A Unique Nickel System having Versatile Catalytic Activity of Biological Significance

A new dinuclear nickel(II) complex, [Ni-2(LH2)(H2O)(2)(OH)(NO3)](NO3)(3) (1), of an "end-off" compartmental ligand 2,6-bis(N-ethylpiperazine-iminomethyl)-4-methyl-phenolato, has been synthesized and structurally characterized. The X-ray single crystal structure analysis shows that the piperazine moieties assume the expected chair conformation and are protonated. The complex 1 exhibits versatile catalytic activities of biological significance, viz. catecholase, phosphatase, and DNA cleavage activities, etc. The catecholase activity of the complex observed is very dependent on the nature of the solvent. In acetonitrile medium, the complex is inactive to exhibit catecholase activity. On the other hand, in methanol, it catalyzes not only the oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC) but also tetrachlorocatechol (TCC), a catechol which is very difficult to oxidize, under aerobic conditions. UV vis spectroscopic investigation shows that TCC oxidation proceeds through the formation of an intermediate. The intermediate has been characterized by an electron spray ionizaton-mass spectrometry study, which suggests a bidentate rather than a monodentate mode of TCC coordination in that intermediate, and this proposition have been verified by density functional theory calculation. The complex also exhibits phosphatase (with substrate p-nitrophenylphosphate) and DNA cleavage activities. The DNA cleavage activity exhibited by complex 1 most probably proceeds through a hydroxyl radical pathway. The bioactivity study suggests the possible applications of complex 1 as a site specific recognition of DNA and/or as an anticancer agent.

A Model of Anomalous Enzyme-Catalyzed Gel Degradation Kinetics

We show that a model of target location involving n noninteracting particles moving subdiffusively along a line segment (a generalization of a model introduced by Sokolov et al. [Biophys. J. 2005, 89, 895.]) provides a basis for understanding recent experiments by Pelta et al. [Phys. Rev. Lett. 2007, 98, 228302.] on the kinetics of diffusion-limited gel degradation. These experiments find that the time t(c) taken by the enzyme thermolysin to completely hydrolyze a gel varies inversely as roughly the 3/2 power of the initial enzyme concentration [E]. In general, however, this time would be expected to vary either as [E](-1) or as [E](-2), depending on whether the Brownian diffusion of the enzyme to the site of cleavage took place along the network chains (1-d diffusion) or through the pore spaces (3-d diffusion). In our model, the unusual dependence of t(c) on [E] is explained in terms of a reaction-diffusion equation that is formulated in terms of fractional rather than ordinary time derivatives.

Sequencing of T-superfamily conotoxins from Conus virgo: Pyroglutamic acid identification and disulfide arrangement by MALDI mass spectrometry

De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivorous cone snail Conus virgo, found off the southern Indian coast, is presented. The peptides, whose masses differ only by 2 Da, possess two disulfide bonds and an amidated C-terminus. Simple chemical modifications and enzymatic cleavage coupled with matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis aided in establishing the sequences of Vi1359, ZCCITIPECCRI-NH2, and Vi1361, ZCCPTMPECCRI-NH2, Which differ only at residues 4 and 6 (Z = pyroglutamic acid). The presence of the pyroglutamyl residue at the N-terminus was unambiguously identified by chemical hydrolysis of the cyclic amide, followed by esterification. The presence of Ile residues in both the peptides was confirmed from high-energy collision induced dissociation (CID) studies, using the observation Of W-n- and d(n)-ions as a diagnostic. Differential cysteine labeling, in conjunction with MALDI-MS/MS, permitted establishment of disulfide connectivity in both peptides as Cys2-Cys9 and Cys3-Cys10. The cysteine pattern clearly reveals that the peptides belong to the class of T-superfamily conotoxins, in particular the T-1 superfamily.

Characterization of Alkali Induced Formation of Lanthionine, Trisulfides, and Tetrasulfides from Peptide Disulfides using Negative Ion Mass Spectrometry

Peptide disulfides are unstable under alkaline conditions, resulting in the formation of products containing lanthionine and polysulfied linkages. Electrospray ionization mass spectrometry has been used to characterize major species obtained when cyclic and acyclic peptide disulfides are exposed to alkaline media. Studies on a model cyclic peptide disulfide (Boc - Cys - Pro - Leu - Cys - NHMe) and an acyclic peptide, oxidized glutathione, bis ((gamma)Glu Cys - Gly - COOH), are described. Disulfide cleavage reactions are initiated by the abstraction of (CH)-H-alpha or (CH)-H-beta protons of Cys residues, with Subsequent elimination of H2S or H2S2. The buildup of reactive thiol species which act on intermediates containing dehydroalanine residues, rationalizes the formation of lanthionine and polysulfide products. In the case of the cyclic peptide disulfide, the formation of cyclic products is facilitated by the intramolecular nature of the Michael addition reaction of thiols to the dehydroalanine residue. Mass spectral evidence for the intermediate species is presented by using alkylation of thiol groups as a trapping method. Mass spectral fragmentation in the negative ion mode of the peptides derived from trisulfides and tetrasulfides results in elimination of S-2. (J Am Soc Mass Spectrom 2009, 20, 783-791) (C) 2009 American Society for Mass Spectrometry.

Characterization of Alkali Induced Formation of Lanthionine, Trisulfides, and Tetrasulfides from Peptide Disulfides using Negative Ion Mass Spectrometry

Peptide disulfides are unstable under alkaline conditions, resulting in the formation of products containing lanthionine and polysulfied linkages. Electrospray ionization mass spectrometry has been used to characterize major species obtained when cyclic and acyclic peptide disulfides are exposed to alkaline media. Studies on a model cyclic peptide disulfide (Boc - Cys - Pro - Leu - Cys - NHMe) and an acyclic peptide, oxidized glutathione, bis ((gamma)Glu Cys - Gly - COOH), are described. Disulfide cleavage reactions are initiated by the abstraction of (CH)-H-alpha or (CH)-H-beta protons of Cys residues, with Subsequent elimination of H2S or H2S2. The buildup of reactive thiol species which act on intermediates containing dehydroalanine residues, rationalizes the formation of lanthionine and polysulfide products. In the case of the cyclic peptide disulfide, the formation of cyclic products is facilitated by the intramolecular nature of the Michael addition reaction of thiols to the dehydroalanine residue. Mass spectral evidence for the intermediate species is presented by using alkylation of thiol groups as a trapping method. Mass spectral fragmentation in the negative ion mode of the peptides derived from trisulfides and tetrasulfides results in elimination of S-2. (J Am Soc Mass Spectrom 2009, 20, 783-791) (C) 2009 American Society for Mass Spectrometry.

Structure determination and biochemical studies on Bacillus stearothermophilus E53Q serine hydroxymethyltransferase and its complexes provide insights on function and enzyme memory

Serine hydroxymethyltransferase (SHMT) belongs to the alpha-family of pyridoxal 5'-phosphate-dependent enzymes and catalyzes the reversible conversion of L-Ser and etrahydrofolate to Gly and 5,10-methylene tetrahydrofolate. 5,10-Methylene tetrahydrofolate serves as a source of one-carbon fragment in many biological processes. SHMT also catalyzes the tetrahydrofolate-independent conversion of L-allo-Thr to Gly and acetaldehyde. The crystal structure of Bacillus stearothermophilus SHMT (bsSHMT) suggested that E53 interacts with the substrate, L-Ser and etrahydrofolate. To elucidate the role of E53, it was mutated to Q and structural and biochemical studies were carried out with the mutant enzyme. The internal aldimine structure of E53QbsSHMT was similar to that of the except for significant changes at Q53, Y60 and Y61. The wild-type enzyme, carboxyl of Gly and side chain of L-Ser were in two conformations in the respective external aldimine structures. The mutant enzyme was completely inactive for tetrahydrofolate-depen dent cleavage of L-Ser, whereas there was a 1.5-fold increase in the rate of tetrahydrofolate-independent reaction with L-allo-Thr. The results obtained from these studies suggest that E53 plays an essential role in tetrahydrofolate/5-formyl tetrahydrofolate binding and in the proper positioning of C beta of L-Ser for direct attack by N5 of tetrahydrofolate. Most interestingly, the structure of the complex obtained by cocrystallization of E53QbsSHMT with Gly and 5-formyl tetrahydrofolate revealed the gem-diamine form of pyridoxal 5'-phosphate bound to Gly and active site Lys. However, density for 5-formyl tetrahydrofolate was not observed. Gly carboxylate was in a single conformation, whereas pyridoxal 5'-phosphate had two distinct conformations. The differences between the structures of this complex and Gly external aldimine suggest that the changes induced by initial binding of 5-formyl tetrahydrofolate are retained even though 5-formyl tetrahydrofolate is absent in the final structure. Spectral studies carried out with this mutant enzyme also suggest that 5-formyl tetrahydrofolate binds to the E53QbsSHMT-Gly complex forming a quinonoid intermediate and falls off within 4 h of dialysis, leaving behind the mutant enzyme in the gemdiamine form. This is the first report to provide direct evidence for enzyme memory based on the crystal structure of enzyme complexes.

The 5-Methyl Group in Thymine Dynamically Influences the Structure of A-Tracts in DNA at the Local and Global Level

DNA sequences containing a stretch of several A:T basepairs without a 5'-TA-3' step are known as A-tracts and have been the subject of extensive investigation because of their unique structural features such as a narrow minor groove and their crucial role in several biological processes. One of the aspects under investigation has been the influence of the 5-methyl group of thymine on the properties of A-tracts. Detailed molecular dynamics simulation studies of the sequences d(CGCAAAUUUGCG) and d(CGCAAATTTGCG) indicate that the presence of the 5-methyl group in thymine increases the frequency of a narrow minor groove conformation, which could facilitate its specific recognition by proteins, and reduce its susceptibility to cleavage by DNase I. The bias toward a wider minor groove in the absence of the thymine 5-methyl group is a static structural feature. Our results also indicate that the presence of the thymine 5-methyl group is necessary for calibrating the backbone conformation and the basepair and dinucleotide step geometry of the core A-tract as well as the flanking CA/TG and the neighboring GC/GC steps, as observed in free and protein-bound DNA. As a consequence, it also fine-tunes the curvature of the longer DNA fragment in which the A-tract is embedded.

Purification and properties of uridine hydrolase from mung-bean (Phaseolus radiatus) seedlings

The occurrence in plants of an enzyme system catalyzing the cleavage of uridine has been demonstrated. The enzyme from Phaseolus radiatus was purified about 132-fold with 24% recovery by a combination of procedures involving mild acid treatment, ammonium sulphate fractionation, negative adsorption on calcium phosphate gel and DEAE-cellulose chromatography. The enzyme cleaves uridine to uracil and ribose in the absence of phosphate indicating that the mechanism of cleavage was hydrolytic rather than phosphorolytic. The enzyme is specific to uridine and does not act on other purine and pyrimidine compounds. The enzyme shows maximum activity at pH 7.4 and has a temperature optimum of 45 °. It does not require metal ions for activity. Inhibition of the enzyme by p-chloromercuribenzoate as well as N-ethylmaleimide and the reversal of p-chloromercuribenzoate inhibition by sulfhydryl agents indicate the probable involvement of readily oxidizable sulfhydryl groups in enzyme activity.

Conversion of isophenoxazine to catechol in Tecoma stans

Isophenoxazine, formed by the condensation of two molecules of o-aminophenol, is reduced by an enzyme system from Tecoma stans leaves to two molecules of catechol. The reaction proceeds well under anaerobic conditions; a 1–2 mole stoichiometry between the substrate disappeared and the product formed was maintained. The enzyme showed maximum activity at pH 5. The substrate at high concentrations caused a diminution in the activity and the optimum concentration of substrate was at 6 × 10−4 Image . The enzyme preparation was able to convert cinnabarinic acid and diphenylene dioxide 2,3-quinone into the corresponding catechol substances. The diphenylene dioxide 2,3-quinone at the same concentration was three times more susceptible to enzymic cleavage than isophenoxazine. Cinnabarinic acid inhibited the enzymic cleavage of isophenoxazine competitively. None of the known electron donors was found to activate the reaction. Inhibition studies suggested that intact sulfhydryl groups are necessary for enzyme activity. Heavy metal ions like Hg++, Ag+, Co++, Fe++, Ni++, and Fe3++ inhibited the reaction. Metal chelating agents did not have any effect on the enzyme.

Applicability of partition ratios, mercuric chloride complexes, and chromatographic behavior for the identification of carotenoids

Partition ratios and M50 values of different carotenoids in hexaneaqueous methanol were determined. Mercuric chloride complexes of 14 epoxy carotenoids were prepared and their absorption maxima in acetone were estimated. The difference in chromatographic behavior of carotenoid epoxides on alumina and magnesium oxide-Celite columns is discussed. It is shown that the magnesium oxide-Celite column behaves as a reverse-phase chromatographic column to alumina column.

Carotenoids In 3 Stages Of Ripening Of Mango

The distribution of carotenoids, both qualitative and quantitative, during 3 stages of ripening of mango has been studied using chromatographic, spectroscopic and chemical methods. There was an increase in content as well as in number of carotenoids during ripening. The present study showed there were 15, 14 and 17 different carotenoids in the unripe, partially ripe and fully ripe mangoes, respectively. Even though phytofluene (39.26%) was the major carotenoid in the partially ripe mango, β-carotene constituted the major carotenoid in the unripe (37.47%) and fully ripe mango (50.64%). cis-β-Carotene was present only in the fully ripe mango. Only the unripe mango contained ζ-carotene, whereas γ-carotene was present in all the 3 stages of ripening. The major xanthophyll present in the unripe mango was mutatoxanthin (9.44%), whereas auroxanthin constituted the major hydroxylated carotenoid of the partially ripe (5.07%) and fully ripe (10.40%) mangoes. The percent of cryptoxanthin dropped to lower levels during ripening. As ripening proceeded, lutein completely is appeared. There were significant quantities of eaxanthin in the partially ripe and fully ripe mango. Epoxy carotenoids such as 5,6-monoepoxy-β-carotene, mutatochrome, cis-violaxanthin, luteoxanthin, mutatoxanthin and auroxanthin were observed in all 3 stages of ripening.

Characterization of two M17 family members in Escherichia coli, Peptidase A and Peptidase B

Escherichia coil encodes two aminopeptidases belonging to the M17 family: Peptidase A (PepA) and Peptidase B (PepB). To gain insights into their substrate specificities, PepA or PepB were overexpressed in Delta pepN, which shows greatly reduced activity against the majority of amino acid substrates. Overexpression of PepA or PepB increases catalytic activity of several aminopeptidase substrates and partially rescues growth of Delta pepN during nutritional downshift and hightemperature stress. Purified PepA and PepB display broad substratespecificity and Leu, Lys, Met and Gly are preferred substrates. However, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and insulin B chain peptide. Importantly, this strategy, i.e. overexpression of peptidases in Delta pepN and screening a panel of substrates for cleavage, can be used to rapidly identify peptidases with novel substrate specificities encoded in genomes of different organisms. (C) 2010 Elsevier Inc. All rights reserved.

Transcription factor GATA4 and apoptotic TRAIL pathway in granulosa cell function and tumors

Normal growth and development require the precise control of gene expression. Transcription factors are proteins that regulate gene expression by binding specific sequences of DNA. Abnormalities in transcription are implicated in a variety of human diseases, including cancer, endocrine disorders and birth defects. Transcription factor GATA4 has emerged as an important regulator of normal development and function in a variety of endoderm- and mesoderm- derived tissues, including gut, heart and several endocrine organs, such as gonads. Mice harboring a null mutation of Gata4 gene die during embryogenesis due to failure in heart formation, complicating the study of functional role of GATA4 in other organs. However, the expression pattern of GATA4 suggests it may play a role in the regulation of ovarian granulosa cell development, function and apoptosis. This premise is supported by in vitro studies showing that GATA4 regulates several steroidogenic enzymes as well as auto-, para- and endocrine signaling molecules important for granulosa cell function. This study assessed the in vivo role of GATA4 for granulosa cell function by utilizing two genetically modified mouse strains. The findings in the GATA4 deficient mice included delayed puberty, impaired fertility and signs of diminished estrogen production. At the molecular level, the GATA4 deficiency leads to attenuated expression of central steroidogenic genes, Steroidogenic acute regulatory protein (StAR), Side-chain cleavage (SCC), and aromatase as a response to stimulations with exogenous gonadotropins. Taken together, these suggest GATA4 is necessary for the normal ovarian function and female fertility. Programmed cell death, apoptosis, is a crucial part of normal ovarian development and function. In addition, disturbances in apoptosis have been implicated to pathogenesis of human granulosa cell tumors (GCTs). Apoptosis is controlled by extrinsic and intrinsic pathways. The intrinsic pathway is regulated by members of Bcl-2 family, and its founding member, the anti-apoptotic Bcl-2, is known to be important for granulosa cell survival. This study showed that the expression levels of GATA4 and Bcl-2 correlate in the human GCTs and that GATA4 regulates Bcl-2 expression, presumably by directly binding to its promoter. In addition, disturbing GATA4 function was sufficient to induce apoptosis in cultured GCT- derived cell line. Taken together, these results suggest GATA4 functions as an anti-apoptotic factor in GCTs. The extrinsic apoptotic pathway is controlled by the members of tumor necrosis factor (TNF) superfamily. An interesting ligand of this family is TNF-related apoptosis-inducing ligand (TRAIL), possessing a unique ability to selectively induce apoptosis in malignant cells. This study characterized the previously unknown expression of TRAIL and its receptors in both developing and adult human ovary, as well as in malignant granulosa cell tumors. TRAIL pathway was shown to be active in GCTs suggesting it may be a useful tool in treating these malignancies. However, more studies are required to assess the function of TRAIL pathway in normal ovaries. In addition to its ability to induce apoptosis in GCTs, this study revealed that GATA4 protects these malignancies from TRAIL-induced apoptosis. GATA4 presumably exerts this effect by regulating the expression of anti-apoptotic Bcl-2. This is of particular interest as high expression of GATA4 is known to correlate to aggressive GCT behavior. Thus, GATA4 seems to protect GCTs from endogenous TRAIL by upregulating anti-apoptotic factors such as Bcl-2.

Integrin trafficking regulated by Rab21 is necessary for cytokinesis

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.

Studies in sesquiterpenes-XVI. Zerumbone, a monocyclic sesquiterpene ketone

The structure, previously assigned to zerumbone, has been found to be untenable. The ketone has been shown to be monocyclic containing three ethylenic linkages, and has been further correlated with humulene. Results from ozonolysis, and base-catalysed cleavage allowed the compound to be formulated as 2,6,9,9-tetramethyl-2,6,10-cyclo-undecatrien-1-one.