Cloning and development of pathotype-specific SCAR marker associated with Sclerospora graminicola isolates from pearl millet

Downy mildew pathogen of pearl millet in India is associated with the spread of the highly virulent Sclerospora graminicola pathotype-1. Twenty-seven S. graminicola isolates were screened using 20 intersimple sequence repeats (ISSR). Dinucleotide repeat primer [17898A-(CA)(6) AC] amplified a similar to 600 bp fragment specific to five isolates of pathotype-1 (Sg 048, Sg 153, Sg 212, DM-11 and DM-90). The ISSR fragment linked with pathotype-1 was cloned successfully and sequenced. To convert ISSR fragments into pathotype-specific sequence characterised amplified region (SCAR) markers, PCR primers were designed using a sequence of the cloned DNA fragment. PCR amplification using SCAR primer pair (UOM3-Sg-Path1-F/R) amplified a single 284 bp band only in isolates of S. graminicola pathotype-1. This SCAR primer pair did not amplify the 284 bp product from the other five S. graminicola pathotypes or a negative control, which demonstrates primer specificity for pathotype-1. The SCAR primer pair (UOM3-Sg-Path1-F/R) obtained in this study will provide a valuable tool for rapid identification and specific detection of S. graminicola pathotype-1.

Characterization of an Evolutionarily Conserved Metallophosphoesterase That Is Expressed in the Fetal Brain and Associated with the WAGR Syndrome

Among the human diseases that result from chromosomal aberrations, a de novo deletion in chromosome 11p13 is clinically associated with a syndrome characterized by Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR). Not all genes in the deleted region have been characterized biochemically or functionally. We have recently identified the first Class III cyclic nucleotide phosphodiesterase, Rv0805, from Mycobacterium tuberculosis, which biochemically and structurally belongs to the superfamily of metallophosphoesterases. We performed a large scale bioinformatic analysis to identify orthologs of the Rv0805 protein and identified many eukaryotic genes that included the human 239FB gene present in the region deleted in the WAGR syndrome. We report here the first detailed biochemical characterization of the rat 239FB protein and show that it possesses metallophosphodiesterase activity. Extensive mutational analysis identified residues that are involved in metal interaction at the binuclear metal center. Generation of a rat 239FB protein with a mutation corresponding to a single nucleotide polymorphism seen in human 239FB led to complete inactivation of the protein. A close ortholog of 239FB is found in adult tissues, and biochemical characterization of the 239AB protein demonstrated significant hydrolytic activity against 2',3'-cAMP, thus representing the first evidence for a Class III cyclic nucleotide phosphodiesterase in mammals. Highly conserved orthologs of the 239FB protein are found in Caenorhabditis elegans and Drosophila and, coupled with available evidence suggesting that 239FB is a tumor suppressor, indicate the important role this protein must play in diverse cellular events.

Presence of nucleoside triphosphates and calcium associated with mycobacteriophage I3

The association of nucleoside triphosphate molecules and calcium ions with purified particles of mycobacteriophage I3 has been documented. The content of nucleoside triphosphate has been determined to be 118 molecules per phage particle by equilibrium dialysis against labelled ATP or 148 molecules per phage particle by the direct determination of labelled nucleoside triphosphate.The concentration of bound Ca2+ exhibited a high degree of variation between different batches, which may be due to the nonspecific binding of Ca2+ by the virus particles. However, the tightly bound Ca2+ not removable by dialysis against calciumspecific chelating agent, showed a constant value of 2985 atoms/phage particle.

Invited paper Application of the least mean fourth (LMF) adaptive algorithm to signals associated with gaussian noise

This paper considers the applicability of the least mean fourth (LM F) power gradient adaptation criteria with 'advantage' for signals associated with gaussian noise, the associated noise power estimate not being known. The proposed method, as an adaptive spectral estimator, is found to provide superior performance than the least mean square (LMS) adaptation for the same (or even lower) speed of convergence for signals having sufficiently high signal-to-gaussian noise ratio. The results include comparison of the performance of the LMS-tapped delay line, LMF-tapped delay line, LMS-lattice and LMF-lattice algorithms, with the Burg's block data method as reference. The signals, like sinusoids with noise and stochastic signals like EEG, are considered in this study.

Possible involvement of a calcium-stimulated ATP-hydrolyzing activity associated with mycobacteriophage I3 in the DNA injection process.

Ca2+ ions are necessary for the successful propagation of mycobacteriophage I3. An assay for the phage DNA release in the presence of an isolated cell wall preparation from the host was established, and in this system Ca2+ ions also stimulated the release of DNA. The inhibition of phage DNA injection caused by Tween 80 (polyoxyethylene sorbitan monooleate), a nonionic detergent routinely used in mycobacterial cultures, was reversed by Ca2+. The presence of a phage-associated ATP-hydrolyzing activity was demonstrated. This enzyme was stimulated by Ca2+ ions and inhibited by Tween 80. From this and the behavior of the two agents at the level of DNA injection, as well as the fact that phage I3 has a contractile tail structure, we conclude that the phage-associated ATPase is involved in the DNA injection process.

Nitrogenase activity (acetylene reduction) associated with leaves of different cultivars of paddy

An investigation was conducted to study the levels of nitrogen fixation on the leaf or sheath surfaces of four cultivars of paddy plants by using acetylene reduction technique. Varying levels of positive nitrogenase activity were observed on all the leaf surfaces. Sheath of IET 1991 cultivar showed a higher rate of fixation than the leaf surface. All the nitrogen-fixing organisms on the leaf or sheath surfaces belonged to the genus Beijerinckia. There was no correlation between the bacterial density and the level of fixation. Scanning electron microscopic data revealed that the upper surface of IET 1991 leaf was highly silicified and the microflora was either scanty or nil while the lower surface appeared quite different and harboured more micro-organisms. Similarly, the inner surface of sheath was devoid of silicification and showed the presence of micro-organisms.

Nitrogenase activity (acetylene reduction) associated with leaves of different cultivars of paddy (Oryza sativa L.)

An investigation was conducted to study the levels of nitrogen fixation on the leaf or sheath surfaces of four cultivars of paddy plants by using acetylene reduction technique. Varying levels of positive nitrogenase activity were observed on all the leaf surfaces. Sheath of IET 1991 cultivar showed a higher rate of fixation than the leaf surface. All the nitrogen-fixing organisms on the leaf or sheath surfaces belonged to the genus Beijerinckia. There was no correlation between the bacterial density and the level of fixation. Scanning electron microscopic data revealed that the upper surface of IET 1991 leaf was highly silicified and the microflora was either scanty or nil while the lower surface appeared quite different and harboured more micro-organisms. Similarly, the inner surface of sheath was devoid of silicification and showed the presence of micro-organisms.

Inhibition of Tyrosine Phosphorylation of Sperm Flagellar Proteins, Outer Dense Fiber Protein-2 and Tektin-2, Is Associated With Impaired Motility During Capacitation of Hamster Spermatozoa

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

Phage lambda beta protein, a component of general recombination, is associated with host ribosomal S1 protein

We have purified phage lambda beta protein produced by a recombinant plasmid carrying bet gene and confirm that it forms a complex with a protein of relative molecular mass 70 kDa. Therefore, beta protein, a component of general genetic recombination, is associated with two functionally diverse complexes; one containing exonuclease and the other 70 kDa protein. Using a number of independent methods, we show that 70 kDa protein is the ribosomal S1 protein of E. coli. Further, the association of 70 kDa protein with beta protein is biologically significant, as the former inhibits joining of the terminal ends of lambda chromosome and renaturation of complementary single stranded DNA promoted by the latter. More importantly, these findings initiate an understanding of an important mode of host- virus interaction in general with specific implication(s) in homologous genetic recombination.

RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L-P complex) catalyses the synthesis of all viral mRNAs in vitro and the abundance of mRNAs follows a gradient of polarity, similar to the occurrence in vivo. In the present work, we demonstrate that the viral mRNAs synthesized in vitro by the recombinant L or purified RNP are capped and methylated at the N-7 guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal of gamma-phosphate from triphosphate-ended 25 nt RNA generated in vitro representing the viral N-terminal mRNA 5' sequence. The L protein forms a covalent enzyme-guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717-2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.

Heat-shock protein 70-2 (HSP70-2) expression in bladder urothelial carcinoma is associated with tumour progression and promotes migration and invasion

Purpose: Testis specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, is essential for the growth of spermatocytes and cancer cells. We investigated the association of HSP70-2 expression with clinical behaviour and progression of urothelial carcinoma of bladder. Experimental design: We assessed the HSP70-2 expression by RT-PCR and HSP70-2 protein expression by immunofluorescence, flow cytometry, immunohistochemistry and Western blotting in urothelial carcinoma patient specimens and HTB-1, UMUC-3, HTB-9, HTB-2 and normal human urothelial cell lines. Further, to investigate the role of HSP70-2 in bladder tumour development, HSP70-2 was silenced in the high-grade invasive HTB-1 and UMUC-3 cells. The malignant properties of urothelial carcinoma cells were examined using colony formation, migration assay, invasion assay in vitro and tumour growth in vivo. Results: Our RT-PCR analysis and immunohistochemistry analysis revealed that HSP70-2 was expressed in both moderate to well-differentiated and high-grade invasive urothelial carcinoma cell lines studied and not in normal human urothelial cells. In consistence with these results, HSP70-2 expression was also observed in superficially invasive (70%) and muscle-invasive (90%) patient's tumours. Furthermore, HSP70-2 knockdown significantly suppressed cellular motility and invasion ability. An in vivo xenograft study showed that inhibition of HSP70-2 significantly suppressed tumour growth. Conclusions: In conclusion, our data suggest that the HSP70-2 expression is associated with early spread and progression of urothelial carcinoma of bladder cancer and that HSP70-2 can be the potential therapeutic target for bladder urothelial carcinoma. (C) 2009 Elsevier Ltd. All rights reserved.

Identification of genes associated with tumorigenesis of meibomian cell carcinoma by microarray analysis

Meibomian cell carcinoma (MCC) is a malignant tumor of the meibomian glands located in the eyelids. No information exists on the cytogenctic and genetic aspects of MCC. There is no report on the gene expression profile of MCC. Thus there is a need, for both scientific and clinical reasons, to identify genes and pathways that are involved in the development and progression of MCC. We analyzed the gene expression profile of MCC by the microarray technique. Forty-four genes were upregulated and 149 genes were downregulated in MCC. Differential expression data were confirmed for 5 genes by semiquantitative RT-PCR in MCC tumors: GTF2H4, RBM12, UBE2D3, DDX17, and LZTS1. We found dysregulation of two major pathways in MCC: MAPK and JAK/STAT. Clusters of genes on chromosomes 1, 12, and 19 were dysregUlated in MCC. The data presented here will facilitate the identification of specific markers and therapeutic targets for the treatment of MCC patients. (c) 2007 Elsevier Inc. All rights reserved.

Evidence for two distinct orthorhombic structures associated with different Tc regimes in LnBa2Cu3O7 − δ (Ln = Nd, Eu, Gd and Dy): A study of the dependence of superconductivity on oxygen stoichiometry

Superconductivity in LnBa2Cu3O7 − δ with Ln = Nd, Eu, Gdand Dy has been investigated as a function of δ, closely following the accompanying changes in crystal structure. Orthorhombic GdBa2Cu3O7 − δ and DyBa2Cu3O7 − δ show a Tc of ≈ 90 K up to δ = 0.2 and a lower Tc plateau (40–50 K) in the δ range 02 to 0.4, similar to that found in YBa2Cu3O7 − δ. The orthorhombic structure II in the lower Tc regions is different from the structure I in the 90 K Tc (low δ) region. The unit cell parameters of the orthorhombic I structure in the high Tc region bear the relationship of a a ≠ b not, vert, similar c/3. This relationship is not seen in the low Tc plateau. The low Tc plateau region does not distinctly manifest itself in NdBa2Cu3O7 − δ just as in LaBa2Cu3O7 − δ.

Bond length distortions associated with torsion potentials; ab-initio studies on methanedio

The 4-31G basis set is used to study the bond length variations as functions of dihedral angels in methanediol. This study is compared with O---C---O bond angle optimization studies by Gorenstein and Kar and the possible reason for bond length shorteing in the trans---trans configuration is analysed.

Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.