Association of pro staglandin-endoperoxide synthase 2 (PTGS2) polymorphisms and Alzheimer's disease in Chinese

Cyclooxygenase-2 (COX-2, encoded by the gene prostaglandin-endoperoxide synthase 2, PTGS2) is a key enzyme in the conversion of arachidonic acid to prostaglandins. The prostaglandins produced by COX-2 are involved in inflammation and pain response in diff

Changes of tissue endothelin-1 and nitric oxide synthase in a sheep model of large intestinal obstruction

Large intestinal obstruction (LIO) in farm animals can cause a ischaemic necrosis of intestinal tissue, eventually leading to death. The roles of endothelin-1 (ET-1) and nitric oxide (NO) are not well understood in the process of LIO, but evidence suggests that endothelial-derived mediators may participate. In the present study, ET-1 concentration and total nitric oxide synthase (NOS) activity were measured in heart, liver, pancreas, lung and kidney in a model of LIO in sheep. Our data demonstrated that ET-1 concentration and NOS activity were altered, with significant increases of ET-1 in heart, lung and kidney and of NOS activity in pancreas and kidney, but a marked decline of NOS activity in liver (p<0.05). It is postulated that these alterations in NOS activity and ET-1 concentration may contribute to the progressive loss of organ function, and finally lead to death in LIO in sheep.

Constitutive expression of thymidylate synthase from LCDV-C induces a transformed phenotype in fish cells

Thymidylate synthase (TS), an essential enzyme in DNA synthesis and repair, plays a key role in the events of cell cycle regulation and tumor formation. Here, an investigation was presented about subcellular location and biological function of viral TS from lymphocystis disease virus from China (LCDV-C) in fish cells. Fluorescence microscopy revealed that LCDV-C TS was predominantly localized in the cytoplasm in fish cells. Cell cycle analysis demonstrated that LCDV-C TS promoted cell cycle progression into S and G2/M phase in the constitutive expressed cells. As a result, the cells have a faster growth rate compared with the control cells as revealed by cell growth curves. For foci assay, the TS-expressed cells gave rise to foci 4-5 weeks after incubation. Microscopic examination of the TS-induced foci revealed multilayered growth and crisscross morphology characteristic of transformed cells. Moreover, LCDV-C TS predisposed the transfected cells to acquire an anchorage-independent phenotype and could grow in 0.3% soft agar. So the data reveal LCDV-C TS is sufficient to induce a transformed phenotype in fish cells in vitro and exhibits its potential ability in cell transformation. To our knowledge, it is the first report on viral TS sequences associated with transforming activity. (C) 2007 Elsevier Inc. All rights reserved.

Ginsenoside R-e increases fertile and asthenozoospermic infertile human sperm motility by induction of nitric oxide synthase

We investigated the effects of Ginsenoside R-e on human sperm motility in fertile and asthenozoospermic infertile individuals in vitro and the mechanism by which the Ginsenosides play their roles. The semen samples were obtained from 10 fertile volunteers and 10 asthenozoospermic infertile patients. Spermatozoa were separated by Percoll and incubated with 0, 1, 10 or 100 mu M of Ginsenoside R-e. Total sperm motility and progressive motility were measured by computer-aided sperm analyzer (CASA). Nitric oxide synthase (NOS) activity was determined by the H-3-arginine to H-3-citrulline conversion assay, and the NOS protein was examined by the Western blot analysis. The production of sperm nitric oxide (NO) was detected using the Griess reaction. The results showed that Ginsenoside R-e significantly enhanced both fertile and infertile sperm motility, NOS activity and NO production in a concentration-dependent manner. Sodium nitroprusside (SNP, 100 nM), a NO donor, mimicked the effects of Ginsenoside R-e. And pretreatment with a NOS inhibitor N-omega-Nitro-L-arginine methyl ester (L-NAME, 100 mu M) or a NO scavenger N-Acetyl-L-cysteine (LNAC, 1 mM) completely blocked the effects of Ginsenoside R-e. Data suggested that Ginsenoside R-e is beneficial to sperm motility, and that induction of NOS to increase NO production may be involved in this benefit.

Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase

The avermectin (Av) polyketide synthase (PKS) and erythromycin (Er) PKS are encoded by modular repeats of DNA, but the genetic organization of the modules encoding Av PKS is more complex than Er PKS. Sequencing of several related DNA fragments from Streptomyces avermitilis that are part of the Av biosynthetic gene cluster, revealed that they encode parts of large multifunctional PKS proteins. The Av PKS proteins show strong similarity to each other, as well as similarity to Er PKS proteins [Donadio et al., Science 252 (1991) 675–679] and fatty acid synthases. Partial DNA sequencing of the 65-kb region containing all the related sequence elements in the avr genes provides evidence for twelve modular repeats encoding FAS-like domains. The genes encoding the Av PKS are organized as two sets of six modular repeats which are convergently transcribed.

Cloning and Comparative Studies of Seaweed Trehalose-6-Phosphate Synthase Genes

The full-length cDNA sequence (3219 base pairs) of the trehalose-6-phosphate synthase gene of Porphyra yezoensis (PyTPS) was isolated by RACE-PCR and deposited in GenBank (NCBI) with the accession number AY729671. PyTPS encodes a protein of 908 amino acids before a stop codon, and has a calculated molecular mass of 101,591 Daltons. The PyTPS protein consists of a TPS domain in the N-terminus and a putative TPP domain at the C-terminus. Homology alignment for PyTPS and the TPS proteins from bacteria, yeast and higher plants indicated that the most closely related sequences to PyTPS were those from higher plants (OsTPS and AtTPS5), whereas the most distant sequence to PyTPS was from bacteria (EcOtsAB). Based on the identified sequence of the PyTPS gene, PCR primers were designed and used to amplify the TPS genes from nine other seaweed species. Sequences of the nine obtained TPS genes were deposited in GenBank (NCBI). All 10 TPS genes encoded peptides of 908 amino acids and the sequences were highly conserved both in nucleotide composition (>94%) and in amino acid composition (>96%). Unlike the TPS genes from some other plants, there was no intron in any of the 10 isolated seaweed TPS genes.

Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

Identification of peptides that bind with thymidylate synthase RNA using mRNA display technique

Using in vitro selection method to isolate nucleic acids, peptides and proteins has been studied intensively in recent years. In vitro mRNA display is a new and effective technique for peptides selection, and the rationale of this technique is that a synthetic mRNA with puromycin could covalently link with the protein that it encodes, thus an mRNA-protein fusion is formed. This approach has been used in identification of many functional peptides. The peptides binding with thymidylate synthase RNA were isolated using mRNA display technique from a large peptide library (>10(13) different sequences). The selection scheme was constructed, and the experimental conditions, including library synthesis, formation of RNA-peptide fusion and RNA immobilization were optimized. Eight cycles have been processed and the results confirmed that the selected peptides could bind with thymidylate synthase mRNA specifically. Compared the amino acid sequences of the selected peptides with those from the initial random library, the basic and aromatic residues in selected peptides were enriched significantly, suggesting these peptide regions may be important in the peptide-TS mRNA interaction. As a novel in vitro selection approach, mRNA display technique would be developed as a powerful tool for isolation of functional peptides and proteins that could interact with immobilized targets with high affinity and specificity.

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (>10(13) different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.

Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio

The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K-m value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.

Biochemical and electron microscopic characterization of the F1F0 ATP Synthase from the hyperthermophilic eubacterium Aquifex aeolicus

The F1F0 ATP synthase has been purified from the hyperthermophilic eubacterium Aquifex aeolicus and characterized. Its subunits have been identified by MALDI-mass spectrometry through peptide mass fingerprinting and MS/MS. It contains the canonical subunits alpha, beta, gamma, delta and epsilon of F-1 and subunits a and c of F-0. Two versions of the b subunit were found, which show a low sequence homology to each other. Most likely they form a heterodimer. An electron microscopic single particle analysis revealed clear structural details, including two stalks connecting F-1 and F-0. In several orientations the central stalk appears to be tilted and/or kinked. It is unclear whether there is a direct connection between the peripheral stalk and the 6 subunit. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Zebrafish thymidylate synthase binds to its own mRNA in vitro and in vivo

Thymidylate synthase (TS), an essential enzyme for DNA de novo synthesis, is a critical therapeutic target in cancer therapy. Previous study has shown that TS was able to bind to its own mRNA in human and E.coli, resulting in translational repression. Zebrafish is the best animal model for vertebrate study. In order to study the regulatory mechanism of zebrafish TS, the enzyme were expressed in E. coli BL21 (DE3) and it was purified to homogeneity. Electrophoretic mobility shift assay (EMSA) was used to detect the interaction of zebrafish TS protein and its own TS transcript in vitro and the results showed that zebrafish TS could bound with its own mRNA specifically. Further study revealed that zebrafish TS was able to interact with its own mRNA in vivo using immunoprecipitation : RT-PCR technique. The results provide evidence that zebrafish may be developed as an useful model for studying the anti-metabolism agents.

Studies on nitric oxide synthase activity in haemocytes of shrimps Fenneropenaeus chinensis and Marsupenaeus japonicus after white spot syndrome virus infection

The nitric oxide synthase (NOS) activity in the haemocytes of shrimps Fenneropenaeus chinensis (Osbeck) and Marsupenaeus japonicus (Bate) was Studied after white spot syndrome virus (WSSV) infection to determine its characteristics in response to virus infection. First, the NOS activity in haemocytes of shrimps was determined by the means of NBT reduction and changes in cell conformation. And the variations of NOS activity in shrimps after challenge with WSSV intramuscularly were evaluated through the analysis Of L-citrulline and total nitrite/nitrate (both as NO derivates) concentrations. The result showed that NOS activity in the haemocytes of F chinensis increased slightly from 0 to 12 h postchallenge, indicated by the variations Of L-Citrulline (from 11.15 +/- 0.10 to 12.08 +/- 0.64 mu M) and total nitrite/nitrate concentrations (from 10.45 +/- 0.65 to 12.67 +/- 0.52 mu M). Then it decreased sharply till the end of the experiment (84 h postchallenge), the concentrations Of L-Citrulline and total nitrite/nitrate at 84 It were 1.58 +/- 0.24 and 2.69 +/- 0.70 mu M, respectively. The LPS-stimulated NOS activity kept constant during the experiment. However, in M. japonicus, the NOS activity kept increasing during the first 72 It postchallenge, the concentrations Of L-Citrulline and total nitrite/nitrate increased from 7.82 +/- 0.77 at 0 h to 10.79 +/- 0.50 mu M at 72 h, and from 8.98 +/- 0.43 at 0 h to 11.20 +/- 0.37 mu M at 72 h, respectively. Then it decreased till the end of the experiment (216 h postchallenge), and the concentrations of L-Citrulline and total nitrite/nitrate at 216 h were 5.66 +/- 0.27 and 4.68 +/- 0.16 mu M, respectively. More importantly, an apparent increase of I-PS-stimulated NOS activity was observed in M japonicus at 48 h postchallenge, which was about 4 times higher than that in the control group of health shrimps. In correspondence with the difference of NOS activity between the two species of shrimps, the Cumulative mortalities of the shrimps were also different. All shrimps of F. chinensis in the mortality experiment died in 66 h, much more quickly than M. japonicus, Whose accumulative mortality reached 100% after 240 h. Data here reported let us hypothesize that NOS activity in the haemocytes of shrimps F chinensis and M. japonicus responses to WSSV infection differently, and this might be one of the reasons for the different susceptibility of F chinensis and M. japonicus to WSSV infection. (c) 2005 Elsevier Inc. All rights reserved.

Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin.

The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Delta mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Delta mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Delta mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Delta starvation-cidal defects in either species, the cidal phenotype was not due to alpha-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Delta starvation viability, it increased Ca. albicans ilv1Delta and ilv2Delta viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Delta mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Delta and ilv2 Delta viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.