Development and validation of a liquid chromatography-tandem mass spectrometric method for the determination of alpha-methyltyrosine in human plasma

A sensitive and selective LC-MS-MS method for the isolation and quantification of alpha-methyltyrosine (AMT) from human plasma is described. The method employs a simple protein precipitation using zinc sulfate and sodium hydroxide. This precipitation procedure produced samples with high aqueous content that could be directly injected into a LC-MS-MS system without compromising reverse-phase chromatographic performance. Chromatographic separation was performed on a MetaChem MonoChrom C(18) column (2.0 mm x 50 mm; 5 microm) at a flow rate of 1 mL/min. Compounds were eluted using a gradient mixture of water-acetic acid (100:0.1, v/v) and acetonitrile-acetic acid (100:0.1, v/v). The structural analog alpha-hydroxymethyltyrosine was used as the internal standard. Mass spectrometric detection was carried out with a triple quadrupole mass spectrometer. The method was validated and used to determine human plasma AMT concentrations, and has been implemented to derive pharmacokinetic parameters.     

Cloning, overexpression, and characterization of a serine/threonine protein kinase pknI from Mycobacterium tuberculosis H37Rv

Protein phosphorylation-dephosphorylation is the principal mechanism for translation of external signals into cellular responses. Eukaryotic-like serine/threonine kinases have been reported to play important roles in bacterial development and/or virulence. The PknI protein is one of the 11 eukaryotic-like serine/threonine kinases in Mycobacterium tuberculosis H37Rv. From the bioinformatic studies, PknI protein has been shown to have an N-terminal cytoplasmic domain followed by a transmembrane region and an extracellular C-terminus suggestive of a sensor molecule. In this study, we have cloned, overexpressed, and characterized the entire coding region and the cytoplasmic domain of PknI as a fusion protein with an N-terminal histidine tag, and used immobilized metal affinity chromatography for purification of recombinant proteins. The purified recombinant proteins were found to be functionally active through in vitro phosphorylation assay and phosphoamino acid analysis. In vitro kinase assay of both proteins revealed that PknI is capable of autophosphorylation and showed manganese-dependent activity. Phosphoamino acid analysis indicated phosphorylation at serine and threonine residues. Southern blot analysis with genomic DNA highlighted the conserved nature of pknI among the various mycobacterial species. In silico analysis revealed a close homology of PknI to Stk1 from Streptococcus agalactiae, shown to have a role in virulence and cell segregation of the organism.     

Crystal structure of a putative CN hydrolase from yeast

The crystal structure of a yeast hypothetical protein with sequence similarity to CN hydrolases has been determined to 2.4 A resolution by the multiwavelength anomalous dispersion (MAD) method. The protein folds as a four-layer alphabetabetaalpha sandwich and exists as a dimer in the crystal and in solution. It was selected in a structural genomics project as representative of CN hydrolases at a time when no structures had been determined for members of this family. Structures for two other members of the family have since been reported and the three proteins have similar topology and dimerization modes, which are distinct from those of other alphabetabetaalpha proteins whose structures are known. The dimers form an unusual eight-layer alphabetabetaalpha:alphabetabetaalpha structure. Although the precise enzymatic reactions catalyzed by the yeast protein are not known, considerable information about the active site may be deduced from conserved sequence motifs, comparative biochemical information, and comparison with known structures of hydrolase active sites. As with serine hydrolases, the active-site nucleophile (cysteine in this case) is positioned on a nucleophile elbow.     

A quantitative structure-activity relationship study of hydroxamate matrix metalloproteinase inhibitors derived from functionalized 4-aminoprolines

A quantitative structure-activity relationship (QSAR) study has been made on the inhibitions of some matrix metalloproteinases (MMPs) by functionalized 4-aminoproline based hydroxamates. Attempts have been made to correlate the inhibition potencies of these hydroxamates with Kier's first-order valence molecular connectivity index ((1)chi(v)) of substituents and electrotopological state (E-state) indices of some atoms. The correlations obtained for the inhibitions of all the enzymes studied, i.e. MMP-1, MMP-2, MMP-3, MMP-7, and MMP-13, were not so uniform, but suggested that in almost all the cases the substituents at the amide nitrogen may be conducive to the activity, though the whole amide group may be sterically unfavourable. Similarly, in most of the cases, the substituens at the phenyl moiety have been found to be beneficial to the inhibition potency and in many cases an electronic role of SO(2) group of the sulfonylphenyl moiety has been indicated.     

Probing the role of a mobile loop in substrate binding and enzyme activity of human salivary amylase

Mammalian amylases harbor a flexible, glycine-rich loop 304GHGAGGA(310), which becomes ordered upon oligosaccharide binding and moves in toward the substrate. In order to probe the role of this loop in catalysis, a deletion mutant lacking residues 306-310 (Delta306) was generated. Kinetic studies showed that Delta306 exhibited: (1) a reduction (>200-fold) in the specific activity using starch as a substrate; (2) a reduction in k(cat) for maltopentaose and maltoheptaose as substrates; and (3) a twofold increase in K(m) (maltopentaose as substrate) compared to the wild-type (rHSAmy). More cleavage sites were observed for the mutant than for rHSAmy, suggesting that the mutant exhibits additional productive binding modes. Further insight into its role is obtained from the crystal structures of the two enzymes soaked with acarbose, a transition-state analog. Both enzymes modify acarbose upon binding through hydrolysis, condensation or transglycosylation reactions. Electron density corresponding to six and seven fully occupied subsites in the active site of rHSAmy and Delta306, respectively, were observed. Comparison of the crystal structures showed that: (1) the hydrophobic cover provided by the mobile loop for the subsites at the reducing end of the rHSAmy complex is notably absent in the mutant; (2) minimal changes in the protein-ligand interactions around subsites S1 and S1', where the cleavage would occur; (3) a well-positioned water molecule in the mutant provides a hydrogen bond interaction similar to that provided by the His305 in rHSAmy complex; (4) the active site-bound oligosaccharides exhibit minimal conformational differences between the two enzymes. Collectively, while the kinetic data suggest that the mobile loop may be involved in assisting the catalysis during the transition state, crystallographic data suggest that the loop may play a role in the release of the product(s) from the active site.     

Protein interactions in undersaturated and supersaturated solutions: a study using light and x-ray scattering

Protein interactions in undersaturated and supersaturated solutions were investigated using static and dynamic light scattering and small angle x-ray scattering. A morphodrom of lysozyme crystals determined at 35 degrees C and pH = 4.6 was used as a guideline in selecting the protein and precipitant concentrations. The osmotic second virial coefficient, B(22), was determined by static and dynamic light scattering. At low ionic strengths for which no crystals were formed, B(22) was positive indicating repulsive interactions between the protein molecules. Negative B(22) at higher ionic strengths corresponds to attractive interactions where crystallization becomes possible. At two extreme salt concentrations, small angle x-ray scattering data were collected and fitted with a statistical mechanical model based on Derjaguin-Landau-Verwey-Overbeek potential using Random Phase Approximation. This model accounted well for the small angle x-ray scattering data at undersaturated condition with constant potential parameters. At very high salt concentration corresponding to supersaturated solution this model seems to fail, possibly due to the presence of non-Derjaguin-Landau-Verwey-Overbeek hydration repulsion between the molecules.     

Evaluation of entomopathogenic nematode, Steinernema feltiae against field population of mustard sawfly, Athalia lugens proxima (Klug) on radish

Mustard sawfly, A. lugens proxima, was found to be highly susceptible to entomopathogenic nematode, S. feltiae under laboratory condition. Application of three different doses of S. feltiae, viz. 1.1 x 10(3), 1.1 x 10(4) and 1.1 x 10(5) infective juveniles/ml, at weekly intervals, significantly reduced the field population of mustard sawfly on radish. The mean larval population of A. lugens proxima in all doses of nematode treated plots ranged from 0.42 to 0.48 larvae per plant as against 2.95 larvae / plant in untreated control plots. Similarly, the yield of radish in all the nematode treated plots was significantly higher by way of recording 2.80 to 2.87 tons/ha as compared to 1.63 tons/ha in the case of control.     

Calcium- and superoxide anion-mediated mitogenic action of substance P on cardiac fibroblasts

Substance P is released from nerve endings in the heart under pathological conditions like ischemia, but its action on cardiac cells has not been investigated. This study tested the hypothesis that substance P is mitogenic to adult cardiac fibroblasts and delineated the underlying mechanism(s). Substance P, acting via neurokinin-1 (NK-1) receptors, stimulated cellular hyperplasia over a range of 1-10 micromol/l. It elicited no change in net collagen production, total protein synthesis, or cell protein content but increased (45)Ca uptake and superoxide generation. EGTA, N-acetyl-cysteine, and superoxide dismutase attenuated the hyperplastic response to substance P. A combination of substance P and EGTA enhanced superoxide generation without an increase in DNA synthesis, showing that an increase in superoxide production does not result in hyperplasia when extracellular Ca(2+) is chelated. Together, the data suggest that substance P may activate, via NK-1 receptors, a hyperplastic but not hypertrophic response in adult cardiac fibroblasts and that alterations in redox state and Ca(2+) homeostasis may act in concert to mediate its mitogenic action.     

Interactive gene expression pattern in prostate cancer cells exposed to phenolic antioxidants

Dietary phenolic compounds are known to elicite vital cellular responses such as cell cycle arrest, apoptosis and differentiation by activating a cascade of molecular events. As there is an increasing interest to improve the efficacy of these compounds for use as potential chemopreventive agents, we wanted to understand the impact of phenolic compounds on target genes in prostate cancer. In this study we used human cDNA microarrays with 2400 clones consisting of 17 prosite motifs to characterize alterations in gene expression pattern in response to the phenolic antioxidants ellagic acid (EA) and resveratrol (RE). Over a 48-hr exposure of androgen - sensitive LNCaP cells to EA and RE, a total of 593 and 555 genes respectively, showed more than a two fold difference in expression. A distinct set of genes in both EA-and RE-treated cells may represent the signature profile of phenolic antioxidant-induced gene expression in LNCaP cells. Although extensive similarity was found between effects of EA - and RE - responsive genes in prostate cancer cells, out of 246 genes with overlapping responses, 25 genes showed an opposite effect. Quantitative RT-PCR was used to verify and validate the differential expression of selected genes identified from cDNA microarrays. In-depth analysis of the data from this study provided insight into the alterations in the p53 - responsive genes, p300, Apaf-1, NF-kBp50 and p65 and PPAR families of genes, suggesting the activation of multiple signaling pathways that leads to growth inhibition of LNCaP cells. This is a first study to look for changes in a large number of human genes in response to dietary compounds.     

Entomopoxvirus of cotton bollworm,Helicoverpa armigera (Hbn.)

Occurrence of an Entomopoxvirus (EPV) from a lepidopteran insect viz;. cotton bollworm, H. armigera (HaEPV) along with gross pathological symptoms is reported for the first time in India. Histopathological study revealed that the fat body being the most favoured site of infection followed by haemocytes and gut epithelium. HaEPV was found to be not cross infective to six of the agricultural lepidopteran insect pests except for the potato black cutworm, Agrotis segetum registering 100% mortality showing typical symptom. Further, safety of HaEPV was shown against beneficial insect like mulberry silkworm, Bombyx mori and an useful insect general predator, Chrysoperla carnea.