Isolation and characterization of the rat proenkephalin gene

The rat proenkephalin gene has been isolated by molecular cloning and characterized by DNA-sequence analysis. The gene exhibits a structural organization similar to that of the human gene. The nucleotide sequence encoding the biologically active opioid peptides which are generated from the proenkephalin precursor as well as the 3' untranslated region of the mRNA are found on a large exon at the 3' end of the gene (Exon III). The nucleotide sequence encoding the N terminus of the mature protein and its signal peptide are located on Exon II while Exon I encodes the 5' untranslated region of the mRNA. The nucleotide sequence of these exons and their flanking regions has been determined and compared to the human proenkephalin gene. Analysis of the nucleotide sequence homology between the human and rat proenkephalin gene reveals the presence of highly conserved regions within both the coding and noncoding portions of the genes. Enkephalin-coding sequences as well as 5' flanking sequences appear to be the most highly conserved. The importance and possible function of these sequences are discussed.     

Real‐time PCR method for the quantification of Burkholderia cepacia complex attached to lung epithelial cells and inhibition of that attachment

Aims: To develop a rapid method to quantify the attachment of the cystic fibrosis pathogen, Burkholderia multivorans, to lung epithelial cells (16HBE14o^?) using real‐time PCR with a view to monitoring potential inhibition of lung cell attachment. Methods and Results: Mammalian and bacterial DNA were purified from bacteria attached to lung epithelial cells. The relative amount of bacteria attached was determined by amplification of the recA gene relative to the human GAPDH gene, in the presence of SYBR Green^®. The method was thoroughly validated and shown to correlate well with traditional plating techniques. Inhibition of bacterial attachment with simple sugars was then evaluated by real‐time PCR. Of the sugars examined, pre‐incubation of B. multivorans with lactose, mannose and xylitol all decreased bacterial adherence to 16HBE14o^? cells, while glucose and galactose had no significant effect. Pre‐incubation with lactose had the greatest effect, resulting in reduced adhesion to 35% of untreated controls. Conclusions: This method can be used to quickly and effectively screen novel agents with higher affinities for bacterial adhesins. Significance and Impact of the Study: This method will enable the rapid development of novel agents to inhibit colonization by this pathogen from the environment.     

Further antigenic analyses of the fish pathogenic bacteriumVibrio anguillarum

TenVibrio anguillarum strains were selected for an immunoelectrophoretic study. Evidence was provided for existence of two new K antigens which displayed cross-reactivity. The importance of an exact characterization of surface antigens inV. anguillarum is considered.     

Properties of phosphorylated NADP+-specific glutamate dehydrogenase fromEscherichia coli

The NADP⁺-specific glutamate dehydrogenase (GDH) fromEscherichia coli strain D₅H₃G₇, an enzyme that catalyzes the interconversion of -ketoglutarate andl-glutamate, has been shown to be phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein is extremely acid labile and is unstable at high pH. Treatment of GDH with diethyl pyrocarbonate (DEP), a histidine-modifying reagent, blocked the incorporation of³²P from [-³²P]ATP. GDH catalytic activity was also inhibited by DEP treatment. Hydroxylamine, a reagent hydrolyzing phosphoramidates, catalyzed the removal of phosphate from phosphorylated GDH, suggesting that GDH may be phosphorylated at a histidine residue(s). A total enzymatic hydrolysis of phosphorylated GDH, which was electroeluted from a native polyacrylamide gel, was analyzed by a Dowex 1-8X anion exchange chromatography. The presence of³²P-labeled 3-phosphohistidine, characterized and identified from this hydrolysate, demonstrates that a histidine residue(s) is the site of phosphorylation.     

ON GUAIACOL SOLUTIONS : I. THE ELECTRICAL CONDUCTIVITY OF SODIUM AND POTASSIUM GUAIACOLATES IN GUAIACOL

1. Measurements on the densities, viscosities, dielectric constants, and specific conductances of pure anhydrous and water-saturated guaiacol at 25°C. are reported. 2. The solubility of water in guaiacol at 25°C., and its effect on the electrical conductivity of a sodium guaiacolate solution is given. 3. Electrical conductivity measurements are reported on solutions of sodium and potassium guaiacolates in water-saturated guaiacol at 25°C. 4. The decrease of electrical conductivity with increasing concentration for these salts is explained on the basis of an ionic equilibrium combined with the interionic attraction theory of Debye and Hückel. 5. The limiting equivalent conductances of sodium and potassium guaiacolates in water-saturated guaiacol at 25°C., the corresponding limiting ionic mobilities, and the dissociation constants are computed from the conductivity measurements. The salts are found to be weak electrolytes with dissociation constants of the order of 5 x 10^–6.     

The relative importance of local phase and local amplitude in patchwise image reconstruction

Natural images were subjected to patchwise Fourier analysis, and the local amplitude and phase spectra were swapped between different images. When the patches were large relative to the image size, the appearance of the reconstructed image was similar to that of the image from which the phase information had been derived, in agreement with previous reports of phase-dominance in the global Fourier Transform. However, when the patch size was made sufficiently small, the appearance of reconstructed images was dominated by amplitude rather than phase. This was not simply due to the DC component of the amplitude spectrum. Prior low-pass filtering of the images enhanced the dominance of amplitude information in the patchwise transform. We conclude that patchwise-reconstructed images contain two quite distinct kinds of information for the human observer. The first is the positional information (local sign) of the patches themselves; the second is the textural information within patches, which is dominated by amplitude rather than phase. The reason why the global Fourier Transform is dominated by phase is that in the absence of any other information about local sign, phase is necessary to reconstruct localised features such as edges.     

The relationship between hydrogen metabolism,sulfate reduction and nitrogen fixation in sulfate reducers

Summary Hydrogenase and nitrogenase activities of sulfate-reducing bacteria allow their adaptation to different nutritional habits even under adverse conditions. These exceptional capabilities of adaptation are important factors in the understanding of their predominant role in problems related to anaerobic metal corrosion. Although the D₂–H⁺ exchange reaction indicated thatDesulfovibrio desulfuricans strain Berre-Sol andDesulfovibrio gigas hydrogenases were reversible, the predominant activity in vivo was hydrogen uptake. Hydrogen production was restricted to some particular conditions such as sulfate or nitrogen starvation. Under diazotrophic conditions, a transient hydrogen evolution was followed by uptake when dinitrogen was effectively fixed. In contrast, hydrogen evolution proceeded when acetylene was substituted as the nitrogenase substrate. Hydrogen can thus serve as an electron donor in sulfate reduction and nitrogen metabolism.     

Effects of external stimuli on environmental bacterial strains harboring analgD-lux bioluminescent reporter plasmid for the study of corrosive biofilms

An alginic acid biosynthesis bioluminescent reporter plasmid, pUTK50, was transconjugated into environmental strains ofPseudomonas putida, Pseudomonas fluorescens, andStenotrophomonas maltophilia. Bioluminescent transconjugates were selected from each strain for investigation of environmental stress factors that promote alginic acid exopolymer biosynthesis in developing biofilms. Environmental stimuli associated with increased levels of alginate synthesis, in a previously developed organism,P. aeruginosa FRD1, were applied to the environmental strains. Increased salt concentrations and higher ratios of nitrate vs ammonium ions as the limiting nitrogen source induced bioluminescence in FRD1 and the environmental strains. However, for environmental strains ofP. putida, P. fluorescens andS. maltophilia, polysaccharides were detected with low uronic acids content and different structural components. When tested within a biofilm,S. maltophilia O46 demonstrated exceptional adhesive and corrosive properties while alginic acid synthesis was not high. In most of the environmental strains, periods of increased bioluminescence were induced by external stimuli, but exopolysaccharides other than alginic acid were expressed. It is hypothesized that the environmental strains have homologous but nonidentical promoter sequences which are responsive to certain environmental stimuli and may control genes necessary for the production of alternative exopolysaccharides.     

Reduction of potassium tellurite by Candidas

Summary A technique for observing the reduction of potassium tellurite byCandidas was developed. Various species of these yeasts reduce it at different concentrations, but that which is most useful for differentiation is 0.02 % added to Sabouraud dextrose agar basal medium. Of the yeasts studied,C. albicans, C. parapsilosis andC. tropicalis all reduced potassium tellurite to the concentration mentioned before, while the growth ofC. krusei, C. parakrusei andC. pseudotropicalis was inhibited. Without exception,C. pseudotropicalis reduced this salt at lower concentrations. The two strains ofC. guilliermondii tested gave contradictory results: one of them grew and reduced potassium tellurite, while the growth of the other was inhibited.Professor of Microbiology.     

Localization of ornithine decarboxylase in the chick embryo during organogenesis

Summary The localization of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis and thus in cell growth, was determined in the 4.5-day-old chick embryo, using two independent methods of analysis. ODC protein was identified by indirect immunofluorescence with a monospecific ODC antibody, and catalytically active ODC was identified by autoradiography with -(5-³H) difluoromethylornithine. Both methods revealed a basically similar distribution of ODC within the embryo. Among the organs, the brain exhibited the highest ODC levels. ODC levels were also high in spinal cord, mesonephric tubules and heart. Similar levels, but confined to limited areas, were found in liver tissue, head mesenchyme, and the oral and pharyngeal regions. Organs that exhibited high ODC levels are all engaged in rapid growth, as well as in extensive tissue remodeling and differentiation.