Antifolate Activity of Plant Polyphenols against Mycobacterium tuberculosis.

With the view of exploring phytochemicals as Mycobacterium tuberculosis (Mtb) dihydrofolate reductase inhibitors, known plant polyphenols from various classes were subjected to detailed docking studies. From this in-silico screening, seven polyphenols were selected and tested against Mtb H37 Rv in whole cell assays. The phytochemicals exhibited potential activity ranging from 3 to 183 µm. These molecules were then tested against the pathogenic and human enzymes in a high-throughput microtitre assay. Epigallocatechin gallate showed the best activity and selectivity. The in-silico analysis was in agreement with the assay results. Of these 7 polyphenols, 5 exhibiting minimum inhibitory concentration values of ≤15 µm were tested for synergistic activity with first line drug Ethambutol and second line folate inhibitor para-amino salicylic acid. Epigallocatechin gallate, Magnolol and Bakuchiol exhibited moderate synergistic association by lowering the minimum inhibitory concentration of these drugs. These simple phytochemicals could hence be considered as leads for further studies, or for preparation of semi-synthetic derivatives to be used in combination therapy, for increased anti-tuberculosis activity after validation in-vivo.

Staphylococcal septicaemia associated with peripheral neuropathy in three different clinical settings.

Among the various etiologies of peripheral neuropathy, S. aureus is a rare cause that is not even mentioned in standard textbooks. Here we like to report three clinical scenarios where patients with different manifestations of S. aureus infection developed peripheral neuropathy presenting as quadriparesis, which subsided gradually with control of infection and supportive care. No other known causes of peripheral neuropathy were present in these cases.

Design, synthesis, and biological evaluation of 4-(5-nitrofuran-2-yl)prop-2-en-1-one derivatives as potent antitubercular agents.

Based on stereoelectronic feature analysis using density functional theory (DFT) at B3LYP/3-21∗G level, a series of 4-(5-nitrofuran-2-yl)prop-2-en-1-one derivatives with low LUMO energies (<-0.10eV); concentrated over the nitro group, furan moiety and α,ß-unsaturated carbonyl bridge were envisaged as potential antitubercular agents. The target compounds were prepared by condensation of 5-nitro-2-furaldehyde with various ketones under acidic condition. The compounds were evaluated for antitubercular activity against Mycobacterium tuberculosis H37Rv and their cytotoxicity in VERO cell line. Several synthesized compounds showed good antitubercular activity of <5µM along with low cytotoxicity. In particular, compound ((E)-3-(5-nitrofuran-2-yl)-1-(4-(piperidin-1-yl)phenyl)prop-2-en-1-one) (3v) was found to be very potent (MIC: 0.19µM) with good selectivity index (MIC(90)/CC(50): >1800). Thus, this study shows the potential of stereoelectronic property analysis in developing improved nitroaromatics as antitubercular agents.

Novel molecular hybrids of cinnamic acids and guanylhydrazones as potential antitubercular agents.

In an attempt to identify potential new agents active against tuberculosis, 20 novel phenylacrylamide derivatives incorporating cinnamic acids and guanylhydrazones were synthesized using microwave assisted synthesis. Activity of the synthesized compounds was evaluated using resazurin microtitre plate assay (REMA) against Mycobacterium tuberculosis H37Rv. Based on empirical structure-activity relationship data it was observed that both steric and electronic parameters play major role in the activity of this series of compounds. Compound 7s (2E)-N-((-2-(3,4-dimethoxybenzylidene) hydrazinyl) (imino) methyl)-3-(4-methoxyphenyl) acrylamide showed MIC of 6.49microM along with good safety profile of >50-fold in VERO cell line. Thus, this compound could act as a potential lead for further antitubercular studies.

Mass spectral analysis of acetylated peptides: Implications in proteomics.

Sequence determination of peptides using mass spectrometry plays a crucial role in the bottom-up approaches for the identification of proteins. It is crucially important to minimise false detection and validate sequence of the peptides in order to correctly identify a protein. Chemical modification of peptides followed by mass spectrometry is an option for improving the spectral quality. In silico-derived tryptic peptides with different N-terminal amino acids were designed from human proteins and synthesized. The effect of acetylation on the fragmentation of peptides was studied. N-terminal acetylation of the tryptic peptides was shown to form b1-ions, improve the abundance and occurrence of b-ions. In some cases, the intensity and occurrence of some y-ions also varied. Thus, it is demonstrated that acetylation plays an important role in improving the de novo sequencing efficiency of the peptides. The acetylation method was extended to tryptic peptides generated from the proteome of an Antarctic bacterium Pseudomonas syringae Lz4W using the proteomics work flow and mass spectra of the peptides were analysed. Comparison of the MS/MS spectra of the acetylated and unacetylated peptides revealed that acetylation helped in improving the spectral quality and validated the peptide sequences. Using this method, 673 proteins of the 1070 proteins identified were validated.

Transcriptional regulation of a mouse Clara cell-specific protein (mCC10) gene by the NKx transcription factor family members thyroid transciption factor 1 and cardiac muscle-specific homeobox protein (CSX).

This report defines the elements between bp -800 and -166 that regulate the quantitative level of mouse CC10 (mCC10) transcription in the lungs. The elements in this promoter domain are the response elements for the NKx2.1 homeobox protein, thyroid transcription factor 1 (TTF1). DNase I footprint analysis identified five binding sites for TTF1 between bp -800 and - 166. These sites are located at bp -344 to -335, - 282 to -273, -268 to -263, -258 to -249, and - 199 to - 190. In addition to these enhancer elements, two TTF1 binding sites were identified in the proximal promoter region (bp - 166 to + 1), at bp -74 to -69 and -49 to -39. An identical footprint of the mCC10 promoter region was also observed with another member of the NKx family, NKx 2.5, the cardiac muscle-specific homeobox protein (CSX). Deletion and linker-scanner mutational analyses of the TTF1 binding sites in the mCC10 distal promoter region with transient cotransfection into CV1 cells with either TTF1 or CSX identified the site located between bp -282 and -273 as the major regulator of CC10 expression, with minor regulation by sites at bp -344 to -335 and -258 to -249. The importance of the NKx binding site at bp -282 to -273 was verified in vivo. Transgenic mice generated with the human growth hormone gene fused to 800 bp of the mCC10 promoter containing a mutation in the TTF1 binding site at bp -282 to -273 showed a reduction in transgene expression equal to that of the mice generated with only 166 bp of 5'-flanking DNA. This report emphasizes the importance of TTF1 or related factors as major regulators of pulmonary gene expression and demonstrates the potential of NKx proteins to bind and activate heterologous target genes.

DNA sequence analysis of the Olir2-76 and Ossr1-92 alleles of the Oli-2 region of the yeast Saccharomyces cerevisiae. Analysis of related amino-acid substitutions and protein-antibiotic interaction.

Petite deletion mapping helped to generate a fine-structure genetic map of the Oli-2 region of the mitochondrial genome of Saccharomyces cerevisiae. Here we report the DNA sequence analysis of the Oli-2 region from two drug-resistant alleles (Olir2-76 and Ossr1-92) which are located in the gene for subunit-6 of mitochondrial ATPase, in agreement with their genetic locations on the mitochondrial genome. An analysis of the corresponding amino-acid substitutions is also presented in the context of protein-antibiotic interactions.

E84G mutation in dihydrofolate reductase from drug resistant strains of Mycobacterium tuberculosis (Mumbai, India) leads to increased interaction with Trimethoprim.

BACKGROUND: Dihydrofolate reductase (DHFR) (dfrA gene) is an essential enzyme for cell survival and an unexplored target in Mycobacterium tuberculosis (Mtb). This study was carried out to analyze mutations in the dfrA gene amongst 20 clinical DNA samples from Mtb isolates obtained from Mumbai, India. METHODS: Sequencing of the PCR amplified dfrA gene from these DNA isolates revealed a point mutation in one strain, leading to a glutamic acid to glycine change. In silico simulation studies revealed a surface alteration in the enzyme due to this E84G mutation. The amplified mutant gene was cloned and expressed. The mutant protein was assessed against known DHFR inhibitors: Methotrexate and Trimethoprim. RESULTS: An increased affinity for inhibitor Trimethoprim and native substrate dihydrofolate was observed with the mutant. Methotrexate did not vary in its activity with both the enzyme forms. CONCLUSIONS: The Glu84Gly point mutation may lead to a variation in the strain which may cause resistance in the future.

Design and Synthesis of a Focused Library of Diamino Triazines as Potential Mycobacterium tuberculosis DHFR Inhibitors.

We report design of a series of 2,4-diamino triazines as Mycobacterium tuberculosis (Mtb) dihydrofolate reductase inhibitors. The synthesized compounds were evaluated against Mtb (H37Rv and Dormant stage H37Ra), their cytotoxicity was assessed (HepG2 and A549 cell lines), and selectivity toward Mtb was evaluated by testing against other bacterial strains. Some derivatives showed promising activity along with low cytotoxicity. The most potent compound in the whole cell assay (MIC 0.325 µM against H37Rv) showed selectivity in the enzyme assay and exhibited synergy with second line anti-TB agent p-amino salicylic acid. This study therefore provides promising molecules for further development as antituberculosis DHFR inhibitors.

A RNA polymerase with transcriptional activity at 0 degrees C from the Antarctic bacterium Pseudomonas syringae.

A DNA-dependent RNA polymerase was purified from the Antarctic psychrotrophic bacterium Pseudomonas syringae. The RNA polymerase showed a typical eubacterial subunit composition with beta, beta', alpha2 and sigma subunits. The subunits cross-reacted with antibodies raised against holoenzyme and the individual subunits of the RNA polymerase of Escherichia coli. However, the enzyme was considered unique, since unlike the RNA polymerase of mesophilic E. coli it exhibited significant and consistent transcriptional activity (10-15%) even at 0 degrees C. But, similar to the enzyme from the mesophilic bacterium, the RNA polymerase from P. syringae exhibited optimum activity at 37 degrees C. The study also demonstrates that the RNA polymerase of P. syringae could preferentially transcribe the cold-inducible gene cspA of E. coli only at lower temperatures (0-22 degrees C). The polymerase was also observed to be relatively more rifampicin-resistant during transcription at lower temperature.

Purification and partial characterization of an erythroagglutinin from the hemolymph of scorpion, Heterometrus bengalensis.

An erythroagglutinin from the hemolymph of the scorpion, Heterometrus bengalensis, has been purified by gel filtration and ion-exchange chromatography. Its homogeneity has been demonstrated by polyacrylamide gel electrophoresis. The purified agglutinin appears to be a monomeric protein having a possible molecular weight between 146,000 and 148,000. It has no divalent cation requirement for erythroagglutination. The erythroagglutination is not inhibited by saccharides, glycoproteins and mucin. Identical erythroagglutination pattern is obtained with normal as well as neuraminidase treated erythrocytes.

Immunohistochemical localization of mouse Clara cell 10-KD protein using antibodies raised against the recombinant protein.

To investigate the developmental regulation of the mouse Clara cell 10-KD protein (mCC10), we raised an antibody against the recombinant mCC10 protein. The coding region for the mature mCC10 protein was placed in frame with the glutathione-S-transferase gene in the pGEX2-T bacterial expression vector. The GST-mCC10 fusion protein was expressed in E. coli DH5 alpha cells. The fusion protein was purified and eluted using glutathione-Sepharose beads. The GST-mCC10 fusion protein was injected into rabbits to raise antibodies. The rabbit anti-mCC10 antibody was tested by immunoblot analysis using both purified protein as well as extracts of lung, liver, and uterus. The antibodies produced were used in immunohistochemistry and immunoelectron microscopy to detect the cellular localization of this protein in the above organs. This anti-mCC10 antibody will be useful for future investigation of the developmental biology of the lung.

Insulin secretion is inhibited by subtype five somatostatin receptor in the mouse.

BACKGROUND: Recently five somatostatin receptor subtypes (SSTRs) were cloned, allowing the development of highly specific agonists to these SSTRs. Previous studies have shown a species specificity phenomenon with respect to the inhibition of insulin secretion by these selective agonists. This study was undertaken to determine which SSTR (2 or 5) is responsible for the inhibitory effect of somatostatin on glucose-stimulated mouse insulin secretion. METHODS: Intact mouse islets (n = 10) were stimulated with D-glucose in the presence or absence of receptor-specific somatostatin agonists. RESULTS: D-glucose (16.7 mmol/L) augmented insulin secretion by 158% above that seen with 3.9 mmol/L D-glucose. In the presence of DC 32-92 (SSTR5) selective agonist, D-glucose (16.7 mmol/L) augmented insulin secretion by 64% above that seen with 3.9 mmol/L D-glucose. The presence of SSTR 5 selective agonist resulted in a significant (P < .05) inhibition of glucose-stimulated insulin secretion. The identification of SSTR5 within the mouse pancreas was established by reverse transcriptase polymerase chain reaction and confirmed by Southern blot analysis. CONCLUSIONS: These results suggest that the inhibitory effect of somatostatin on insulin secretion is mediated through the subtype 5 receptor within the mouse islet.

The Cre-loxP system: a versatile tool for targeting genes in a cell- and stage-specific manner.

Gene-targeted mice, derived from embryonic stem cells, are useful tools to study gene function during development. However, if the inactivation of the target gene results in embryonic lethality, the postdevelopmental function of the gene cannot be further studied. The Cre recombinase-loxP (Cre-loxP) system was developed to overcome this limitation as well as to confine the inactivation of the target gene in a cell- or tissue-specific manner. This system allows for the inactivation of the target gene in a single cell type, thereby allowing the analysis of physiological and pathophysiological consequences of the genetic alteration in mature animals. A unique property of the insulin gene to be expressed only in pancreatic beta cells has allowed using the beta-cell-specific rat insulin promoter (RIP) for Cre recombinase expression to inactivate genes in beta cells. The RIP has been used to inactivate genes in beta cells and analysis of these genetically altered mice has provided important information regarding the role of potential transcription factors and the receptors in vivo, for regulation of insulin gene transcription and in the development of beta cells. The Cre-loxP system is at a relatively early stage of development, and the ability of this technique to virtually target any gene in any tissue at any stage of development makes the study of gene function in a single cell type in vivo an attainable goal. It is anticipated that the continued experience with this system will provide an important tool to determine the role of the transcription factors involved in insulin gene regulation and islet cell differentiation and ultimately provide the basis for novel therapy to treat diabetes.

Studies on the cytoplasmic protein tyrosine kinase activity of the Antarctic psychrotrophic bacterium Pseudomonas syringae.

The Antarctic psychrotrophic bacterium Pseudomonas syringae contains a 66-kDa cytoplasmic protein which was found to by phosphorylated on a tyrosine residue [Ray, M.K. et al. (1994) FEMS Microbiol. Lett. 122, pp. 49-54]. To investigate the nature of the cytoplasmic protein tyrosine kinase and its role in the bacterial physiology, we carried out some biochemical studies of the enzyme in vitro in the presence of exogenous peptide substrates and expression studies in vivo at low and high temperature during various phases of growth. The results suggest that the protein tyrosine kinase associated with the cytoplasmic fraction of the bacterium has certain similarities and dissimilarities with the known eukaryotic tyrosine kinases. The protein tyrosine kinase could phosphorylate exogenous substrate corresponding to the N-terminal peptide of p34cdc2 kinase but could not do so on poly(Glu:Tyr). The enzyme could not be inhibited by genistein, staurosporine and dimethyl aminopurine, but could be inhibited by piceatannol which is a known competitive inhibitor of the peptide binding site of mammalian protein tyrosine kinases. The enzyme activity in the cytoplasm is uniquely inhibited by sodium orthovanadate (IC50 = 20 microM) which is a known protein tyrosine phosphatase inhibitor. The expression studies show that the enzyme is produced more at a higher temperature (22 degrees C) of growth than at lower temperature (4 degrees C) and during the stationary phase of growth of P. syringae.

Occurrence and expression of cspA, a cold shock gene, in Antarctic psychrotrophic bacteria.

The homologue of cold shock gene cspA of Escherichia coli was detected in various isolates of Antarctic psychrotrophs representing both Gram-positive and Gram-negative bacteria. The Northern hybridization study indicated that the transcript size of cspA in the psychrotrophic Gram-positive bacterium Arthrobacter protophormiae and Gram-negative Pseudomonas fluorescens was similar to that of E. coli and that the cspA homologues in these two psychrotrophs were expressed constitutively at a low level both at 4 degrees C and 22 degrees C. In P. fluorescens, the expression of cspA mRNA was inducible after shift of temperature from 22 to 4 degrees C and the maximum level of induction occurred after 1 h which correlated with the time-lag required for growth of the culture after temperature shift.

Histidine utilisation operon (hut) is upregulated at low temperature in the antarctic psychrotrophic bacterium Pseudomonas syringae.

The antarctic psychrotrophic bacterium Pseudomonas syringae was mutagenised using a transposon Tn5-OT182 which facilitates identification of promoter fusions expressing the reporter gene (lacZ) for beta-galactosidase. Most mutants expressed beta-galactosidase both at optimal growth temperature (20-22 degrees C) and at low temperature (4 degrees C). But a small percentage of the mutants (approximately 5%) were unique in that they expressed beta-galactosidase activity predominantly at low temperature. One such mutant was found to have an insertion in the gene for urocanase (hutU) of the histidine utilisation (hut) operon. Direct assay of urocanase and histidase activity in wild-type cells of various antarctic psychrotrophic strains including P. syringae, P. fluorescens and P. putida also suggested that the hut operon is expressed at an elevated level at low temperature.

Differential inhibition of insulin and islet amyloid polypeptide secretion by intraislet somatostatin in the isolated perfused human pancreas.

Islet amyloid polypeptide (IAPP) and insulin are co-stored and generally secreted in parallel; however, studies have demonstrated that the IAPP/insulin molar secretory ratio may be altered in response to certain stimuli. Because we previously demonstrated that intraislet somatostatin is an inhibitory regulator of basal insulin secretion in the isolated perfused human pancreas, this study was designed to determine the relative influence on the regulation of IAPP versus insulin secretion. Single-pass perfusion was performed in pancreata obtained from cadaveric organ donors with continuous perfusion of a modified Krebs media with the glucose level maintained at constant 3.9 mM. Intraislet somatostatin was immunoneutralized by the infusion of either a highly sensitive monoclonal somatostatin antibody (SAb) or its FAb fragment (SFAb). Sequential test periods separated by basal periods were performed by infusion of either of the following: glucose, SAb, SFAb, or appropriate controls. IAPP/insulin molar secretory ratio decreased by 33% in response to infusion of either SAb or the SFAb, respectively (p < 0.01), and decreased by 67% in response to glucose infusion (p < 0.01). An alteration of the IAPP/insulin secretory ratio is seen in response to infusion of exogenous glucose or in response to the neutralization of intraislet somatostatin.

Gastrointestinal polyposis syndromes.

Well recognized cutaneointestinal syndromes in which colonic polyps are a constant and defining feature include Gardner's syndrome (familial adenomatous polyposis), Peutz-Jeghers syndrome, and Cronkhite-Canada syndrome. Colonic polyps are also found with increased frequency in Cowden's disease, the Muir-Torre syndrome, and neurofibromatosis. The Ruvalcaba-Myhre-Smith syndrome is a newly defined entity in this category. The relationship between acrochordons and colonic polyps remains a controversial issue, awaiting additional studies.

Role of iron in the enhancement by Agrobacterium tumefaciens infection in mice.

Microorganisms require iron for their growth and usually compete with their host for available iron from the system. Iron supplementation to host causes an increase of available iron both to host and to potential microbial invaders and favours the latter more than the former as the bacteria release siderophores which are responsible for iron transport mechanism. In view of this observation a study was done to deal with the distribution of storage and injected iron given as an overload within a physiological pool, taking mice as the host, with a correlation to its utilization by Agrobacterium tumefaciens and with bacterial growth and multiplication. The results obtained help in understanding the host--parasite relationships, regarding bacterial virulence and infection and the growth-promoting effect of iron, as iron promoted the development and progression of serum-exposed A. tumefaciens in mice.