatocytes. The lipids composition of enriched fractions of spermatids and spermatocytes, isolated from rat testicular tissue, has been investigated. More than 20% of the total fatty acids of spermatids but only 10% of those of spermatocytes, isolated from testes of mature rats, was 4,7,10,13,16-docosapentaenoic acid. Spermatocyte-enriched fractions isolated from testes of immature rats had fatty acid compositions similar to those isolated from testes of mature rats. On the other hand, spermatids isolated from immature rats had a level of docosapentaenoic acid which was intermediate between the level found in spermatocytes and that of spermatids from mature rats. Major phospholipid classes and the triacylglycerols of spermatids contained much more of the docosapentaenoic acid than the corresponding lipid types from spermatocytes. Differences in content of total phospholipids, individual classes of phospholipids and triacylglycerols among spermatocytes, spermatids and late spermatids were also observed.


The effect of nutritional state on the lipoprotein lipase activity of isolated fat cells. The lipoprotein lipase activity of rat epididymal adipose tissue falls on starvation and increases on refeeding. Studies with fat cells isolated from this tissue have shown that increases in the activity of the enzyme occur under appropriate incubation conditions in vitro. The present study compares the responses of cells isolated from the adipose tissue of fed and 48-h starved rats. Fat cells from rats starved for 48 h display a lower initial lipoprotein lipase activity than cells from fed rats. When cells from rats in both nutritional states are incubated in a suitable medium at 25 degrees C, there is a progressive increase in the medium lipoprotein lipase activity. The absolute increase in the total activity of the incubation system during incubations of cells from 48-h starved rats is significantly less than during incubations of cells from fed rats. However, when expressed as a percentage of the initial cell activity, the rises in total activity are similar in the two nutritional states. Cycloheximide has no significant effect on the increase in activity of lipoprotein lipase that occurs with cells from 48-h starved rats. However, it does partially block the increase in activity seen with cells from fed rats and in a manner similar to that previously reported for cells from 24-h starved rats. The significance of the results is discussed in relation to previous studies with both intact adipose tissue and isolated fat cells.


Structures and fatty acid compositions of neutral glycosphingolipids of human plasma. Major neutral glycosphingolipids were isolated from human plasma and their structures and fatty acid compositions studied. The four neutral glycosphingolipids of plasma were characterized as Glc beta(1 leads to 1)ceramide, Gal beta(1 leads to 1)- ceramide, Gal beta(1 leads to 4) Glc beta (1 leads to 1)ceramide, Gal alpha(1 leads to 4) Gal beta(1 leads to 4) Glc beta(1 leads to 1)ceramide and GalNAc beta(1 leads to 3) Gal (1 leads to 4) Gal (1 leads to 4) Glc beta(1 leads to 1)-ceramide. The glycosphingolipids contained mostly short chain fatty acids of which most prominent was C16. Erythrocyte glucosylceramide and lactosylceramide exhibited similar fatty acid compositions as their plasma counterparts. Triglycosylceramide and globoside of erythrocytes contained almost exclusively long-chain fatty acids. In lactosylceramide obtained from "p" erythrocytes, an accumulation of long-chain fatty acids was found; this accumulation was not observed, however, in lactosylceramide isolated from "p" plasma. It was concluded that plasma and erythrocyte glycosphingolipids are synthesized at separate sites where short- and long-chain fatty acids, respectively, are available. Plasma and erythrocyte glucosylceramide, and probably a fraction of lactosylceramide, exchange between plasma and erythrocyte pools. The latter conclusion is discussed in the light of the relative roles of carbohydrate and lipid moieties of the glycosphingolipids in maintaining their association with erythrocyte membranes.


Animal carotenoids 15. Carotenoid distribution and carotenoprotein of Asterias rubens. A clear differentiation in localization according to functional groups in the carotenoids of the starfish, Asterias rubens, is reported. Only the free alpha-ketols, 7,8,7',8'-tetradehydroastaxanthin, 7,8-didehydroastaxanthin and astaxanthin, are present in the purified carotenoprotein. A post mortem liberated slime contained beta,beta-carotene, free and esterified alloxanthin and esterified alpha-ketols. Evidence suggesting the existence of an alloxanthin protein complex was obtained. The carotenoprotein, asteriarubin, accounts for approx. 10% of the protein extracted by low salt dialysis from the purple-blue part of the top skin of A. rubens and exhibits an absorbance maximum at 554 nm in buffer solution. Asteriarubin is a glycoprotein with an equivalent Stoke's radius corresponding to that of globular proteins of molecular weight 8--10 10(4) and contains 20 microgram carotenoid per mg asteriarubin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified asteriarubin disclosed two major components, one of which is a glycopeptide.


The metabolism of 4-androstene-3,17-dione by isolated rat hepatocytes. Maintenance of the sex differences in culture. Hepatocytes from adult rats were isolated and cultivated as primary monolayers for 3 days in a medium containing only 1% homologous serum. The metabolism of 4-androstene-3,17-dione was followed in hepatocytes from male, female and hypophysectomized animals. It was found that immediately after preparation, 5alpha-reductase and 16alpha-hydroxylase activities were present in the cells in amounts comparable to those found in microsomal preparations from liver homogenates. After 3 days in culture, these enzyme activities had decreased to about half the values measured on day 0. During this time the sexual differences in steroid metabolism in the cells were stable, i.e. there was no detectable induction of 16alpha-hydroxylase in cells from female animals, and the relative sex difference in 5alpha-reductase activity persisted.


Intralysosomal hydrolysis of cholesterol esters of varying fatty acid composition in cultured human skin fibroblasts. Intralysosomal accumulation of low density lipoprotein-cholesterol ester was achieved during incubation of cultured human skin fibroblasts in the presence of chloroquine and the subsequent hydrolysis of the cholesterol ester was studied after removal of chloroquine. The fall in the ratio of cholesterol linoleate to cholesterol oleate during recovery from chloroquine inhibition suggested preferential hydrolysis of cholesterol linoleate. To verify this assumption, low density lipoprotein (labeled with [3H] cholesterol linoleate, oleate, palmitate or stearate) was sequestered in the lysosomes as described above. The rate of hydrolysis of the different cholesterol esters was determined 24 and 48 h after removal of chloroquine from the medium and was found to be similar for the four cholesterol esters studied. These findings indicate that enrichment in cholesterol oleate in atheromatous lesions does not result from preferential hydrolysis of intralysosomal cholesterol linoleate, but rather could be due to preferential utilization of oleic acid for the esterification in the cytoplasm of free cholesterol released from the lysosomal compartment.


Enzymatic sulphation of bile salts in human liver. An enzyme catalyzing the transfer of the sulphate group from 3' -phosphoadenosine-5' -phosphosulphate to lithocholate and glycolithocholate is identified in the cytosol of human liver. The rate of sulphation was greatest with unconjugates lithocolate. Km values for lithocholate and glycolithocholate were 2 . 10(-6) and 3.3 . 10(-6) M, respectively. No enzyme activity was found in human kidney cytosol. A simple method for quantitative assay of the enzyme in percutaneous liver biopsy specimens is described.


Long-chain acyl-CoA synthetase and "outer" carnitine long-chain acyltransferase activities of intact brown adipose tissue mitochondria. 1. The activities of long-chain acyl-CoA synthetase (acid: CoA ligase (AMP-forming), EC 6.2.1.3) and the "outer" carnitine long-chain acyltransferase (palmitoyl-CoA: L-carnitine O-palmitoyltransferase, EC 2.3.1.21) have been estimated in intact brown adipose tissue mitochondria. The assay of both enzymes is based on a coupled reaction in which the intramitochondrial (matrix) CoASH is the final acyl acceptor and the oxidation-reduction state of the flavoproteins in the acyl-CoA dehydrogens pathway is used to determine the intramitochondrial level of acyl-CoA. 2. Using endogenous fatty acids as the substrate, the progress curve of acyl-CoA synthetase activity was in most mitochondrial preparations linear within the first 30 s. When initial rates were measured, the Km value for CoASH (2.4 micron) was lower than previously determined for the acyl-CoA synthetase in brown adipose tissue mitochondria as well as in mitochondria of other tissues. The pH activity curve indicates that the unprotonated form of the fatty acids represents the substrate of acyl-CoA synthetase, i.e. similar to the effect of pH on the binding of fatty acids to bovine serum albumin. 3. Experimental evidence is presented that at temperatures higher than the transition temperature of the acyl-CoA synthetase (i.e. Tt = 19 degrees C), this enzymic reaction is rate-limiting in the sequence of coupled reactions leading to beta-oxidation in the mitochondrial matrix. 4. The initial rate of the long-chain acyl-COA synthetase reaction was estimated to v = 119 +/- 16 nmol . min-1 . mg-1 protein (mean +/- S.D., n = 5) at an optimal concentration of palmitate which exceeds that of rat heart mitochondria by a factor of 10.


Enzymes catalyzing the hydrolysis of long-chain monoacyglycerols in rat adipose tissue. Acetone-ether preparations of epididymal fat pads from fasted or fed rats contained two enzymes catalyzing the hydrolysis of long-chain monoacylglycerols. The enzymes were identified as monoacylglycerol lipase (Tornqvist, H. and Belfrage, P., (1976) J. Biol Chem. 251, 813--819) and lipoprotein lipase by their apparent pI values after electrofocusing in non-ionic detergent, selective inhibition properties, substrate specificity and positional specificity. It was estimated that monoacylglycerol lipase accounted for about 90% of the total monoacylglycerol-hydrolyzing activity in acetone-ether preparations from fasted and 70% from fed rats. Its enzyme activity did not change with the nutritional state in contrast to that of lipoprotein lipase. The latter enzyme hydrolyzed 2-monoacylglycerols at a much lower rate than the 1(3)-isomers. Monoacylglycerol lipase was located almost entirely in the adipocytes, thus most of the enzyme activity towards monoacylglycerols in the adipose tissue was found in this site. Fractionated sucrose homogenates of rat epididymal fat pads also contained a third enzyme with monoacylglycerol-hydrolyzing activity, identified as hormone-sensitive lipase by its pI, selective inhibition properties and substrate specificity. It was estimated that hormone-sensitive lipase accounted for less than 20% of the total activity against monoacylglycerols in these tissue preparations from fasted rats. Over-all quantitative estimations emphasized the dominant role of monoacylglycerol lipase over the other two enzymes in the hydrolysis of monoacylglycerols.


[Effect of molecular structures on the probabilities of radiating and non-radiating transitions in molecules derived from chlorophyll]. The values of luminescence quantum yield and duration have been measured for rhodoporphyrin XV, deoxophyllerythrin, phyllerythrin, etiochlorin, chlorin e6 and their zinc complexes as well as for bacteriochlorin and isobacteriochlorin. The effect of the structural elements, which are characteristic of the chlorophyll molecule, on the probabilities (rate constants) of primary photophysical processes has been traced. It has been shwon, in particular, that all these elements enhance the radiationless de-excitation of the lowest triplet state.


[Molecular oxygen quenching of the singlet and triplet states of poryphyrins]. Rate constants of molecular oxygen quenching in solutions of singlet and triplet states of chlorophyll porphyrines molecules and their complexes with metals were measured with the help of the methods of laser photolysis, impulse fluorometry and luminescence. It has been shown that the quenching of fluorescence results from the intensification of intercombinational transition into the triplet state. The mechanism of quenching of the triplet state is discussed.


[Quaternary structure of histidine decarboxylase according to small-angle x-ray diffraction and electron microscopic findings]. The data on small angle X-ray scattering with histidine decarboxilase (HDC) from Micrococcus sp. n. were analysed and a line of succesively improving approximations of the molecule shape was found: by oblate ellipsoid a:b:c = 1:10.63, by continuous cylinder and hollow cylinder with H = 50 A, 2R = 76 A, 2r = 8A. Biochemical data and electron micrographs of HDC obtained made possible to distinguish subunits and thus to increase resolution of the model. The model of the enzyme molecule consisting of three subunits is suggested, whose X-ray small angle scattering curve well agrees with the experimental one up to value S = 0.21 A-1.


[Effect of physiologic concentrations of local anesthetics of the procaine series on the structural and functional state of cerebral synaptosome membranes]. Studies are presented of the effect of procaine group anesthetics on rat brain synaptosome stability to dodecyl sulfate and on catalytic properties of the membrane bound alkaline phosphatase. The dose curves of detergent stability are characterized by two maxima, one at 3.10(-4) M for tetracaine, 2.10(-6) M for lidocaine and 5.10(-5) M for procaine; the other being at 10(-3) M for all anesthetics. The curves of Vmax and KM versus procaine concentration to exhibit the minimum at 5.10(-7) M and maximum at 3,2.10(-6) M. Procaine at 5.10(-7) M increases enthalpy and enthropy of membraneous alkaline phosphatase. It is suggested that interactions between anesthetics and centers of high affinity lead to synaptosome structural rearrangements, which affect the properties of membraneous enzymes.


[2 types of soluble complexes of bovine serum albumin with polyelectrolytes]. It has been shown by high-velocity sedimentation that BSA forms soluble complexes with polyacrylic acid (PAA) and sodium polystyrene sulfonate (PSSNa) with neutral water solutions. Factors responsible for polyanion binding of similarly charged BSA molecules are analysed. It has been found that occupation of PAA and PSSNa chains with BSA globules proceeds steadily. BSA-PAA and BSA-PSSNa complexes are compared with the earlier described products of BSA sorption on poly-4-vinyl-N-ethylpyridine bromide, where the occupation of polycations with protein globules proceeds unevenly. Reasons for the existence of two types of soluble BSA-polyelectrolyte complexes are described; probable structural model of BSA complex with polyacids and polyanions is suggested.


[Use of statistical analysis in studying the conformational oscillations of chloroplasts]. Conformational oscillations of chloroplasts have been studied by the statistic analysis technique. Statistic characteristics of the parameters describing the process of chloroplast cross-section oscillations in time are given. According to the statistic analysis results it can be suggested that conformational oscillations of chloroplasts have the multilevel structure. The suggestions concerning causative factors of that multilevel organization of oscillating process are also advanced.


[Effect of exposure to "stress" on the electrical properties of BLM]. The influence of high potential and hydrostatic pressure gradients on BLM has been studied. It has been found that short exposures of BLM to the above factors, which are nearly as strong as those rupturing BLM, often induce in the membrane unusual states characterized by drastic increase in the current fluctuations with simultaneous increase of its average value. The level of fluctuations and the average current on individual membranes subjected to intermittent exposures vary appreciably. Of greatest interest are the membranes for which the average current value is not much higher than the background level. Such membranes can fluctuate steadily for several hours without any appreciable change in their behavior. In these cases the minimum current value during fluctuations often corresponds to the background current. Usually the current fluctuations on membranes excited by potential or hydrostatic pressure gradients cease abruptly. The electrical properties of such membranes are virtually the same as those of the unstressed membranes. The qualitative studies of the influence of the solution composition and temperature showed that in the range 25--40 degrees C in different solutions the excitation effects of BLM from general lipids of brain and a lecithin-cholesterol mixture are qualitatively similar and resemble those due to some biologically active substances diminishing the mechanical stability of BLM. Some possible mechanisms of excitation of BLM exposed to stress factors are discussed.


[Nonlinear resistance in a diffuse trigger system. Biological barrier]. Mathematical model of antagonistic interrelations of biological species in which the migration is taken into account is considered. The ecological nish, occupied by one of the antagonistic species cannot be populated by the other if the initial quantity of the new species does not exceed the particular value. This is considered as the demonstration of the ecological barrier. The approach to the investigation of the nonlinear stability for spatially distributed systems can be applied to other biophysical problems.


[Role of electrical interference between adjacent portions of the myocardium in the origin of arrhythmias]. Excitation propagation was studied in the papillary muscle of cat's heart and on a mathematical model of homogenous fibre, considerable inhomogeneity being created in the duration of the action potential (DAP) in the left and right part of the object studied. Gradual changes in DAP were observed during excitation propagation period, therefore rough differences in DAP of neighbouring myocardial areas may be excluded. No arrhythmia was observed. It is shwon that the arrhythmia which occurred previously [7] with an increase of DAP in one part of the preparation after EDTA addition was not connected with DAP differences in the neighbouring myocardial areas.


[Impulse circulation in an excitable medium. Critical size of the closed circuit]. Periodical regimes of impulse circulation are studied for the system of two differential equations Exixi-vE-F(E)-g=0, vg=epsilon(phi(E)-g),(epsilon less than 1), which describes stationary propagation of the impulse with the velocity upsilon. Analytical expressions are obtained for the minimal size of the closed circuit (lambdamin) and minimal circulation velocity (upsilonmin). It has been found that when the circuit size was close to lambdamin, the impulse became relaxed; it means that the plateau lenght was close to the front length. It was shown that lambdamin was practically independent of the small parameter epsilon. In case of the approximation of f(E) by the piecewise N-shaped function with an incident region --Kf(E--alpha), and phi(E) by the linear function KgE, the increase of lambdamin takes place with an increase of alpha and Kg parameters, and a decrease of Kf (fig. 4). Such a change in the parameters brings about a decrease (instead of an intuitively expected increase!) of the stationary velocity of normal impulse propagation.


[Theoretical analysis of an experiment with motor neuron voltage clamping. Proof of the N-shape of the steady-state volt-ampere characteristic of the dendrite membrane]. Current--voltage relation (I--V) in motoneurons [6] is discussed on the basis of the hypothesis [1] about N-shaped steady-state I--V for the dendritic membrane. A spontaneous discharge of motorneurons after clamp-off is interpreted as direct evidence in favour of the hypothesis. Some indirect evidence and possible ways for further testing of the hypothesis are also presented.


[Water content in thin films of nucleic acid nitrogen bases. Ultramicroquantitative determination by means of a quartz resonator]. A method of quartz resonator is proposed for accurate micro- and ultramicroquantitative analysis of water in various biological systems. Sensitivity of this method may be about 10(-9) g. Some advantages and specific applications of quartz resonator are shown by "weighing" water rigidly bound to homogenous dry films of cytosine at great changes of their mass. It is suggested to use the quartz resonator with a specific sorbent applied to it as an efficient detector of small quantities of gases and microadmixtures of solved substances.


[Relationship between ion channel properties and the structure of the lactone ring of polyene antibiotic molecules]. Properties of ionic channels created by amphoterecin B, nystatine and mycoheptin in phospholipid--cholesterol bilayer have been compared. Ionic conductivity and selectivity of channels as well as the frequency of transitions between an open state and a closed one depend on lacton ring structure. Appearance of one more carbonyl group in mycoheptin molecule leads to a decrease of channel anion permeability. Any pair of these antibiotics being added into different aqueous solutions create combined channels. These data confirm hypothesis that polyene antibiotics create channels of two half-pores formed in different monolayers of the membrane.


[Chlorophyll phosphorescence in leaves and cells of algae]. Phosphorescence and delayed fluorescence of chlorophyll have been observed in frozen suspensions of green, blue-green and red alga, normal and mutant leaves of pea, kidney bean and maize at the temperature range from --196 to --70 degrees C. The phosphorescence maximum lies at 960--980 nm, excitation maximum--at 674+/-2 nm, lifetime is 1.5--2.5 msec, quantum yield vaires in the range of 0.03--3 x 10(-6), being much lower than that of chlorophyll phosphorescence in solutions. The weakest phosphorescence was found in normal leaves, the strongest one--in cells of Scendesmus and leaves of xi-carotene mutants of maize. The data suggest that phosphorescence belongs to antenna chlorophyll and phosphorescence quenching is due to energy migration from singlet and triplet states of antenna to non-phosphorescent forms of chlorophyll and carotenoids.


[Thermal structural transitions in the membranes of fat cells]. Studies have been carried out of the structural state of plasma membranes of epididimal fat cells from rats at 5--40 degrees C. The temperature dependence of the fluorescence intensity bound ANS and the rate of solubilization by 0.2% sodium dodecyl sulfate have shown sharp changes at 19--24 degrees C. At the same temperature intervals there was a break in the Arrhenius curve for activity of the membrane 5'-nucleotidase. ANS fluorescence and light scattering of liposomes from membrane lipids have revealed discontinuities at 21 degrees and 29--30 degrees C. The data are interpreted as thermal rearrangements initiated in the lipid membrane phase.


Circular dichroism investigation of the (L lysine)n-Cu(II) (1:1) systems with n = 4 and n = 25. The study of the (L Lys)n-Cu(II) (1:1) system with n = 4 and n = 25 using circular dichroism (CD) data has provided evidence indicating the formation of two complexes in a two-step process. In the first of these complexes, obtained at pH 6.6, the alpha-amino terminal group and the adjacent deprotonated amide nitrogen are bound to the metal. In the second, additional amino nitrogens of side chains lie at the other two corners of the coordination square. A comprehensive investigation of changes occuring in spectral patterns when coordination takes place enables the assignment of three bands that are characteristic to each type of nitrogen coordination.


[Precise relationships for calculating the binding of regulatory proteins and other lattice ligands in double-stranded polynucleotides]. The binding of long multisite ligands to double-stranded nucleic acids is considered. The ligand is taken as a lattice of AT- and GC-specific reaction centres the sequence of which is complementary to the base pair sequence in the specific ligand interaction site on DNA. Exact equations are derived for the two cases: that when a ligand binds in a fixed orientation relative to the DNA and that when it can be attached to DNA in the two alternative orientations related by two-fold rotation. Included in the formulation are two alternative orientations related by two-fold rotation. Included in the formulation are the ligand size effects as well as cooperative effects for which the interactions between the nearest neighbour adsorbed molecules are responsible. In particular, if cooperative interactions are allowed only between the adjacent ligand molecules related by two-fold rotation symmetry, the ligands would tend to associate into symmetrical "dimers" upon binding to DNA. This model enables one to explain the recognition of specific base sequences which are related by two-fold rotation symmetry and are complementary to the sequence of ligand reaction centres. Numerical calculations are carried out for several specific cases and for various values of parameters involved in the theoretical model.


Similarity in metabolic patterns of different chemical species of vanadium in the rat. To gain information about the influence of the oxidation state of vanadium on its metabolic behavior, different 48V-labeled vanadium compounds, such as cationic VO2+(V), VO2+(IV), V3+(III), and anionic V4O12(3-)(V), VS4(3-)(V) species were prepared and intravenously injected into rats. The 48V radioactivity was measured in whole tissues as well as in nuclei, mitochondria, lysosomes, microsomes, and cytosols from liver and kidney homogenates. The distribution of 48V radioactivity between the plasma components was investigated using gel filtration of the 48V-labeled plasma. The findings indicate that there are common pathways of the different chemical forms of vanadium in animals. The similarities are referred to the distribution in different tissues and their intracellular distribution as well as to the transport in the blood, in which 48V was always found in the plasma bound to transferrin. The results obtained tend to exclude a possible influence of the oxidation state of vanadium on its metabolism and support the existence in the body of two mechanisms of conversion of different chemical forms of vanadium ions to one with the same valence.


[B-form as the absolute energy minimum for DNA. Entropy nature of the B--A transition]. The B-form is characterizing by lower potential energy than A-form, especially if electrostatic term is added. For realistic values of epsilon (4; 1) spontaneous A--B-transition occurs during minimization procedure. The conclusion arises about entropy mechanism of A-form stabilization. The B--A-transition is impossible lower certain temperature for certain salt concentrations. The strong and mobile dehydratant must stimulate the A-form realization. The strong and polymeric dehydrant must stimulate the compactization of macromolecule on the basis of B-form.


Osmium-labeled polynucleotides: reaction of osmium tetraoxide, with poly-1,N6-ethenoadenylic acid. Osmium tetraoxide, in the presence of ligands such as pyridine and bipyridine, adds across the etheno bridge of 1,N6-etheno-9-methyladenine and poly-1,N6-ethenoadenylic acid. The Os:P ratio in the labeled polynucleotide was approximately equal to 1 when bipyridine was used as the stabilizing ligand. A similar study with polycytidylic acid, which had been partially modified with chloroacetaldehyde so that some bases were converted to 3,N4-ethenocytosine, gave an OS:P ratio of approximately equal to 1.3. Calf-thymus DNA, in which the adenine and cytosine bases were modified by chloroacetaldehyde, gave an Os:P ratio of approximately equal to 1 after 24 h. These results suggest that 3,N4-ethenocytosine will add two Os labels.


[Evolution of the gene in energy space]. General idea of a gene as a part of the hereditary molecule DNA permits to correlate various alleles states of a gene to various energy states of the hereditary molecule. Hereditary molecule under the action of a random force moves along its energy states. So far as the number of individuals in a population usually is very big, such motion of a gene in energy space resembles random motion of the Brownian particle in liquid. Here the process of striving hereditary molecule for minimum potential energy will correspond to the slow precipitation of particles under the effect of the force of gravity. Therefore, gene diffusion in energy space is described by the Focker--Plank's equation. The solution of this equation shows the time evolution of the distribution of genes alleles states within the limits of an isolated population.


Interaction of antibiotic lasalocid A (X537A) with praseodymium(III) in methanol. The binding of lasalocid A (X537A) to Pr(III) in methanol has been studied by lasalocid fluorescence, circular dichroism, and 1H and 13C NMR spectroscopy. It is clear that in addition to a mono complex, bis and tris complexes are also formed. Values of the binding constants and spectral properties of the various complexes have been determined by computer fitting of the binding isotherms. The Pr (III) ion binds only at the salicylic "head" of the lasalocid A, in stark contrast with other known structures. The lasalocid A appears to have an "open" conformation in these complexes. The relevance of these results to the structure and conformation of the Ca(II) complex is discussed. The first order rate constant for the dissociation of the tris complex has also been determined.


Ionization and divalent cation dissociation constants of nalidixic and oxolinic acids. The ionization constants and some divalent cation dissociation constants of nalidixic and oxolinic acids, both specific inhibitors of bacterial DNA replication, have been determined. The carboxylic pKa' values are 6.1 and 6.9 at 25 degrees for nalidixic and oxolinic acids, respectively. These values indicate that intramolecular hydrogen-bonding stabilizes the un-ionized form of these compounds in aqueous solution. Both compounds bind divalent cations; the divalent cation dissociation constants for oxolinic acid are somewhat smaller that those for nalidixic acid. We suggest that both compounds may act by forming a complex in situ with a divalent cation in a metalloprotein involved in DNA replication. The evidence that both drugs inhibit at the same target site is briefly reviewed.


Binding sites between platinum (II) and uracil derivatives. The complexes of triammineplatinum with uracil, 6-methyluracil, or uridine were prepared in aqueous solution at pH 7. The reaction of uracil with triammineplatinum gave two complexes at the same time. One was a complex in which triammineplatinum displaced a proton from uracil and coordinated to the N(3) position, and the other the complex coordinated to the N(1) position by displacing a proton. When triammineplatinum was treated with 6-methyluracil or uridine in aqueous solution at pH7, only a complex coordinated to the N(3) position was obtained. The ultraviolet (UV), NMR, and infrared (IR) spectral data provide useful information for determining the binding site of these complexes. The UV and IR spectral behaviors of the complex coordinated to the N(3) of uracil are very similar to those of 3-methyluracil. The NMR spectrum of the complex coordinated to the N(1) of uracil exhibits satellite peaks of 195Pt-proton and its coupling constant (39 Hz) gives good evidence for determining the binding site at the N(1) position.


Selenium protection against mercury toxicity: high binding affinity of methylmercury by selenium-containing ligands in comparison with sulfur-containing ligands. In determining the protection of selenium against mercury toxicity, the binding affinity of methylmercury by various selenium-containing ligands was investigated by proton magnetic resonance (PMR) spectroscopy. The most striking feature was the small J199Hg-1H value of the selenocysteamine- and selenocysteine-methylmercury complexes, namely, the high affinity of the selenohydryl group to the mercury in comparison with those of the sulfhydryl and amino groups. The order of binding affinity of the coordination groups toward methylmercury is clearly SeH greater than SH greater than or equal to Se-Se greater than NH2 greater than S-S, SeCH3, SCH3. A definite correlation was found to exist between the mercury-proton coupling constants and the chemical shifts of methyl groups of the methylmercury complexes. A relationship between the order (Se greater than S greater than NH2) of affinity for methylmercury and the basicity (or electronegativity and covalent radius) of the donor groups was also discussed. These results suggest the high covalency of the CH3Hg-Se bond, which involves dpi-dpi back bonding.


Induction by alpha-mercapto-beta-arylacrylic acids of low-molecular-weight zinc-binding protein in rat liver. Intraperitoneal injections of alpha-mercapto-beta-(2-furyl)acrylic acid (MFA) induced incorporation of zinc into a low-molecular-weight metalloprotein fraction in rat-liver cytosol. Cytosol of control rats contained little or none of this material. alpha-Mercapto-beta-(2-thienyl)acrylic acid induced less incorporation of zinc in this fraction than did MFA. Twenty-two hours after administration of 65ZnSO4 (50 mumol/kg) and MFA (200 mumol/kg), serum concentrations of both total and radioisotopic zinc were markedly elevated, compared to serum concentrations in rats administered only 65ZnSO4. Further, in livers of MFA-treated rats, radioisotopic zinc concentration was significantly greater in all cytosol zinc-containing proteins resolved by gel chromatography, but total zinc was elevated only in the metallothionein fraction and not in proteins of higher molecular weight. This appears to be the first example of induction of a metallothionein-like protein by a procedure not involving food restriction or administration of metal salts.


Arsenic and cancer: effects of joint administration of arsenite and selenite on the genesis of mammary adenocarcinoma in inbred female C3H/St mice. The joint administration of 2 ppm of arsenic as arsenite and of 2 ppm of selenium as selenite in the drinking water of inbred female C3H/St mice increases the incidence of spontaneous mammary adenocarcinoma significantly over the simultaneous controls and animals receiving 2 ppm As as arsenite or 2 ppm Se as selenite alone, indicating that arsenic abolishes the anticarcinogenic effect of selenium. Arsenite caused a significant increase of the tumor growth rates and raised the incidence of multiple tumors. The tumor growth rates in the As-Se group were also increased, but the incidence of multiple tumors was lower than in the 2 ppm As group.


Design of iron (III) chelates in oral treatment of anemia: solution properties and absorption of iron (III) acetohydroxamate in anemic rats. The design and use of iron(III) chelates in the oral treatment of iron-deficiency anemia is discussed in terms of the following criteria: (1) the ability of a chelate to exist as a monomeric species even at physiological pH values, (2) the rate of transfer of iron from the chelate to the iron-transporting protein-apotransferrin, (3) the rate of depolymerization of ferric citrate polymer by the free ligand, and (4) its nontoxicity and ability to regenerate hemoglobin levels in anemic rats. Detailed species distribution studies, stability constants, and kinetic data for iron(III) acetohydroxamate (criteria 1-3 above) show that it remains monomeric at physiological pH values and undergoes very rapid iron transfer with apotransferrin. Detailed animal studies show a significant increase in regeneration of hemoglobin in rats fed 2 ml of 4 mM Fe(III) acetohydroxamate daily when compared to rats fed similarly with Fe(III) citrate. A strong potential is thus indicated for Fe(III) acetohydroxamate as a source of iron in the anemic animal.


On the interaction of histone Hl and Hl peptides with DNA. Sedimentation, thermal denaturation and solubility studies. The interactions in buffered 0.14 M NaCl (pH 7.0) of DNA, native or sonicated, with histone Hl and with its fragments N-Hl (residues 1-72) and C-Hl (residues 73-COOH), have been studied by using the techniques of sedimentation, thermal denaturation and solubility. Histone Hl shows a preferential affinity for high molecular weight DNA in comparison with sonicated DNA. The binding of histone Hl to sonicated DNA in 0.14 M NaCl is reversible and the reversibility decreases with time. These findings are consistent with the view that Hl is cooperatively distributed along DNA molecules and that this distribution is energetically more favoured for long DNA than for sonicated DNA. It has been found that under conditions of moderate salt concentration, the C-terminal region is the main one responsible for the interaction of Hl with DNA, and also for a cooperative distribution of the histone along the DNA molecules. Addition of urea to the solution produces a decrease of solubility of Hl-DNA complexes. This effect reflects an additional condensation of the complex, which is probably related to the unfolding of the globular part of the histone. It is suggested that this globular region does not play a substantial role in the condensation of Hl-DNA complexes. A model which takes into account the presence of free lysyl and phosphate groups within the complex is discussed.


Comparative structural analysis of myosin after limited tryptic hydrolysis by use of two-dimensional gel electrophoresis. Polypeptides obtained by limited tryptic digestion of several myosins have been analyzed by a two-dimensional electrophoresis technique. The different maps thus obtained exhibit some common and distinct features specific of the myosins studied. Myosins from rabbit, fast and slow muscle and cardiac tissue, as well as beef cardiac myosin, have been compared. The polypeptides obtained vary in molecular weight from 120 000 60 15 000. The light chains LC1 and LC2, have disappeared. A peptide which comigrates with fast type LC3 is however found. The fast myosin hydrolyzate is very different from that obtained by the hydrolysis of slow and cardiac myosin. Numerous peptides are common to cardiac and slow myosins. However a few peptides are specific of the two myosin types. In the hydrolyzate from fetal calf myosin, some of the typical slow, fast and cardiac peptides can be found. However several apparently unique fetal peptides are also present. The comparison of the fetal calf tissue myosin hydrolyzate and that of [35S] methionine labeled myosin from myotubes in cultures shows qualitatively a very great homology. Thus the protein synthesized by cultured cells seems to be very similar to or the same as that of the embryonic tissue.


A new method of preparation and some properties of 3-hydroxyproline. The hydrolyzate of Delonix Regia seed extract is fractionated sequentially on Dowex 50 X 8 resin and on QAE Sephadex A 25. Purification is completed by recrystallisation from ethyl alcohol. 3-hydroxyproline is destroyed by NO2H and by chloramine T, which prevents from using most of the colorimetric reactions in use for 4-hydroxyproline. For its characterization, 3-hydroxyproline may be clearly separated from 4-hydroxyproline by TLC chromatography and by high voltage paper electrophoresis.


Conformation of snake toxic polypeptides studied by a method of prediction and circular dichroism. Short and long neurotoxins as well as cardiotoxins belong to three distinct families of homologous toxic polypeptides extracted from cobra venoms. A study of their conformation was undertaken by using the method of Chou and Fasman for prediction of secondary structures of proteins. To improve the reliability of this method, an averaging scheme was developed. The data obtained showed that all toxins have a predominant trend for beta-sheet nucleation. Moreover, predicted beta-sheet strands fitted well those actually observed from X-ray data. Thus, it seems that all toxins share similarities in their secondary structure. This proposition was supported by a comparative study of the CD spectra of a set of toxins. Nevertheless, the present data suggest also that each type of toxins possesses localized structural individualities which might be responsible for the biological and/or immunological specificities.


[Ephedrine, salsoline and cytisine derivatives as substrates and inhibitirs of cholinesterases]. 25 iodomethylates of acetic, propionic, butyric, isobutyric and valeric esters of N-(beta-hydroxyethyl)-derivatives of ephedrine (I) pseudo-ephedrine (II), salsoline (III), salsolidine (IV) and cytisine (V) are studied as substrates and inhibitors of acetylcholine esterase (EC 3.1.1.8) from human erythrocytes and butyrylcholine esterase (EC 3.1.1.8) from horse serum. Butyrylcholine esterase found to increase the hydrolysis rate of all the alkaloid esters studied with the increase of acyl radical either to valerates (for ephedrine and pseudo-ephedrine derivatives), or to butyrates (for the rest alkaloids) and then it did not considerably change under further elongation of carbon chain up to valerate. Isobutyrates were observed to be similar to propionates in their hydrolysis rates. Acetylcholine esterase hydrolyzed acetates with the highest rate, while butyrates of ephedrine and pseudoephedrine derivatives were hydrolyzed by the enzyme 2,5-3-fold as slow as acetates. The rate of choline esterase hydrolysis decreased in the row: ephedrine--salsoline--cytisine with the volumetric increase of the cationic group. The decrease was almost 10-fold for butyrylcholine esterase, while a transition from "poor" substrates to reversible inhibitors was observed for acetylcholine esterase (3 of 5 cytisine esters were reversible inhibitors of the enzyme). The data obtained are compared with literary data on other cyclic choline esterase substrates; they are discussed from the viewpoint of unproductive binding hypothesis and on the basis of the structure of active centres of acetyl- and butyrylcholine esterases.


[Changes in activities of DNA-dependent RNA-polymerases from rat liver under influence of glycerol, dimethyl sulfoxide and polyethylene glycol-400]. Effects of glycerol, dimethyl sulfoxide (DMSO) and polyethylene glycol (PEG) 400 on transcription of isolated nuclei and DNA in rat liver under the action of homologous RNA-polymerases I and II were studied. Addition of these organic compounds to the reaction mixture before initiation altered RNA synthesis on native DNA matrix. Addition of glycerol or DMSO to the synthesising system 1 and 5 minutes after initiation of RNA synthesis had no effect on the rate of transcription. PEG-400 inhibited incorporation of 3H-UTP into newly-synthesized independently of the time of its introduction to the reaction mixture. The data obtained suggest that the compounds under study may impair the formation of initiation complex of RNA polymerase with DNA and alter RNA synthesis in isolated nuclei of rat liver.


[Properties of 2 fibrin monomer forms which differ in the degree of thrombin activation. Relation of these 2 forms to specific polymerization inhibitors]. The inhibitory effect of fibrinogen and its fragment D on the clotting of two fibrin monomer species has been studied. One of them (f0) lacks peptides A and B, the other (fB) preserves peptides B. The inhibitors retard the clotting of f0 but fail to influence fB polymerization. This means that the peptide B removal and appearance of the active site B in the central (E) domain of the fibrin molecule is a prerequisite for the inhibitory effect of fibrinogen or fragment D. The specificity of this effect suggests that fragment D and the periferal D-domains of fibrinogen possess a special site (B') which reacts selectively with the fibrin active site B to block polymerization. The present investigation has demonstrated the importance of the H-bond system formation for B-B' sites interaction.


[Preparation and characteristics of carboxymethylated lysozyme derivatives]. Hen egg-white lysozyme (EC 3.2.1.17) was carboxymethylated with iodoacetic acid and the individual products of various degree of modification were isolated by column chromatography on Amberlite CG-50. Peptide mapping of the products obtained reveals that His-15, Lys-1, -33, -96 (or -97) residues are blocked; the Lys-13 and -116 residues are unmodified. Salt activation of the carboxymethylated derivatives is facilitated with the increase of the number of modified groups; this fact is consistent with the increased lytic activity and affinity to substrate of the derivatives at lowered ionic strength.


[Determination of a primary structure of abnormal D-like hemoglobin isolated from a blood donor]. Abnormal hemoglobin with contents 40% was isolated from the blood of a donor. It was purified, its chains were separated, and the defect was found to locate at beta-chain. Abnormal betaT13 peptide was found under the separation of tryptic hydrolysate. Data on its amino acid composition, electrophoretic mobility as well as attempts to determine its N-terminal amino acid and N-terminal sequence made possible to identify the location and the character of the amino acid substitution in the abnormal hemoglobin. The first case of the presence of hemoglobin D Penjab alpha2beta2(121Glu leads to Gln) among the Russian population is described.


[Analysis of the interaction of glucocorticoid receptors obtained from rat liver with DNA in a aqueous dextran-polyethylene glycol biphasic system]. The binding of the receptor-glucocorticoid complexes from rat liver cells to DNA has been studied in an aqueous dextran-polyethylene glycol biphasic system. It has been shown that the capacity of DNA for binding hormone-receptor complexes is very large, and that no saturation is observed at physiological concentrations of the glucocorticoid receptor. The linear binding curve is indicative of low affinity of the DNA acceptor sites for the complexes. Cell cytosol contains substances which inhibit the binding of the receptor-dexamethasone complexes to homologous DNA.


[Effect of detergents on the main respiratory chain and cyanide-resistant pathway of electron tranfer of Candida lipolytica yeast mitochondria]. Effects of detergents--triton X-100 and deoxycholate--on the main respiratory chain and cyanide-resistant electron transfer pathway in the mitochondria of Candida lipolytica yeast were studied. Triton X-100 and deoxycholate were shown to completely inhibit the activity of alternative oxidase at concentrations of 0.7 and 1.2 mM, respectively. At these concentrations the detergents did not inhibit the activity of the main respiratory chain. The inhibiting effect of triton X-100 was exerted both in the presence of Mg2+ and without them, whereas the inhibiting action of deoxycholate was observed in the presence of Mg2+. The addition of asolektine in the presence of Mg2+ prevented the inhibiting effect of detergents. These data lead us to the conclusion that inhibition of electron transfer through the alternative pathway is caused by separation of alternative oxidase from the main respiratory chain with the disturbance of structural integrity of inner mitochondrial membrane under the action of detergents. Inhibition of electron transfer in the alternative pathway does not decrease the rate of oxygen consumption by mitochondria. Therefore, the main respiratory chain in the presence of detergents can provide for transfer of all reducing equivalents which were earlier transferred both by the respiratory chain itself and by alternative oxidase.


[Post-transcriptional association of RNP particles with chromatin in Zajdela ascities hepatoma cells]. The interrelationship between nuclear RNP particles and chromatin in Zajdela hepatoma cells double labelled with 3H-thymidine and 14C-uridine was investigated by three independent methods (Nucleoprotein-Celite chromatography, sucrose density and CsCl band centrifugations). All nuclear RNP particles were found to be associated with the chromatin. Some of chromatin-associated RNA molecules are polyadenylated, thus indicating the post-transcriptional character of this association. DNA and RNA molecules in these complexes are bound through protein rather than being connected directly. The site of contact between RNP particle and chromatin is relatively resistant to the action of DNase I and pancreatic RNase. The experiments with exogeneous labelled RNP particles added to isolated nuclei do not reveal the formation of artificial RNP-chromatin complexes. The results obtained are discussed in the light of current views on the nucleus-to-cytoplasm transport of RNA molecules.


[Cleavage of bacteriophage PM2 DNA by S1 nuclease]. The kinetics of cleavage of superhelical PM2 DNA by the single strand-specific S1 nuclease is studied at various salt concentrations (0.01--1 MNaCl) by electrophoresis in neutral and alkaline agarose gels. Cleavage of different DNA forms (superhelical DNA I, relaxed circular DNA II and linear DNA III) is described by the kinetic equations of the first order, and respective rate constants k1, k2, and k3 are determined at all salt concentrations used. It is shown that a high salt concentration (not lower than 0.2 MNaCl) is necessary for high specificity of S1 nuclease action on DNA regions with impaired base pairing. Examination of the S1 nuclease preparation action on superhelical PM2 DNA may be used as a convenient and reliable assay of its specificity.


[Kinetics of (C14) thymidine metabolism in hepatomas and tissues from normal and tumor-bearing animals]. The incorporation of 14C-thymidine into DNA and the acid-soluble fraction of mouse hepatomas 22A and 48 and into DNA of rat Zajdela hepatoma as well as into corresponding fractions of liver, spleen and thymus of normal organisms and tumor-bearing animals, was studied. Ion exchange chromatography on DEAE-cellulose disks was used to study distribution of the 14C-thymidine label between nucleosides, nucleotides, and nucleoside di- and triphosphates of the cell pool. The distribution of the label as early as 5 to 10 minutes after administration of 14C-thymidine  was found to be specific for each tissue studied and remains unchanged for at least 1 hour. Specific radioactivity (per mg DNA) of the acid-soluble fraction in 22A hepatoma is declined from the original high level very rapidly from the first minutes following administration of labelled thymidine with a corresponding increase in the specific radioactivity of DNA. The character of the both curves depends on the growth rate of hepatomas studied. Systemic effect of the highly malignant hepatomas manifests itself in their successful competition with the host's tissues for the vital precursor--thymidine. This phenomenon entails a drastic suppression of the incorporation of thymidine into the immunocompetent host's organs--spleen of mice and thymus of rats.


[Nature of changes in the fluorescence process of P700-enriched chloroplast fragments]. Spectral and photochemical properties of P700-enriched chloroplast fragments, obtained by ether treatment of liophylized digitonin fragments, were studied. It was shown that time course of fluorescence changes of isolated fragments (in contrast to digitonin fragments) at 20 degrees does not correspond to time course of absorption changes at 700 nm. Differences in low temperature fluorescence spectra of fragments, initially distinguished by redox states of photosystem 1 reaction centers were found. However, the fragments under study were incapable of light-induced changes of fluorescence yield at--196 degrees, independently of spectral region of measured fluorescence (lambda greater than 660 nm or lambda greater than 710 nm), though these fragments reveal phototransformation of P700. Thus changes in the low temperature fluorescence spectra cannot be accounted for by redox changes of P700. The fragments, isolated by ether treatment at 20 degrees as well at--196 degrees do not reveal light-induced fluorescence changes caused by redox changes of P700. The fluorescence changes observed may be due to accessory photoprocesses of chlorophyllprotein complex.


[Distribution of 5-methylcytosine in pyrimidine oligonucleotides of higher plant DNA]. The level of pyrimidine clusters (isopliths) and distribution of 5-methylcytosine in DNAs of some archegonial (ferns, ginkgo) and flowering (cordyline, tulip, wheat) plants has been studied. DNA from Cordyline australis (Liliaceae) is one of the GC-type (GC = 57.5 mol.%), has very low methylation level (amount of m5C is about 1 mol.%) and significantly differs from other species DNAs studied in terms of pyrimidine distribution patterns, i.e. amount of dipyrimidine fragments is higher than that of monopyrimidine ones. All other plant DNAs under study are of the AT-type, have similar pyrimidine distribution patterns and are characterized by high degree of pyrimidine blocking; specific differences in the level (approximately 10-fold) and type of genome methylation are found. In all DNAs studied 5-methylcytosine is unevenly distributed in pyrimidine isopliths: the degree of cytosine methylation [m5C(C+m5C)] is decreased with an increase in the length of isopliths, irrespective of the ratio of thymine and cytosine residues. 5-methylcytosine is mainly accumulated in mono- and dipyrimidine fragments (60--68%). Specific differences in 5-methylcytosine content in trinucleotides and long-chain pyrimidine oligonucleotides are revealed.


Umbilical vein-artery differences of plasma amino acids in the last trimester of human pregnancy. The plasma levels of 20 free amino acids in the umbilical veins and umbilical arteries of 8 premature (29--36 weeks gestation) and 16 mature (38--42 weeks gestation) newborn infants were measured at delivery. In premature newborn infants, most of the 20 amino acids were significantly higher in the umbilical vein than in the umbilical artery. Only glutamic acid was significantly lower in the umbilical vein than in the umbilical artery. In mature newborn infants, 7 (Ala, Lys, Leu, Val, Ile, Phe and His) of the 20 plasma amino acids were significantly higher and 4 (Glu, Gly, Ser and Orn) were significantly lower in the umbilical vein than in the umbilical artery. These results indicate that the relative contribution of individual amino acids to the placental supply of nitrogen to the human fetus discernibly changes with increasing fetal age during the last trimester of gestation.


Balance of net base in the rat. III. Effects of oral sodium bicarbonate and sodium citrate loading. The balance of net base in groups of male weanling Wistar rats exposed to sustained oral loads of net base were studied. In response to dietary loads of sodium bicarbonate (approx. 50 mmol.kg-1.day-1) an average increment in the mean rate of retention of net base of 21.8 mmol.kg-1.day-1 was observed in the absence of significant changes in the blood 'base excess'. Following withdrawal of sodium bicarbonate, mean rates of gastrointestinal absorption, renal excretion, and retention of net base promptly returned to control values, accumulated net base being retained in the body, presumably as skeletal sodium carbonate. In response to equimolar loads of net base in the form of trisodium citrate, a similar, albeit less pronounced, rise in the rate of retention of net base occurred.


Perinatal methadone exposure in rats. Effects on body and organ development. The effects of maternal methadone (5 mg/kg methadone-HCl) administration during gestation and/or lactation on gross body measurements, absolute organ weights, dry weights, and tissue water content were studied in 21-day-old Sprague-Dawley rats. Offsprings perinatally exposed to methadone had significantly lower body weights, crown to rump lengths, tail lengths and head diameters. In contrast to age-control values, absolute organ weights of the adrenals, brain, heart, kidneys, liver, lungs, muscle, and testes were reduced in all drug-treated groups; the spleen, thymus, and ovaries of some methadone groups were also lighter in weight. Organ:body weight ratios revealed fewer significant differences than recorded for absolute weights. Dry weights and tissue water content of the kidneys, liver and muscle were affected in all drug-treated animals; other organs were markedly affected by at least one schedule of methadone administration. These results suggest that perinatal methadone exposure has detrimental effects on preweaning organ and body development that may be of long-term functional consequence to the animal.


Relationship between fatty acids and lipid peroxidation in lungs of neonates. Triglycerides from the lungs of neonatal rats and mice were found to contain large amounts of the polyunsaturated fatty acids, arachidonate (20:4) and docosahexaeonoate (22:6). These fatty acids were diminished or absent in the triglycerides from the lungs of adult rats and mice and both neonatal and adult guinea pigs. No age-related changes were observed in the fatty acid composition of lung phospholipids in any of these species. The presence of arachidonic acid and docosahexaenoic acid in lung triglycerides correlated with the ability of these lungs to peroxidize lipids in vitro in all species. Depletion of lung triglycerides in neonatal rats by fasting abolished this lipid peroxidizing activity.


Comparative toxicity of caffeine and aminophylline (theophylline ethylenediamine) in young and adult rats. The toxicity of aminophylline and caffeine was studied in adult and 2-day-old rats following a single subcutaneous injection of the respective drug. Following the injection of high doses of either methylxanthine, adult rats developed convulsions, tremors, lethargy and licking of lips. In adult rats, the LD50 of caffeine and aminophylline was the same after 24 h and after 1 week of observation: caffeine 265 mg/kg, and aminophylline 202 mg/kg (theophylline base 172 mg/kg). In young rats, the LD50 was greater when the observation was carried out for 1 week than at 24 h after the injection; at 24 h: caffeine 220 mg/kg, and aminophylline 169 mg/kg (theophylline base 144 mg/kg); at 1 week: caffeine 155 mg/kg, and aminophylline 140 mg/kg (theophylline base 119 mg/kg). Young rats failed to gain weight at a normal rate after administration of either methylxanthine. The greater toxicity of both methylxanthines in newborn animals may be at least partly due to the extremely slow elimination of theophylline and caffeine in the neonate.


Possible involvement of growth hormone in the pathogenesis of early neonatal hypocalcemia in infants of diabetic mothers. The concentrations of plasma calcium and plasma growth hormone were determined up to 30 h of age in 10 infants of diabetic mothers (IDM) and 9 healthy infants. Plasma calcitonin was determined in all IDM and 5 control infants. The results are discussed in relation to the pathogenesis of early neonatal hypocalcemia.


Proteolytic activity of maternal serum on newborn IgD-positive lymphocytes. Study with protease inhibitors. We studied the effect of maternal serum on the surface IgD of newborn lymphocytes in the presence of protease inhibitors (epsilon-aminocaproic acid and Trasylol). Contact with maternal serum caused a significant reduction (p less than 0.000001) in the percentage of neonatal IgD-positive cells. The addition of either epsilon-aminocaproic acid or Trasylol significantly reduced this action of maternal serum (p l-ess than 0.00001 and p less than 0.0001), without difference between the effects of the two inhibitors (p greater than 0.05). We suggest, therefore, that activity of maternal serum is due, at least in part, to proteolytic enzymes, such as plasmin. The relative role of plasminogen and alpha2-macroglobulin in the incapacity of the newborn serum to reduce the number of IgD-positive lymphocytes is discussed.


Cardiac myopathies in neonatal lambs: histological and histochemical studies. White muscle disease (WMD), a selenium-responsive cardiac myopathy in neonatal lambs has been described from southern Iran. 15 lambs in terminal condition were referred to our lab. All the lambs were observed with the affected hearts. The lambs examined were between the postnatal ages of 1 week and 3 months. Gross changes included chalky-white appearance of entire endocardium of right ventricle and subendocardial plaques in the interventricular septum and left ventricular wall. Histologically the affected myofibrils showed swelling, acidophilia, fragmentation, segmental and patchy myonecrosis, round muscle cell nuclei, loss of sarcoplasm and sarcolemmal collapse. Purkinje fibers were relatively unaffected. Histochemical studies including Von Kossa and other stains indicated marked deposition of calcium salts in the cytoplasm of the damaged myofibrils. Calcium salts appeared as uniform, fine granules in relation to individual fiber striations. It appears that rapid accumulation of calcium into the damaged cells possibly interferes with the mitochondrial activity. As mitochondria bind excessive calcium, their capacity to respire and phosphorylate is depressed thus causing myocardial necrosis. The cardiac myopathy noted in our investigation is probably due to selenium deficiency in the soil of southern Iran. When selenium was given, it resulted in the fast recovery of the affected neonatal lambs. Moreover, no further cases of this disease (WMD) were reported after such treatments.


The supply of citrate to the sheep fetus. In situ placental perfusion has been used to study the role of the sheep placenta in fetal citrate metabolism. The perfusate citrate concentrations rose steadily to values higher than in fetal plasma. This rise was not influenced by intravenous administration of citrate to the ewe. Concentration and electropotential gradients indicate that transplacental passage is unlikely and the rise is probably due to placental synthesis. However, the quantity of citrate supplied to the fetus was calculated to be small compared with the fetal metabolic rate. Comparison with the guinea pig shows close similarity in fetal plasma concentrations and the amount of citrate formed per unit weight of fetus.


Cardiovascular responses to autonomic blockade in brain-injured fetal lambs. The cardiovascular responses to autonomic blockade were investigated in 15 late-gestation fetal lambs in utero. 4 animals had normal brains and 11 had hydranencephalic-like cerebral injury secondary to bilateral carotid artery obstruction (BCO). Following atropine administration, heart rate increased only 0.7 +/- 0.7% in the BCO lambs compared to 25.7 +/- 5.2% in the controls. alpha-Blockade with phentolamine resulted in a small decrease in systolic (2.2 +/- 0.8%) and diastolic (1.8 +/- 1.4%) blood pressure, and beta-blockade with propranolol produced a small decrease in heart rate (2.5 +/- 0.8%) as compared to control fetuses. Post mortem examination revealed extensive destruction of the cerebrum but normal hypothalamus and brain stem. These data suggest that supramedullary modification of autonomic cardiovascular control involves the cerebrum.


Prediction of the conformation of the cow and sheep kappa-caseins. The secondary structures of cow and sheep kappa-caseins were established according to the predictive rules of Chou and Fasman. The diagrams derived from this treatment allowed us to study the chymosin sensitive bond (milk-clotting process), as well as the glycosylation and phosphorylation sites, found to be situated in beta-turns. Despite a high variability between the primary structures of the COOH-terminal part (caseinoglycopeptide) of cow, sheep, and also other caseins, the secondary structures of the biologically important sites were found to be conserved.


Formation of "solvent-free" black lipid bilayer membranes from glyceryl monooleate dispersed in squalene. A simple technique for forming "black" lipid bilayer membranes containing negligible amounts of alkyl solvent is described. The membranes are formed by the method of Mueller et al (Circulation. 1962. 26:1167.) from glyceryl monooleate (GMO) dispersed in squalene. The squalene forms an annulus to satisfy the boundary conditions of the planar bilayer but does not appear to dissolve noticeably in the bilayer itself. The specific geometric capacitance (Cg) of the membranes at 20 degrees C formed by this technique is 0.7771 +/- 0.0048 muF/cm2. Theoretical estimates of Cg for solvent-free bilayers range from 0.75 to 0.81 muF/cm2. Alkane-free GMO bilayers formed from n-octadecane by the solvent freeze-out method of White (Biochim. Biophys. Acta. 1974. 356:8) have values of Cg = 0.7903 +/- 0.0013 muF/cm2 at 20.5 degrees C. The agreement between the various values of Cg strongly suggests that the bilayers are free of squalene. DC potentials applied to the bilayers have no detectable effect on the value of Cg, as expected for solvent-free films. The ability to form bilayers essentially free of the solvent used in the forming solution makes it possible to determine the area per molecule of the surface active lipid in the bilayer. The area per molecule of GMO at 20 degrees C is estimated to be 37.9 +/- 0.2 A2.


Drug tolerance and biomembranes.

Drug tolerance and biomembranes. Our understanding of the structure and function of biomembranes is now at a stage where we can begin to examine drug actions on the membrane bilayer. Cells need a certain optimal degree of fluidity in their membranes and some organisms can adapt their membrane composition to maintain the appropriate fluidity under changing environmental conditions. Chronic administration of membrane-disrupting drugs may evoke such an adaptive process, which would show itself in vivo as drug tolerance.


Laser Raman light-scattering observations of conformational changes in myosin induced by inorganic salts. The Raman spectra of aqueous solutions of myosin and mixtures of myosin in solutions of the salts CaCl(2), MgCl(2), and LiBr have been taken. The spectrum of the solvent background has been subtracted by means of a computer, leaving only the Raman peaks of the protein. From an analysis of the Raman bands in the regions at 900, 940, 1,240-1,300, and 1,650-1,670 cm(-1), it seems likely that CaCl(2) effects an alpha-to beta-transition in myosin, probably owing to the interaction of the Ca(2+) ion, LiBr appears to denature the protein leading to increased random coil structure, and MgCl(2) appears to have an effect intermediate between the two other salts. These results are reported for concentrations as low as 10(-5) M of CaCl(2) and MgCl(2).This investigation indicates the usefulness of the Raman light-scattering technique for the study of protein conformational changes.


Selective deficiencies in complement component : a family with hereditary C2 deficiency. We report herein a new case of C2 deficiency in a patient with systemic lupus. The subject and one of her brothers, who shows no clinical manifestations, are hymozygous C2 deficient. All other family members are heterozygous for the C2 deficiency. Gene for C2 deficiency (C2d) was shown to be inherited with HLA-A9, B7/Bfs and HLA-A10, B27/Bfs haplotypes. This association has not previously been described.


Primary intermediates in the photochemical cycle of bacteriorhodopsin. Picosecond studies of the primary photochemical events in the light-adapted bacteriorhodopsin, bR570, indicate that the first metastable intermediate K610 is formed with a rise time of 11 ps. Difference spectra obtained at 50 ps after excitation show that K610 is the same species as that trapped in low temperature glasses. A precursor species (S) of the K610 intermediate has been observed which is red shifted with respect to K610 and is formed within the 6-ps time width of the excitation pulse. The formation of the precursor has no observable thermal dependence between 298 degrees and 1.8 degrees K. The formation of K610 has a very low thermal barrier and at very low temperatures, the rate of formation becomes practically temperature independent which is characteristic of a tunneling process. The rate of formation becomes practically temperature independent which is characteristic of a tunneling process. The rate of formation of K610 has a moderate deuterium isotope effect of kH/kD approximately 1.6 at 298 degrees K and 2.4 at 4 degrees K. The mechanism for formation of K610 is found to involve a rate-limiting proton transfer which occurs by tunneling at low temperatures.


Picosecond and steady state, variable intensity and variable temperature emission spectroscopy of bacteriorhodopsin. The bacteriorhodopsin emission lifetime at 77 degrees K has been obtained for different regions of the emission spectrum with single-pulse excitation. The data under all conditions yield a lifetime of 60 +/- 15 ps. Intensity effects on this lifetime have been ruled out by studying the relative emission amplitude as a function of the excitation pulse energy. We relate our lifetime to previously reported values at other temperatures by studying the relative emission quantum efficiency as a function of temperature. These variable temperature studies have indicated that an excited state with an emission maximum at 670 nm begins to contribute to the spectrum as the temperature is lowered. Within our experimental error the picosecond data seem to suggest that this new emission may arise from a minimum of the same electronic state responsible for the 77 degrees K emission at 720 nm. A correlation is noted between a 1.0-ps formation time observed in absorption by Ippen et al. (Ippen, E.P., C.V. Shank, A. Lewis, and M.A. Marcus. 1978. Subpicosecond spectroscopy of bacteriorhodopsin. Science [wash. D.C.]. 200:1279-1281 and a time extrapolated from relative quantum efficiency measurements and the 77 degrees K fluorescence lifetime that we report.


Fluorescein excitation and emission polarization spectra in living cells: changes during the cell cycle. Changes in the fluorescein fluorescence emission and excitation polarization spectra in synchronized cultured S3 fibroblasts at G1, mid-S, and mitosis, as well as in human lymphocytes before and after stimulation with mitogens, were studied. In contrast to those measured in aqueous solutions the emission and excitation polarization spectra in living cells exhibit a wavelength dependence characteristic for the state of the cell cycle. Changes in the temperature and in the amount of intracellular water result in quantitative wavelength-independent changes in the polarization spectra. Possible mechanisms for the qualitative wavelength-dependent changes in the fluorescein emission and excitation polarization spectra during the cell cycle are discussed.