Putrescine-Sensitive (Artifactual) and Insensitive (Biosynthetic) S-Adenosyl-L-Methionine Decarboxylase Activities of Lathyrus sativus Seedlings

The crude extracts of 3-day-old etiolated seedlings of Lathyrus sativus contained two S-adenosyl-L-methionine decarboxylase activities. The artifactual putrescine-dependent activity was due to the H2O2 generated by diamine oxidase (EC 1.4.3.6) of this plant system and was inhibited by catalase. This observation was confirmed by using an electrophoretically and immunologically homogeneous preparation of L. sativus diamine oxidase. In the presence of putrescine, diamine oxidase, in addition to S-adenosylmethionine, decarboxylated L-lysine, L-arginine, L-ornithine, L-methionine and L-glutamic acid to varying degrees. The decarboxylation was not metal-ion dependent. The biosynthetic S-adenosylmethionine decarboxylase (EC 4.1.1.21) was detected after removing diamine oxidase specifically from the crude extracts by employing an immunoaffinity column. This Mg2+ -dependent decarboxylase was not stimulated by putrescine or inhibited by catalase. The enzyme activity was inhibited by semicarbazide, 4-bromo-3-hydroxybenzoylamine dihydrogen phosphate and methylglyoxal-bis (guanylhydrazone). It was largely localized in the shoots of the etiolated seedlings and was purified 40-fold by employing a p-hydroxymercuribenzoate/AH-Sepharose affinity column, which also separated the decarboxylase activity from spermidine synthase.

Salmonella pathogenicity island 2 mediates protection of intracellular Salmonella from reactive nitrogen intermediates.

Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid. A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is essential for virulence in mice, as well as survival and multiplication within macrophages. Reactive nitrogen intermediates (RNI) synthesized by inducible nitric oxide synthase (iNOS) are involved in the control of intracellular pathogens, including S. typhimurium. We studied the effect of Salmonella infection on iNOS activity in macrophages. Immunofluorescence microscopy demonstrated efficient colocalization of iNOS with bacteria deficient in SPI2 but not wild-type Salmonella, and suggests that the SPI2 system interferes with the localization of iNOS and Salmonella. Furthermore, localization of nitrotyrosine residues in the proximity was observed for SPI2 mutant strains but not wild-type Salmonella, indicating that peroxynitrite, a potent antimicrobial compound, is excluded from Salmonella-containing vacuoles by action of SPI2. Altered colocalization of iNOS with intracellular Salmonella required the function of the SPI2-encoded type III secretion system, but not of an individual "Salmonella translocated effector." Inhibition of iNOS increased intracellular proliferation of SPI2 mutant bacteria and, to a lesser extent, of wild-type Salmonella. The defect in systemic infection of a SPI2 mutant strain was partially restored in iNOS(-/-) mice. In addition to various strategies to detoxify RNI or repair damage due to RNI, avoidance of colocalization with RNI is important in adaptation of a pathogen to an intracellular life style.

Nitric Oxide Is Involved in Mycobacterium bovis Bacillus Calmette-Guerin-Activated Jagged1 and Notch1 Signaling.

Pathogenic mycobacteria have evolved unique strategies to survive within the hostile environment of macrophages. Modulation of key signaling cascades by NO, generated by the host during infection, assumes critical importance in overall cell-fate decisions. We show that NO is a critical factor in Mycobacterium bovis bacillus Calmette-Guérin-mediated Notch1 activation, as the generation of activated Notch1 or expression of Notch1 target genes matrix metalloproteinase-9 (MMP-9) or Hes1 was abrogated in macrophages derived from inducible NO synthase (iNOS) knockout (iNOS(-/-)), but not from wild-type, mice. Interestingly, expression of the Notch1 ligand Jagged1 was compromised in M. bovis bacillus Calmette-Guérin-stimulated iNOS(-/-) macrophages, and loss of Jagged1 expression or Notch1 signaling could be rescued by NO donors. Signaling perturbations or genetic approaches implicated that robust expression of MMP-9 or Hes1 required synergy and cross talk between TLR2 and canonical Notch1-PI3K cascade. Further, CSL/RBP-Jk contributed to TLR2-mediated expression of MMP-9 or Hes1. Correlative evidence shows that, in a murine model for CNS tuberculosis, this mechanism operates in vivo only in brains derived from WT but not from iNOS(-/-) mice. Importantly, we demonstrate the activation of Notch1 signaling in vivo in granulomatous lesions in the brains of Mycobacterium tuberculosis-infected human patients with tuberculous meningitis. Current investigation identifies NO as a pathological link that modulates direct cooperation of TLR2 with Notch1-PI3K signaling or Jagged1 to regulate specific components of TLR2 responses. These findings provide new insights into mechanisms by which Notch1, TLR2, and NO signals are integrated in a cross talk that modulates a defined set of effector functions in macrophages.

Metabolic enzymes as potential drug targets in Plasmodium falciparum.

Plasmodium falciparum causes the most severe form of malaria that is fatal in many cases. Emergence of drug resistant strains of P. falciparum requires that new drug targets be-identified. This review considers in detail enzymes of the glycolytic pathway, purine salvage pathway, pyrimidine biosynthesis and proteases involved in catabolism of haemoglobin. Structural features of P. falciparum triosephosphate isomerase which could be exploited for parasite specific drug development have been highlighted. Utility of P. falciparum hypoxanthine-guanine-phosphoribosyltransferase, adenylosuccinate synthase, dihydroorotate dehydrogenase, thymidylate synthase-dihydrofolate reductase, cysteine and aspartic proteases have been elaborated in detail. The review also briefly touches upon other potential targets in P. falciparum

H-1, C-13, N-15 assignments of the dimeric regulatory subunit (ilvN) of the E. coli AHAS I

Acetohydroxyacid synthase (AHAS) is an enzyme involved in the biosynthesis of the branched chain amino acids viz, valine, leucine and isoleucine. The activity of this enzyme is regulated through feedback inhibition by the end products of the pathway. Here we report the backbone and side-chain assignments of ilvN, the 22 kDa dimeric regulatory subunit of E. coli AHAS isoenzyme I, in the valine bound form. Detailed analysis of the structure of ilvN and its interactions with the catalytic subunit of E. coli AHAS I will help in understanding the mechanism of activation and regulation of the branched chain amino acid biosynthesis.

Involvement of heme in the transcriptional activation of CYPIIB1/B2 gene by phenobarbitone in rat liver—Studies with succinylacetone

Earlier studies in this laboratory had implicated heme to function as a positive modulator of phenobarbitonemediated activation of CYPIIB1/B2 gene transcription in rat liver. However, recent reports have indicated that succinylacetone, a specific inhibitor of δ-aminolevulinate dehydrase, does not affect this process. The present studies indicate that succinylacetone does inhibit the phenobarbitone-mediated increase in CYPIIB1/B2 mRNAs and their transcription in rat liver at early time points (45 min to 3 h), but the inhibition is not pronounced at later time points (16 h). Succinylacetone is a weaker inhibitor of heme biosynthesis than CoCl2, 3-amino-1,2,4-triazole, or thioacetamide used earlier in this laboratory. Succinylacetone induces δ-aminolevulinate synthase, whereas the other compounds depress the levels of the enzyme. There is a good correlation between the amount of freshly synthesized nuclear heme pool and the activation of CYPIIB1/B2 transcription by phenobarbitone. A model implicating a nuclear heme pool regulating the transcription of δ-aminolevulinate synthase, CYPIIB1/ B2, and heme oxygenase genes is proposed.

The primary structure of sheep liver cytosolic serine hydroxymethyltransferase and an analysis of the evolutionary relationships among serine hydroxymethyltransferases

The complete amino-acid sequence of sheep liver cytosolic serine hydroxymethyltransferase was determined from an analysis of tryptic, chymotryptic, CNBr and hydroxylamine peptides. Each subunit of sheep liver serine hydroxymethyltransferase consisted of 483 amino-acid residues. A comparison of this sequence with 8 other serine hydroxymethyltransferases revealed that a possible gene duplication event could have occurred after the divergence of animals and fungi. This analysis also showed independent duplication of SHMT genes in Neurospora crassa. At the secondary structural level, all the serine hydroxymethyltransferases belong to the alpha/beta category of proteins. The predicted secondary structure of sheep liver serine hydroxymethyltransferase was similar to that of the observed structure of tryptophan synthase, another pyridoxal 5'-phosphate containing enzyme, suggesting that sheep liver serine hydroxymethyltransferase might have a similar pyridoxal 5'-phosphate binding domain. In addition, a conserved glycine rich region, G L Q G G P, was identified in all the serine hydroxymethyltransferases and could be important in pyridoxal 5'-phosphate binding. A comparison of the cytosolic serine hydroxymethyltransferases from rabbit and sheep liver with other proteins sequenced from both these sources showed that serine hydroxymethyltransferase was a highly conserved protein. It was slightly less conserved than cytochrome c but better conserved than myoglobin, both of which are well known evolutionary markers. C67 and C203 were specifically protected by pyridoxal 5'-phosphate against modification with [C-14]iodoacetic acid, while C247 and C261 were buried in the native serine hydroxymethyltransferase. However, the cysteines are not conserved among the various serine hydroxymethyltransferases. The exact role of the cysteines in the reaction catalyzed by serine hydroxymethyltransferase remains to be elucidated.

Vasomodulatory effect of novel peroxovanadate compounds on rat aorta: Role of rho kinase and nitric oxide/cGMP pathway

The present study was undertaken to assess the role of reactive oxygen species (ROS) in rat aortic ring vasoreactivity and integrity by using various peroxovanadate (pV) compounds. All the pV compounds (1 nM-300 mu M) used in the present study exerted concentration-dependent contractions on endothelium intact rat aortic rings. All compounds with an exception of DPV-asparagine (DPV-asn) significantly altered vascular integrity as shown by diminished KCl responses. Phenylephrine (PE)-mediated contractions (3 nM-300 mu M) were unaltered in the presence of these compounds. Acetylcholine (Ach)-mediated relaxation in PE (1 mu M) pre-contracted rings was significantly reduced in presence of diperoxovanadate (DPV), poly (sodium styrene sulfonate-co-maleate)-pV (PSS-CoM-pV) and poly (sodium styrene 4-sulfonate)-pV (PSS-pV). However, no significant change in Ach-mediated responses was observed in the presence of poly (acrylate)-pV (PM-pV) and DPV-asn. DPV-asn was thus chosen to further elucidate mechanism involved in peroxide mediated modulation of vasoreactivity. DPV-asn (30 nM-300 mu M) exerted significantly more stable contractions, that was found to be catalase (100 U/ml) resistant in comparison with H(2)O(2) (30 nM-300 mu M) in endothelium intact aortic rings. These contractile responses were found to be dependent on extracellular Ca(2+) and were significantly inhibited in presence of ROS scavenger N-acetylcysteine (100 mu M). Intracellular calcium chelation by BAPTA-AM (10 mu M) had no significant effect on DPV-asn (30 nM-300 mu M) mediated contraction. Pretreatment of aortic rings by rho-kinase inhibitor Y-27632 (10 mu M) significantly inhibited DPV-asn-mediated vasoconstriction indicating role of voltage-dependent Ca(2+) influx and downstream activation of rho-kinase. The small initial relaxant effect obtained on addition of DPV-asn (30 nM-1 mu M) in PE (1 mu M) pre-contracted endothelium intact rings, was prevented in the presence of guanylate cyclase inhibitor, methylene blue (10 mu M) and/or nitric oxide synthase (NOS) inhibitor, L-NAME (100 mu M) suggesting involvement of nitric oxide and cGMP. DPV-asn, like H(2)O(2), exerted a response of vasoconstriction in normal arteries and vasodilation at low concentrations (30 nM-1 mu M) in PE-pre contracted rings with overlapping mechanisms. These findings suggest usefulness of DPV-asn having low toxicity, in exploring the peroxide-mediated effects on various vascular beds. The present study also convincingly demonstrates role of H(2)O(2) in the modulation of vasoreactivity by using stable peroxide DPV-asn and warrants future studies on peroxide mediated signaling from a newer perspective. (C) 2011 Published by Elsevier Ltd.

Crystal structures of open and closed forms of D-serine deaminase from Salmonella typhimurium - implications on substrate specificity and catalysis

Metabolism of D-amino acids is of considerable interest due to their key importance in cell structure and function. Salmonella typhimurium D-serine deaminase (StDSD) is a pyridoxal 5' phosphate (PLP) dependent enzyme that catalyses degradation of D-Ser to pyruvate and ammonia. The first crystal structure of D-serine deaminase described here reveals a typical Foldtype II or tryptophan synthase beta subunit fold of PLP-dependent enzymes. Although holoenzyme was used for crystallization of both wild-type StDSD (WtDSD) and selenomethionine labelled StDSD (SeMetDSD), significant electron density was not observed for the cofactor, indicating that the enzyme has a low affinity for the cofactor under crystallization conditions. Interestingly, unexpected conformational differences were observed between the two structures. The WtDSD was in an open conformation while SeMetDSD, crystallized in the presence of isoserine, was in a closed conformation suggesting that the enzyme is likely to undergo conformational changes upon binding of substrate as observed in other Foldtype II PLP-dependent enzymes. Electron density corresponding to a plausible sodium ion was found near the active site of the closed but not in the open state of the enzyme. Examination of the active site and substrate modelling suggests that Thr166 may be involved in abstraction of proton from the C alpha atom of the substrate. Apart from the physiological reaction, StDSD catalyses a, b elimination of D-Thr, D-Allothr and L-Ser to the corresponding alpha-keto acids and ammonia. The structure of StDSD provides a molecular framework necessary for understanding differences in the rate of reaction with these substrates.

Phylogenetic Predictions on Grids

A phylogenetic or evolutionary tree is constructed from a set of species or DNA sequences and depicts the relatedness between the sequences. Predictions of future sequences in a phylogenetic tree are important for a variety of applications including drug discovery, pharmaceutical research and disease control. In this work, we predict future DNA sequences in a phylogenetic tree using cellular automata. Cellular automata are used for modeling neighbor-dependent mutations from an ancestor to a progeny in a branch of the phylogenetic tree. Since the number of possible ways of transformations from an ancestor to a progeny is huge, we use computational grids and middleware techniques to explore the large number of cellular automata rules used for the mutations. We use the popular and recurring neighbor-based transitions or mutations to predict the progeny sequences in the phylogenetic tree. We performed predictions for three types of sequences, namely, triose phosphate isomerase, pyruvate kinase, and polyketide synthase sequences, by obtaining cellular automata rules on a grid consisting of 29 machines in 4 clusters located in 4 countries, and compared the predictions of the sequences using our method with predictions by random methods. We found that in all cases, our method gave about 40% better predictions than the random methods.

Binding of BIS like and other ligands with the GSK-3 beta kinase: a combined docking and MM-PBSA study

Binding of several bisindolylmaleimide (BIS) like (BIS-3, BIS-8 and UCN1) and other ligands (H89, SB203580 and Y27632) with the glycogen synthase kinase-3 (GSK-3 beta) has been studied using combined docking, molecular dynamics and Poisson-Boltzmann surface area analysis approaches. The study generated novel binding modes of these ligands that can rationalize why some ligands inhibit GSK-3 beta while others do not. The relative binding free energies associated with these binding modes are in agreement with the corresponding measured specificities. This study further provides useful insight regarding possible existence of multiple conformations of some ligands like H89 and BIS-8. It is also found that binding modes of BIS-3, BIS-8 and UCN1 with GSK-3 beta and PDK1 kinases are similar. These new insights are expected to be useful for future rational design of novel, more potent GSK-3 beta-specific inhibitors as promising therapeutics.

Photocytotoxic organometallic compounds

Organometallic compounds have recently found applications in medicinal chemistry and as diagnostic tools in chemical biology. Naturally occurring biomolecules, viz., cobalamine, NiFe hydrogenase, Acetyl-CoA synthase, etc., also contain metal-carbon bonds. Among organometallic compounds having medicinal importance, (arene)ruthenium complexes, radioactive technetium complexes and ferrocene conjugates are notable ones. Applications of photoactive organometallic complexes or metal complexes conjugated with an organometallic moiety are of recent origin. Photodynamic therapy (PDT) is a promising method to treat cancer cells in presence of light. This review primarily focuses on different aspects of the chemistry of organometallic complexes showing photocytotoxic activities. Half-sandwich tungsten, iron or ruthenium complexes are known to show photonuclease and/or photo-crosslinking activity. Photoinduced organometallic CO releasing molecules also exert photocytotoxic activity. Attempts have been made in this review to highlight the photocytotoxic behavior of various metal complexes when conjugated with a photoactive organometallic moiety, viz., ferrocene.

Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus

Background: Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines. Methods: Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-gamma production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus. Results: We identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-alpha and IFN-gamma) in T cells from non-human primates (NHPs) after BCG vaccination. Conclusions: PBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

Hypoxia-Mediated Impairment of the Mitochondrial Respiratory Chain Inhibits the Bactericidal Activity of Macrophages

In infected tissues oxygen tensions are low. As innate immune cells have to operate under these conditions, we analyzed the ability of macrophages (M phi) to kill Escherichia coli or Staphylococcus aureus in a hypoxic microenvironment. Oxygen restriction did not promote intracellular bacterial growth but did impair the bactericidal activity of the host cells against both pathogens. This correlated with a decreased production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and inducible NO synthase (NOS2) double-deficient M phi revealed that in E. coli- or S. aureus-infected cells the reduced antibacterial activity during hypoxia was either entirely or partially independent of the diminished PHOX and NOS2 activity. Hypoxia impaired the mitochondrial activity of infected M phi. Inhibition of the mitochondrial respiratory chain activity during normoxia (using rotenone or antimycin A) completely or partially mimicked the defective antibacterial activity observed in hypoxic E. coli-or S. aureus-infected wild-type M phi, respectively. Accordingly, inhibition of the respiratory chain of S. aureus-infected, normoxic PHOX-/- NOS2(-/-) M phi further raised the bacterial burden of the cells, which reached the level measured in hypoxic PHOX-/- NOS2(-/-) M phi cultures. Our data demonstrate that the reduced killing of S. aureus or E. coli during hypoxia is not simply due to a lack of PHOX and NOS2 activity but partially or completely results from an impaired mitochondrial antibacterial effector function. Since pharmacological inhibition of the respiratory chain raised the generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded as the mechanism underlying the antimicrobial activity of mitochondria.

Retrobiosynthetic Approach Delineates the Biosynthetic Pathway and the Structure of the Acyl Chain of Mycobacterial Glycopeptidolipids

Glycopeptidolipids (GPLs) are dominant cell surface molecules present in several non-tuberculous and opportunistic mycobacterial species. GPLs from Mycobacterium smegmatis are composed of a lipopeptide core unit consisting of a modified C-26-C-34 fatty acyl chain that is linked to a tetrapeptide (Phe-Thr-Ala-alaninol). The hydroxyl groups of threonine and terminal alaninol are further modified by glycosylations. Although chemical structures have been reported for 16 GPLs from diverse mycobacteria, there is still ambiguity in identifying the exact position of the hydroxyl group on the fatty acyl chain. Moreover, the enzymes involved in the biosynthesis of the fatty acyl component are unknown. In this study we show that a bimodular polyketide synthase in conjunction with a fatty acyl-AMP ligase dictates the synthesis of fatty acyl chain of GPL. Based on genetic, biochemical, and structural investigations, we determine that the hydroxyl group is present at the C-5 position of the fatty acyl component. Our retrobiosynthetic approach has provided a means to understand the biosynthesis of GPLs and also resolve the long-standing debate on the accurate structure of mycobacterial GPLs.