Solution conformation of d-CTCGAGCTCGAG by two-dimensional NMR: conformational heterogeneity at XhoI cleavage site

Nonexchangeable proton resonances in the 500-MHz NMR spectrum of d-CTCGAGCTCGAG have been assigned by using two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY). 1H-1H coupling constants (J) in the deoxyribose rings have been measured by analyzing intensity and multiplet patterns in the phase-sensitive omega 1-scaled COSY spectra. A modification of the J-resolved technique, called amplitude-modulated J-resolved spectroscopy, has been described and used to increase the accuracy of J measurements. Absorption mode omega 1-scaled NOESY spectra at mixing times in the range 50-200 ms have been analyzed to monitor spin diffusion. A 50-ms spectrum has been used to estimate several interproton distances. The coupling constant and distance data have been used to arrive at sequence-specific sugar geometries and glycosidic torsion angles. The backbone structure has been refined by model building using the FRODO program, employing the sugar geometries and glycosidic torsion angles discussed above. The molecule shows interesting sequence-dependent variations in the structure. The cleavage site of the restriction enzyme XhoI exhibits unique differences in the sugar geometry and backbone torsion angles.     

Eliminating a Set of Four Penicillin Binding Proteins Triggers the Rcs Phosphorelay and Cpx Stress Responses in Escherichia coli

Penicillin binding proteins (PBPs) are responsible for synthesizing and modifying the bacterial cell wall, and in Escherichia coli the loss of several nonessential low-molecular-weight PBPs gives rise to abnormalities in cell shape and division. To determine whether these proteins help connect the flagellar basal body to the peptidoglycan wall, we surveyed a set of PBP mutants and found that motility in an agar migration assay was compromised by the simultaneous absence of four enzymes: PBP4, PBP5, PBP7, and AmpH. A wild-type copy of any one of these restored migration, and complementation depended on the integrity of the PBP active-site serine. However, the migration defect was caused by the absence of flagella instead of improper flagellar assembly. Migration was restored if the flhDC genes were overexpressed or if the rcsB or cpxR genes were deleted. Thus, migration was inhibited because the Rcs and Cpx stress response systems were induced in the absence of these four specific PBPs. Furthermore, in this situation Rcs induction depended on the presence of CpxR. The results imply that small changes in peptidoglycan structure are sufficient to activate these stress responses, suggesting that a specific cell wall fragment may be the signal being sensed. The fact that four PBPs must be inactivated may explain why large perturbations to the envelope are required to induce stress responses.     

Purification and biological characterization of a halophilic thermostable protease from <Emphasis Type="Italic">Haloferax lucentensis</Emphasis> VKMM 007

An extremely halophilic archaeon Haloferax lucentensis VKMM 007, isolated from a solar saltern, was found to produce a protease. This extracellular enzyme consisted of a single polypeptide chain of 57.8 kDa as determined by SDS–PAGE and was purified by a combination of ultrafiltration, bacitracin–Sepharose affinity chromatography and Sephadex G-100 gel filtration. The purified protein was stable in a wide range of temperatures (20–70°C), NaCl concentrations (0.85–5.13 M) and pH (5.0–9.0) with maximal activity observed at 60°C, 4.3 M NaCl and pH 8.0. Proteolytic activity was enhanced by Ca²⁺, K⁺, Mg²⁺, Na⁺, and Fe²⁺ ions and the protein was classified as a trypsin-like serine protease. Further assays indicated highest degree of specificity when hemoglobin was used as an enzyme substrate. Most importantly, the proteolytic activity remained stable or only marginally inhibited in the presence of various polar and non-polar solvents, surfactants and reducing agents thus emphasizing the biotechnological potential of this novel halophilic protease.     

Length-weight relationship of six demersal fish species from Gulf of Mannar,Bay of Bengal,Eastern Indian Ocean

Length-weight relationship (LWR) parameters were analysed for six demersal finfish species from the Gulf of Mannar coast, Bay of Bengal. Fishes were sampled monthly from the landings of trawlers operated along the coast of Tuticorin and Rameshwaram with a cod-end mesh size of 35 mm at the depth of 60–80 m. Fish specimens were sampled by measuring the total length (TL) and total weight (TW) with precision to 0.1 cm and 0.1 g respectively. The present study also recorded a new maximum total length for Engyprosopon macrolepis, Torquigener brevipinnis and Leiognathus brevirostris.     

Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology

The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.     

Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence

The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly⁻⁸² to Gly³¹⁶), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca²⁺ ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.     

Purification and characterization of a highly active chromate reductase from endophytic Bacillus sp. DGV19 of Albizzia lebbeck (L.) Benth. actively involved in phytoremediation of tannery effluent-contaminated sites

Phytoremediation using timber-yielding tree species is considered to be the most efficient method for chromium/tannery effluent-contaminated sites. In this study, we have chosen Albizzia lebbeck, a chromium hyperaccumulator plant, and studied one of its chromium detoxification processes operated by its endophytic bacterial assemblage. Out of the four different groups of endophytic bacteria comprising Pseudomonas, Rhizobium, Bacillus, and Salinicoccus identified from A. lebbeck employed in phytoremediation of tannery effluent-contaminated soil, Bacillus predominated with three species, which exhibited not only remarkable chromium accumulation ability but also high chromium reductase activity. A chromate reductase was purified to homogeneity from the most efficient chromium accumulator, Bacillus sp. DGV 019, and the purified 34.2-kD enzyme was observed to be stable at temperatures from 20°C to 60°C. The enzyme was active over a wide range of pH values (4.0–9.0). Furthermore, the enzyme activity was enhanced with the electron donors NADH, followed by NADPH, not affected by glutathione and ascorbic acid. Cu²⁺ enhanced the activity of the purified enzyme but was inhibited by Zn²⁺ and etheylenediamine tetraacetic acid (EDTA). In conclusion, due to its versatile adaptability the chromate reductase can be used for chromium remediation.     

Extracellular enzymes of mycobacteria

Abstract Extracellular enzymes were studied in different mycobacteria using a plate substrate assay. All the pathogenic mycobacteria included in the study showed the presence of protease, while lipase, ribonuclease, mucinase and β-lactamase could also be detected in some strains. In contrast, no protease was detected in the 3 saprophytic mycobacteria studied. DNase was not detected in any of the species studied. Thus, the demonstration of extracellular enzymes, in particular of protease, in mycobacteria may be relevant in understanding their role in pathogenicity.     

Factors related to iron absorption by enzymically isolated leaf cells

The rate of Fe absorption by cells enzymically isolated from tobacco leaves is correlated with the age of the leaves from which the cells are derived. The cells obtained from younger leaves absorb Fe more rapidly than those from older ones. Ca inhibits Fe and Mn absorption. Fe and Mn are mutually antagonistic in their absorption by leaf cells. Ca enhances the inhibition of Mn absorption by Fe, but reduces the inhibition of Fe absorption by Mn. The affinity constant for Fe absorption by leaf cells is low. The chelate EDDHA (ethylenediamine di(o-hydroxyphenylacetate) competitively inhibits Fe absorption.