Sulfuryl transfer catalyzed by phosphokinases.

Adenosine 5'-sulfatopyrophosphate is a substrate for nucleoside diphosphate kinase. The reaction appears to proceed through a ping-pong mechanism analogous to the physiological reaction involving ATP, presumably by way of a sulfohistidine intermediate. Unlike the phosphoryl transfer reactions, the corresponding sulfuryl transfers catalyzed by nucleoside diphosphate kinase do not have a strict divalent metal requirement. The estimated rate constants for the metal- and nonmetal-catalyzed sulfuryl transfers differ by less than an order of magnitude and are approximately 1000-fold slower than the corresponding phosphate transfers. These results suggest that the role of the metal ion in nucleoside diphosphate kinase is to coordinate the alpha, beta-phosphates of the substrate. Sulfuryl and phosphoryl transfer probably occur through dissociative transition states.     

Optimal design of feedback control by inhibition

The local stability of unbranched biosynthetic pathways is examined by mathematical analysis and computer simulation using a novel nonlinear formalism that appears to accurately describe biochemical systems. Four factors affecting the stability are examined: strength of feedback inhibition, equalization of the values among the corresponding kinetic parameters for the reactions of the pathway, pathway length, and alternative patterns of feedback interactions. The strength of inhibition and the pattern of feedback interactions are important determinants of steady-state behavior. The simple pattern of end-product inhibition in unbranched pathways may have evolved because it optimizes the steady-state behavior and is temporally most responsive to change. Stability in these simple systems is achieved by shortening pathway length either physically or, in the case of necessarily long pathways, kinetically by a wide devergence in the values of the corresponding kinetic parameters for the reactions of the pathway. These conclusions are discussed in the light of available experimental evidence.     

Cytokinin Production by Bradyrhizobium japonicum

Although there is considerable circumstantial evidence for the involvement of cytokinins in legume nodulation, the cytokinins produced by rhizobia have not been well characterized. Bradyrhizobium japonicum 61A68, a bacterium which nodulates soybean (Glycine max [L.] Merr.), was grown in defined medium. Cytokinins were purified from the culture medium by Amberlite XAD-2 chromatography and fractionated by column chromatography on Sephadex LH-20 in 35% ethanol. Pooled fractions from the Sephadex column were analyzed for cytokinin activity with the tobacco callus bioassay. Cytokinin activity was observed in fractions corresponding to the elution volumes of zeatin, ribosylzeatin, and methylthiozeatin. No activity corresponding to the elution volumes of isopentenyladenine or its riboside was found. Total cytokinin activity in the B. japonicum culture filtrate was equivalent to approximately 1 microgram of kinetin per liter. Transfer RNA was isolated from B. japonicum cells by phenol extraction, followed by potassium acetate extraction, cetyltrimethylammonium bromide precipitation, and DEAE cellulose chromatography. Transfer RNA was enzymically hydrolyzed to nucleosides. High performance liquid chromatographic analysis of cytokinin nucleosides showed peaks corresponding to the retention times of trans-ribosylzeatin, methylthioribosylzeatin, isopentenyladenosine, and methylthioisopentenyladenosine. Analysis of the tRNA hydrolysate by Sephadex LH-20 chromatography and tobacco bioassay showed cytokinin activity in fractions corresponding to ribosylzeatin, methylthioribosylzeatin, and isopentenyladenosine. The presence of the trans isomer of ribosylzeatin was also determined by enzyme immunoassay.     

Conformation of poly(sodium ethacrylate) in solution studied by small-angle X-ray scattering

The pH-induced conformational transition of poly(sodium ethacrylate) PNaEA in aqueous solution, which occurs between a compact form at low charge-density and an extended coil at high charge-density, was studied by small-angle X-ray scattering and the structure at an each conformational state was analyzed and compared with the corresponding one of poly(sodium methacrylate) PNaMA. The conformational transition for PNaEA induced a remarkable change in the scattering data plotted in the form of the Kratky plot. By comparing the scattering data with theoretical scattering functions, it was clarified that the structures of the compact form and the extended coil are well mimicked by a swollen gel having a network structure and by a wormlike chain, respectively. Although such a structure of the extended coil of PNaEA is similar to the corresponding one of PNaMA, the structure of the compact form of PNaEA is different from the corresponding one of PNaMA, which is still represented by a wormlike chain in a Theta medium.     

Correlation model of object recognition by echolocating animals]

The model proposed in an attempt to find out physical bases of object perception during echolocation. It is shown that echolocational perception can be provided with correlational treatment of corresponding signals. The character of objects is determined by the comparison by echo probing accepted in the given cycle with typical distortions remembered in the course of individual experience of the animal. The distortions take place during the reflection of the probing impulse from these or those objects. "Binding" of the objects according to distance may be carried out by using the choice of typical distortions for corresponding correction of the copy of probing impulse, serving as a bearing signal of distance correlometer. The response of correlometer to the echo from correctly perceived target increases. The block-scheme of such correlation perception during echolocation is given. Performance of some experiments allowing to check and refine the model considered.     

Phosphorylation of actin-binding proteins by casein kinases 1 and 2

Filamin and vinculin from chicken gizzards were significantly phosphorylated in vitro by casein kinases 1 and 2, but not by alpha-actinin. Antisera raised against these actin-binding proteins immunoprecipitated the phosphorylated proteins corresponding to filamin and vinculin, but no phosphoprotein corresponding to alpha-actinin was detected. These results suggest that filamin and vinculin are phosphorylated in vivo but alpha-actinin is not.     

Inhibition of translation and cell growth by minigene expression

A random five-codon gene library was used to isolate minigenes whose expression causes cell growth arrest. Eight different deleterious minigenes were isolated, five of which had in-frame stop codons; the predicted expressed peptides ranged in size from two to five amino acids. Mutational analysis demonstrated that translation of the inhibitory minigenes is essential for growth arrest. Pulse-labeling experiments showed that expression of at least some of the selected minigenes results in inhibition of cellular protein synthesis. Expression of the deleterious minigenes in cells deficient in peptidyl-tRNA hydrolase causes accumulation of families of peptidyl-tRNAs corresponding to the last minigene codon; the inhibitory action of minigene expression could be suppressed by overexpression of the tRNA corresponding to the last sense codon in the minigene. Experimental data are compatible with the model that the deleterious effect of minigene expression is mediated by depletion of corresponding pools of free tRNAs.     

Oxidation of 4-tert-butylcatechol and dopamine by hydrogen peroxide catalysed by horseradish peroxidase.

The catalytic cycle of horseradish peroxidase (HRP; donor:hydrogen peroxide oxidoreductase; EC 1.11.1.7) is initiated by a rapid oxidation of it by hydrogen peroxide to give an enzyme intermediate, compound I, which reverts to the resting state via two successive single electron transfer reactions from reducing substrate molecules, the first yielding a second enzyme intermediate, compound II. To investigate the mechanism of action of horseradish peroxidase on catechol substrates we have studied the oxidation of both 4-tert-butylcatechol and dopamine catalysed by this enzyme. The different polarity of the side chains of both o-diphenol substrates could help in the understanding of the nature of the rate-limiting step in the oxidation of these substrates by the enzyme. The procedure used is based on the experimental data to the corresponding steady-state equations and permitted evaluation of the more significant individual rate constants involved in the corresponding reaction mechanism. The values obtained for the rate constants for each of the two substrates allow us to conclude that the reaction of horseradish peroxidase compound II with o-diphenols can be visualised as a two-step mechanism in which the first step corresponds to the formation of an enzyme-substrate complex, and the second to the electron transfer from the substrate to the iron atom. The size and hydrophobicity of the substrates control their access to the hydrophobic binding site of horseradish peroxidase, but electron density in the hydroxyl group of C-4 is the most important feature for the electron transfer step.     

Temperature dependence of photovoltages generated by bacteriorhodopsin.

The temperature dependence of the photovoltage developed by a model membrane containing bacteriorhodopsin (BR) is studied. The model membrane is formed by first coating a thin Teflon sheet with lipid and then fusing BR vesicles to it. The time course of the photoresponse is resolved down to 1 microsecond. The photoresponse is taken to be a sum of exponentials. Exponential time constants and amplitudes are determined by an analysis of the photoresponse with a photovoltage vs. log time plot, correlation filter, and nonlinear least-squares routine. The photovoltage is taken to be the sum of three exponentials but only two of the three time constants are resolved. Both are temperature dependent and indicate a thermally activated transport process. The corresponding activation energies are 55 kJ/mol and 62 kJ/mol. Since the photovoltage is proportional to charge times displacement the corresponding charge displacements are 11 and 34 A assuming a total displacement of 45 A. The remaining exponential term corresponds to a small negative transient in the photovoltage that has a rise time less than 1 microsecond even at -20 degrees C. The calculated charge displacement is estimated to be less than 2 A.     

Characterization of new human c-myc mRNA species produced by alternative splicing

Characterization of two human c-myc cDNAs corresponding to the mRNAs 2.5 and 3.1 kb in length transcribed from PO previously demonstrated the existence of alternative acceptor sites at the end of intron 1 and intron 2, respectively [Bentley, D.L. and Groudine, M. (1986) Mol. Cell. Biol. 6, 3481–3489]. We investigated the use of these alternative acceptor sites in each c-myc mRNA species. We characterized cDNAs corresponding to c-myc mRNAs transcribed in the SW613-S human carcinoma cell line. The use of the alternative acceptor site at the end of intron I was demonstrated in two out of 10 cDNAs corresponding to the 3.1-kb mRNA transcribed from PO and in three out of 10 cDNAs corresponding to the mRNAs transcribed from P1 or P2. The use of this acceptor site is therefore not restricted to the 2.5-kb mRNA transcribed from PO. The mRNAs resulting from the use of this acceptor site would encode for a variant form of the p67 polypeptide lacking one amino-acid residue. Conversely, the use of the alternative acceptor site at the end of intron 2 was not found in any of the cDNAs corresponding to the mRNAs transcribed from PO (0/10), from P1 or P2 (0/10) and from P3 (0/10). In the course of this study, we isolated a cDNA corresponding to another new c-myc mRNA species. This mRNA is produced by alternative splicing within intron 1 and encodes only for p64.     

Determination of monoclonal antibody specificity by mixed precipitation ingel

The enzyme-immunodiffusion technique is advanced which permits testing monoclonal antibodies included in the precipitate line formed in gel by polyclonal antibodies with the corresponding antigen. Rat or mouse monoclonal antibodies were mixed with polyclonal rabbit antiserum to the antigen at issue. The precipitate formed by immunogen and rabbit polyclonal antibodies included monoclonals.     

Inhibition of Alcoholic Fermentation of Grape Must by Fatty Acids Produced by Yeasts and Their Elimination by Yeast Ghosts

In a complete nutritive medium rich in sugar, such as grape must, the inhibition of alcoholic fermentation is caused by substances produced by the yeast which, acting synergistically with ethanol, are toxic to the yeasts themselves. Among these are decanoic and octanoic acids and their corresponding ethyl esters. Their adsorption by yeast ghosts permits the prevention and treatment of fermentation stoppages.     

Early stages of LDL oxidation: apolipoprotein B structural changes monitored by infrared spectroscopy

Changes in the conformation of apoliprotein B-100 in the early stages of copper-mediated low density lipoprotein oxidation have been monitored by infrared spectroscopy. During the lag phase no variation in structure is observed, indicating that copper binding to the protein does not significantly affect its structure. In the propagation phase, while hydroperoxides are formed but the protein is not modified, no changes in secondary structure are observed, but the thermal profile of the band corresponding to alpha-helix is displaced in frequency, indicating changes in tertiary structure associated with this conformation but not with beta-sheet components. When aldehyde formation starts, a decrease of approximately 3% in the area of bands corresponding to alpha-helix and beta-sheet is produced, concomitantly with an increase in beta-turns and unordered structure. The two bands corresponding to beta-turns vary as well under these conditions, indicating changes in these structures. Also at this stage the thermal profile shows variations in frequency for the bands corresponding to both alpha-helix and beta-sheet.The results are consistent with the hypothesis that as soon as the polyunsaturated fatty acids from the particle core are modified, this change is reflected at the surface, in the alpha-helical components contacting the monolayer.     

Aldehyde production by alcohol oxidation with Gluconobacter oxydans

The microbial oxidation of various primary alcohols to the corresponding aldehydes has been investigated. A focused screening performed amongst some acetic acid bacteria showed that a newly isolated strain of Gluconobacter oxydans oxodizes various short-chain aliphatic alcohols to the corresponding aldehydes with negligible acid production. 3-Methyl-1-butanol (isoamyl alcohol) proved to be the better substrate with high yields (more than 90%) without by-product formation. This biotransformation also occurs with continuous or semicontinuous addition of substrate since the volatile product is removed from the medium under vigorous aeration conditions. Product recovery is attained either by the use of cold traps or by reversible complex formation.     

Enhancement of allele discrimination by introduction of nucleotide mismatches into siRNA in allele-specific gene silencing by RNAi

Allele-specific gene silencing by RNA interference (RNAi) is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi), the design and assessment of small interfering RNA (siRNA) duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wild-type reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs) against mutant alleles of the human Prion Protein (PRNP) gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the 'seed region' of microRNAs. Due to the essential role of the 'seed region' of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs), of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3'-ends of sense-strand siRNA elements, which possibly increase the assembly of antisense-strand (guide) siRNAs into RNA-induced silencing complexes (RISCs), may enhance ASP-RNAi in the case of inert siRNA duplexes. Therefore, the data presented here suggest that structural modification of functional portions of an siRNA duplex by base substitution could greatly influence allele discrimination and gene silencing, thereby contributing to enhancement of ASP-RNAi.     

Formation of N-nitrosodiphenylamine from 1,1-diphenylhydrazine by rat liver microsomal preparations

The present study provides the first evidence for in vitro metabolic conversion of a 1,1-disubstituted hydrazine to the corresponding nitrosamine. The study shows that superoxide radical which is generated by NADPH-cytochrome c reductase is involved in the oxidation of 1,1-diphenylhydrazine to N-nitrosodiphenylamine catalyzed by rat liver microsomes.     

Foetomaternal relationships of serum bile acid pattern estimated by high-pressure liquid chromatography.

The bile acid patterns in the maternal and umbilical vein and artery serum samples were analysed by a two-step chromatographic method involving group separation by piperidinohydroxypropyl-Sephadex LH-20 and high-pressure liquid chromatography using immobilized 3 alpha-hydroxy steroid dehydrogenase. Glycochenodeoxycholate predominates in the maternal blood and taurochenodeoxycholate in the umbilical blood. In cases where a free bile acid was detected in the maternal blood, the same bile acid was also demonstrated in the corresponding cord blood. The concentrations of taurocholate and taurochenodeoxycholate were found to be significantly higher in the umbilical artery than in the corresponding umbilical vein. Our data suggest that there is a bidirectional placental transfer of free bile acids and that there is a transfer of taurine-conjugated primary bile acids from the foetus to the mother.     

Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae

Recombinant hirudin was produced by the yeast Saccharomyces cerevisiae using the alpha-pheromone prepro sequence to direct its secretion into the culture medium. The secreted hirudin was isolated to greater than or equal to 95% purity as measured by 205-nm absorbance integration from a reverse-phase chromatogram. One major activity peak corresponding to the complete, correctly processed molecule and two minor activity peaks corresponding to C-terminally truncated forms were identified. The primary structure of the major peak, determined by N-terminal sequencing of tryptic peptides, was that predicted from the cDNA sequence, and the molecular mass analyzed by fast atom bombardment mass spectrometry (FAB-MS) was 6892.6 (calculated 6892.5). UV spectral analysis suggested that, in contrast to the natural molecule, recombinant hirudin produced by S. cerevisiae is not sulfated.     

Wetting of Planar Surfaces by a Gay-Berne Liquid Crystal

Molecular simulation of the wetting of an unstructured attractive wall by Gay-Berne liquid crystals are reported. Simulations are performed in the grand canonical ensemble on a wide pore at constant temperatures of T *=0.53 and 0.56, corresponding to temperatures below and above the nematic-isotropic-vapor triple point. Close to the coexistence chemical potential, a thick liquid film wets the solid surface. The film is composed of stratified layers of molecules parallel to the solid surface, which follow to a nematic domain at the lower temperature and an isotropic one at the higher temperature. In both cases, the film is in equilibrium with the corresponding vapor phase. Close to the liquid-vapor interface there is a manifest tendency for the molecules to orient themselves parallel to the interface. The adsorption on the wall varies continuously with the thermodynamic parameters considered and no evidence of a first order prewetting transition is observed.