VopA inhibits ATP binding by acetylating the catalytic loop of MAPK kinases

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors. One of these effectors, Vibrio outer protein A (VopA), inhibits MAPK signaling via a novel mechanism, distinct from those described for other bacterial toxins, that disrupts this signaling pathway. VopA is an acetyltransferase that potently inhibits MAPK signaling pathways not only by preventing the activation of MAPK kinases (MKKs) but also by inhibiting the activity of activated MKKs. VopA acetylates a conserved lysine found in the catalytic loop of all kinases and blocks the binding of ATP, but not ADP, on the MKKs, resulting in an inactive phosphorylated kinase. Acetylation of this conserved lysine inhibits kinase activity by a new mechanism of regulation that has not been observed previously. Identifying the target of VopA reveals a way that the reversible post-translational modification of lysine acetylation can be used to regulate the activity of an enzyme.     

Patterns of nucleotide sequence variation in <Emphasis Type="Italic">ICAM1</Emphasis> and <Emphasis Type="Italic">TNF</Emphasis> genes in twelve ethnic groups of India: roles of demographic history and natural selection

We have studied DNA sequence variation in and around the genes ICAM1 and TNF, which play functional and correlated roles in inflammatory processes and immune cell responses, in 12 diverse ethnic groups of India, with a view to investigating the relative roles of demographic history and natural selection in shaping the observed patterns of variation. The total numbers of single nucleotide polymorphisms (SNPs) detected at the ICAM1 and TNF loci were 29 and 12, respectively. Haplotype and allele frequencies differed significantly across populations. The site frequency spectra at these loci were significantly different from those expected under neutrality, and showed an excess of intermediate-frequency variants consistent with balancing selection. However, as expected under balancing selection, there was no significant reduction of F _ST values compared to neutral autosomal loci. Mismatch distributions were consistent with population expansion for both loci. On the other hand, the phylogenetic network among haplotypes for the TNF locus was similar to expectations under population expansion, while that for the ICAM1 was as expected under balancing selection. Nucleotide diversity at the ICAM1 locus was an order of magnitude lower in the promoter region, compared to the introns or exons, but no such difference was noted for the TNF gene. Thus, we conclude that the pattern of nucleotide variation in these genes has been modulated by both demographic history and selection. This is not surprising in view of the known allelic associations of several polymorphisms in these genes with various diseases, both infectious and noninfectious.     

Potential application of cyclic lipopeptide biosurfactants produced by Bacillus subtilis strains in laundry detergent formulations

AIMS: Crude cyclic lipopeptide (CLP) biosurfactants from two Bacillus subtilis strains (DM-03 and DM-04) were studied for their compatibility and stability with some locally available commercial laundry detergents. METHODS AND RESULTS: CLP biosurfactants from both B. subtilis strains were stable over the pH range of 7.0-12.0, and heating them at 80 degrees C for 60 min did not result in any loss of their surface-active property. Crude CLP biosurfactants showed good emulsion formation capability with vegetable oils, and demonstrated excellent compatibility and stability with all the tested laundry detergents. CONCLUSION: CLP biosurfactants from B. subtilis strains act additively with other components of the detergents to further improve the wash quality of detergents. The thermal resistance and extreme alkaline pH stability of B. subtilis CLP biosurfactants favour their inclusion in laundry detergent formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has great significance because it is already known that microbial biosurfactants are considered safer alternative to chemical or synthetic surfactants owing to lower toxicity, ease of biodegradability and low ecological impact. The present study provides further evidence that CLP biosurfactants from B. subtilis strains can be employed in laundry detergents.     

The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein

Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem–loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA subunits, namely the RPA70 kDa and RPA32 kDa, were isolated from pea and their roles were validated in a yeast system in which MYMIV DNA replication has been modelled. Here, we present evidences that only the RPA32 kDa subunit directly interacted with the carboxy terminus of MYMIV-Rep both in vitro as well as in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating its nicking and closing activities. The possible role of these modulations in the context of viral DNA replication has been discussed. Finally, we showed the positive involvement of RPA32 in transient replication of the plasmid DNA bearing MYMIV replication origin using an in planta based assay.     

Magnesium promotes structural integrity and conformational switching action of a calcium sensor protein

Calcium binding proteins carry out various signal transduction processes upon binding to Ca2+. In general, these proteins perform their functions in a high background of Mg2+. Here, we report the role of Mg2+ on a calcium sensor protein from Entamoeba histolytica (EhCaBP), containing four Ca2+-binding sites. Mg2+-bound EhCaBP exists as a monomer with a conformation different from that of the holo- and apo-EhCaBP. NMR and biophysical data on EhCaBP demonstrate that Mg2+ stabilizes the closed conformation of the apo form. In the presence of Mg2+, the partially collapsed apo-EhCaBP gains stability and structural integrity. Mg2+ binds to only 3 out of 4 calcium binding sites in EhCaBP. The Ca2+ binding affinity and cooperativity of the conformational switching from the "closed" to the "open" state is significantly modulated by the presence of Mg2+. This fine-tuning of the Ca2+ concentration to switch its conformation is essential for CaBPs to carry out the signal transduction process efficiently.     

Inhibition of choline transport by redox-active cholinomimetic bis-catechol reagents

Both N,N'-(2,3-dihydroxybenzyl)-N,N,N',N'-tetramethyl-1,6-hexanediamine dibromide (DTH, 6) and N,N'-(2,3-dihydroxybenzyl)-N,N,N',N'-tetramethyl-1,10-decanediamine dibromide (DTD, 7), which are symmetrical bis-catechol substituted hexamethonium and decamethonium analogues, respectively, were found to inhibit high-affinity choline transport in mouse brain synaptosomes. Inhibitory properties were evaluated using an extraordinarily sensitive capillary electrophoresis method employing electrochemical detection at an enzyme-modified microelectrode. Dose-response curves were generated for each inhibitor and IC(50) values were determined to be 76 microM for 6 and 21 microM for 7. Lineweaver-Burk analysis revealed that both molecules inhibit high-affinity choline uptake by a mixed inhibition mechanism. The K(I) values for 6 and 7 were determined to be 73+/-1 and 31+/-2 microM, respectively. The inhibition properties were further compared to a series of mono-catechol analogues, 3-[(trimethylammonio)methyl]catechol (1), N,N-dimethylepinephrine (4) and 6-hydroxy-N,N-dimethylepinephrine (5), as well as the well-characterized hemicholinium inhibitors, hemicholinium-15 (HC-15, 8) and hemicholinum-3 (HC-3, 9).     

E,E,E-1-(4-Arylamino-4-oxo-2-butenoyl)-3,5-bis(arylidene)-4-piperidones: a topographical study of some novel potent cytotoxins

A series of E,E,E-3,5-bis(arylidene)-1-(4-arylamino-4-oxo-2-butenoyl)-4-piperidones 4 (phenylidene) and 5 (4-nitrophenylidene) were prepared in order to explore the structural features of the N-acyl group which affects the cytotoxic potency. Evaluation toward human Molt 4/C8 and CEM T-lymphocytes revealed that many of the IC(50) figures were submicromolar and lower than melphalan. Marked inhibitory potencies toward murine leukemia L1210 cells were also noted. When evaluated against a panel of human tumor cell lines, three representative compounds in series 4 displayed selective toxicity to leukemia and colon cancer cell lines and were significantly more potent than the reference drug melphalan. Molecular modeling of representative compounds in both series 4 and the analogs, in which the configuration of the olefinic double bond was changed from E to Z (series 3), revealed that the torsion angles of the arylidene aryl rings and locations of the terminal arylaminocarbonyl groups may have contributed to the greater cytotoxic properties displayed in 3. Compounds 4c (3,4-dichlorophenylamino), d (4-methylphenylamino) and 5c (3,4-dichlorophenylamino), d (4-methylphenylamino) inhibited the activity of human N-myristoyltransferase by approximately 50% at concentrations of 50-100 microM. The compounds in series 4 and 5 were well tolerated in a short-term toxicity study in mice.     

A newly discovered post-translational modification--the acetylation of serine and threonine residues

Recent studies on a bacterial virulence factor, YopJ of Yersinia, have led to the realization that the acetylation of serine and threonine residues could be an important form of post-translational modification in eukaryotes. Although the identification of the machinery used for the addition and removal of acetyl groups on serine or threonine residues is in its infancy, the enzymes thus-far studied provide early insight into the mechanism of this newly discovered post-translational modification, and hint at its potential importance. For example, acetylation can compete with phosphorylation targeted to the same residues and could, therefore, alter the course of signaling pathways. What are the implications for signal transduction in eukaryotes and how widespread could acetylation of serine and threonine prove to be?     

Differential utilization of pyrene as the sole source of carbon by Bacillus subtilis and Pseudomonas aeruginosa strains: role of biosurfactants in enhancing bioavailability

AIMS: Our goal is to compare the efficiency of utilization of pyrene as the sole source of carbon for growth and energy by two nonactinomycetous groups of bacteria viz., Bacillus subtilis DM-04 and Pseudomonas aeruginosa mucoid (M) and nonmucoid (NM) strains, isolated from a petroleum-contaminated soil sample of north-east India. METHODS AND RESULTS: Bacillus subtilis DM-04 and P. aeruginosa M and NM bacterial strains were capable of secreting biosurfactant in the culture medium while growing on pyrene and their pyrene utilizing efficiency was demonstrated by correlating the bacterial growth in the presence of pyrene as the sole source of carbon along with a concomitant decrease in pyrene content from the culture medium with respect to time. The biosurfactant secreted by the respective bacterial strains enhanced the apparent solubility of pyrene by factors of 5-7 and influenced the bacterial cell surface hydrophobicity resulting in higher uptake and utilization of pyrene by bacteria. The growth of B. subtilis DM-04 and P. aeruginosa M and NM strains at the expense of pyrene after 96 h showed an assimilation of about 48.0 +/- 1.1% (mean +/- SD) and 32.0 +/- 0.6% (mean +/- SD) of pyrene carbon, respectively, showing differences in metabolism of pyrene by these bacterial strains. CONCLUSIONS: Bacillus subtilis DM-04 strain exhibited higher utilization and cellular assimilation of pyrene compared with P. aeruginosa M and NM strains. Further, the biosurfactants produced by the bacteria under study are capable of enhancing the solubility of pyrene in aqueous media and can influence the cell surface hydrophobicity of the biosurfactant-producing strains that results in a higher uptake of pyrene. SIGNIFICANCE AND IMPACT OF THE STUDY: It may be suggested that the bacteria used in this study are suitable candidates for practical field application for effective in situ bioremediation of pyrene-contaminated sites.