Modulation of the sensitivity of FimB recombination to branched-chain amino acids and alanine in Escherichia coli K-12

Phase variation of type 1 fimbriae of Escherichia coli requires the site-specific recombination of a short invertible element. Inversion is catalyzed by FimB (switching in either direction) or FimE (inversion mainly from on to off) and is influenced by auxiliary factors integration host factor (IHF) and leucine-responsive regulatory protein (Lrp). These proteins bind to sites (IHF site II and Lrp sites 1 and 2) within the invertible element to stimulate recombination, presumably by bending the DNA to enhance synapses. Interaction of Lrp with a third site (site 3) cooperatively with sites 1 and 2 (termed complex 1) impedes recombination. Inversion is stimulated by the branched-chain amino acids (particularly leucine) and alanine, and according to a current model, the amino acids promote the selective loss of Lrp from site 3 (complex 2). Here we show that the central portion of the fim invertible element, situated between Lrp site 3 and IHF site II, is dispensable for FimB recombination but that this region is also required for full amino acid stimulation of inversion. Further work reveals that the region is likely to contain multiple regulatory elements. Lrp site 3 is shown to bind the regulatory protein with low affinity, and a mutation that enhances binding to this element is found both to diminish the stimulatory effects of IVLA on FimB recombination and to inhibit recombination in the absence of the amino acids. The results obtained emphasize the importance of Lrp site 3 as a control element but also highlight the complexity of the regulatory system that affects this site.     

Genetic characterization of high-level gentamicin-resistant strains of Enterococcus faecalis in Iran

The prevalence of resistance to high levels of gentamicin among 182 isolates of Enterococcus faecalis from 2 Iranian hospitals was 42%. Gentamicin resistance was associated with conjugative plasmids (>70 kb) in most strains. Fingerprinting using EcoRI and HindIII showed genetic variation among these plasmids and gave evidence of nosocomial outbreaks and persistence of infection in different wards of the study hospitals, as well as transfer of plasmids between genetically diverse isolates. Using EcoRI, hospital-based specific plasmid fingerprints were detected for the isolates that had previously proved to be unrelated by multilocus enzyme electrophoresis, suggesting the persistence of related plasmids at each hospital, though minor changes in these related plasmids could be detected with HindIII.     

Novel anti-viability ceramide analogs: Design,synthesis, and structure–activity relationship studies of substituted (S)-2-(benzylideneamino)-3-hydroxy-N-tetradecylpropanamides

A group of novel l-serinamides, substituted (S)-2-(benzylideneamino)-3-hydroxy-N-tetradecylpropanamides (3a–o) and substituted (S)-2-(benzylamino)-3-hydroxy-N-tetradecyl propanamides (4c, 4i, 4l, and 4o), were synthesized as potential anti-tumor lead compounds. In vitro cell viability assay results indicate treatment with 3a–o compounds resulted in significant inhibition of cell viability in the chemoresistant breast cancer cell line, MCF-7TN-R. Compounds 3i and 3l, both ortho-substituted analogs, show the greatest efficacy with IC₅₀ values of 10.3 μM and 12.5 μM, respectively. The SAR analysis indicate that the imine functional group of 3a–o is critical for the cellular anti-viability effect, and the partial atomic charge (PAC) value of imine C atom is a valuable structural parameter for predicting the activity of these ceramide analogs.     

Using the concept of Chou's pseudo amino acid composition for risk type prediction of human papillomaviruses

High-risk types of human papillomaviruses (HPVs) are the etiological agents in nearly all cases (99.7%) of cervical cancer, and the HPV E6 protein is one of the two viral oncoproteins which is expressed in virtually all HPV-positive cancers. Therefore, classifying the risk type of HPVs is very useful and necessary for diagnosis and remedy of cervical cancer. To predict and to classify the risk types of HPV by bioinformatics analysis, 96 E6 protein sequences from available databases were obtained. To investigate the risk type of these sequences, PseAAC server, ROC curves and statistical analysis were applied. Our classification was based on some characters of HPV E6 proteins, such as hydrophobicity, hydrophilicity, side chain mass, PK of the α-COOH group, PK of the α-NH3⁺ group and PI at 25 °C. Risk type of 4 unknown HPV types and 25 non-reported HPV types were also predicted. These results show that bioinformatics based theoretical approaches can direct and simplify experimental studies.     

Structural and functional characterization of a mutant of Pseudocerastes persicus natriuretic peptide

We hereby report on a mutational analysis of a novel natriuretic peptide (PNP), recently isolated by us from the Iranian snake venom. The PNP variant (mutPNP) with four substitutions (G16T, K18S, R21S, G23R) and a disulfide bonded ring shortened by 3 residues. mutPNP peptide was expressed in pET32 and purified by affinity separation on nickel resin followed by RP-HPLC chromatography. The conformation of mutPNP was characterized in solution by 1H nuclear magnetic resonance spectroscopy, where it was found that the 14-residue disulfide bonded ring, like the 17-residue ring in PNP, retains a high degree of conformational flexibility. The conformation of mutPNP bound to NPR-C receptor was predicted by homology protein structure modeling. When injected intravenously into rats, mutPNP, in contrast to PNP had no physiological effect on blood pressure or on diuresis. The loss of physiological activity is explained in terms of the modeled bound conformation and the ensemble of solution conformations obtained using the NMR constraints.     

The cytosolic Na+ : K+ ratio does not explain salinity-induced growth impairment in barley: a dual-tracer study using 42K+ and 24Na+

It has long been believed that maintenance of low Na+ : K+ ratios in the cytosol of plant cells is critical to the plant's ability to tolerate salinity stress. Direct measurements of such ratios, however, have been few. Here we apply the non-invasive technique of compartmental analysis, using the short-lived radiotracers 42K+ and 22Na+, in intact seedlings of barley (Hordeum vulgare L.), to evaluate unidirectional plasma membrane fluxes and cytosolic concentrations of K+ and Na+ in root tissues, under eight nutritional conditions varying in levels of salinity and K+ supply. We show that Na+ : K+ ratios in the cytosol of root cells adjust significantly across the conditions tested, and that these ratios are poor predictors of the plant's growth response to salinity. Our study further demonstrates that Na+ is subject to rapid and futile cycling at the plasma membrane at all levels of Na+ supply, independently of external K+, while K+ influx is reduced by Na+, from a similar baseline, and to a similar extent, at both low and high K+ supply. We compare our results to those of other groups, and conclude that the maintenance of the cytosolic Na+ : K+ ratio is not central to plant survival under NaCl stress. We offer alternative explanations for sodium sensitivity in relation to the primary acquisition mechanisms of Na+ and K+.     

Molecular markers in medicinal plant biotechnology: past and present

Plant based medicines have gained popularity worldwide due to their almost negligible side effects. In India, the three traditional medicinal systems, namely homeopathy, Ayurveda and Siddha rely heavily on plants for medicinal formulations. To prevent the indiscriminate collection of these valuable medicinal plants and for their proper authentication and conservation, it is imperative to go for sustained efforts towards proper germplasm cataloguing and devising conservation strategies. For this purpose, molecular markers have a significant role, as they provide information ranging from diversity at nucleotide level (single nucleotide polymorphisms) to gene and allele frequencies (genotype information), the extent and distribution of genetic diversity, and population structure. Over the past twenty years, the molecular marker field has completely transformed the meaning of conservation genetics which has emerged from a theory-based field of population biology to a full-fledged pragmatic discipline. In this review, we have explored the transition and transformation of molecular marker technologies throughout these years.