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High Homology (high + homology)
Selected AbstractsProtection of red sea bream Pagrus major against red sea bream iridovirus infection by vaccination with a recombinant viral proteinMICROBIOLOGY AND IMMUNOLOGY, Issue 3 2010Hajime Shimmoto ABSTRACT Megalocytivirus infections cause serious mass mortality in marine fish in East and Southeast Asian countries. In this study the immunogenicity of crude subunit vaccines against infection by the Megalocytivirus RSIV was investigated. Three capsid proteins, 18R, 351R and a major capsid protein, were selected for use as crude subunit vaccines. High homology among Megalocytivirus types was found in the initial sequence examined, the 351R region. Red sea bream (Pagrus major) juveniles were vaccinated by intraperitoneal injection of recombinant formalin-killed Escherichia coli cells expressing these three capsid proteins. After challenge infection with RSIV, fish vaccinated with the 351R-recombinant bacteria showed significantly greater survival than those vaccinated with control bacteria. The 351R protein was co-expressed with GAPDH from the bacterium Edwardsiella tarda in E. coli; this also protected against viral challenge. A remarkable accumulation of RSIV was observed in the blood of vaccinated fish, with less accumulation in the gills and spleen tissues. Thus, the 351R-GAPDH fusion protein is a potential vaccine against Megalocytivirus infection in red sea bream. [source] Identification and characterization of novel senescence-associated genes from barley (Hordeum vulgare) primary leavesPLANT BIOLOGY, Issue 2008N. Ay Abstract Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed. [source] pMesogenin1 and 2 function directly downstream of Xtbx6 in Xenopus somitogenesis and myogenesisDEVELOPMENTAL DYNAMICS, Issue 12 2008Shunsuke Tazumi Abstract T-box transcription factor tbx6 and basic-helix-loop-helix transcription factor pMesogenin1 are reported to be involved in paraxial mesodermal differentiation. To clarify the relationship between these genes in Xenopus laevis, we isolated pMesogenin2, which showed high homology with pMesogenin1. Both pMesogenin1 and 2 appeared to be transcriptional activators and were induced by a hormone-inducible version of Xtbx6 without secondary protein synthesis in animal cap assays. The pMesogenin2 promoter contained three potential T-box binding sites with which Xtbx6 protein was shown to interact, and a reporter gene construct containing these sites was activated by Xtbx6. Xtbx6 knockdown reduced pMesogenin1 and 2 expressions, but not vice versa. Xtbx6 and pMesogenin1 and 2 knockdowns caused similar phenotypic abnormalities including somite malformation and ventral body wall muscle hypoplasia, suggesting that Xtbx6 is a direct regulator of pMesogenin1 and 2, which are both involved in somitogenesis and myogenesis including that of body wall muscle in Xenopus laevis. Developmental Dynamics 237:3749,3761, 2008. © 2008 Wiley-Liss, Inc. [source] Aberrant promoter methylation of the TPEF gene in esophageal squamous cell carcinomaDISEASES OF THE ESOPHAGUS, Issue 7 2008B.-J. Zhao SUMMARY., Aberrant methylation of tumor suppressor genes plays an important role in the development of esophageal squamous cell carcinoma (ESCC). The purpose of the present study was to identify the epigenetic changes in ESCC. Methylation-sensitive arbitrarily primed polymerase chain reaction (MS AP-PCR) analysis was used on 22 matched ESCC tumors and adjacent normal tissues. Through this screen we identified a frequently methylated fragment that showed a high homology to the 5,-CpG island of the gene encoding a transmembrane protein containing epidermal growth factor and follistatin domains (TPEF). The methylation status of the TPEF gene was then detected by bisulfite sequencing and the levels of TPEF mRNA were detected by RT-PCR. In addition, the effects of a methylation inhibitor 5-aza-2,-deoxycytidine on TPEF mRNA expression was determined in cells of ESCC cell lines. Hypermethylation of the 5,-CpG island of TPEF was found in 12 of 22 (54.5%) primary tumors. Reverse transcription PCR analysis demonstrated that TPEF mRNA expression was significantly lower in tumors than in adjacent normal tissues, which is associated with promoter hypermethylation. In addition, treatment of ESCC cell lines with 5-aza-2,-deoxycytidine led to re-expression of the TPEF transcript. In conclusion, we observed promoter of TPEF gene is frequently hpermethylated, and is associated with the loss of TPEF mRNA expression in ESCC samples. Promoter hypermethylation of TPEF gene may play a role in the development of ESCC. [source] Novel member of the mouse desmoglein gene family: Dsg1-,EXPERIMENTAL DERMATOLOGY, Issue 1 2003L. Pulkkinen Abstract: Desmosomes are major intercellular adhesion junctions that provide stable cell,cell contacts and mechanical strength to epithelial tissues by anchoring cytokeratin intermediate filaments of adjacent cells. Desmogleins (Dsg) are transmembrane core components of the desmosomes, and belong to the cadherin supergene family of calcium-dependent adhesion molecules. Currently, there are three known isoforms of Dsgs (Dsg1, Dsg2, and Dsg3), encoded by distinct genes that are differentially expressed to determine their tissue specificity and differentiation state of epithelial cells. In this study, we cloned a novel mouse desmoglein gene sharing high homology to both mouse and human Dsg1. We propose to designate the previously published mouse Dsg1 gene as Dsg1- , and the new gene as Dsg1-,. Analysis of intron/exon organization of the Dsg1-, and Dsg1-, genes revealed significant conservation. The full-length mouse Dsg1-, cDNA contains an open reading frame of 3180 bp encoding a precursor protein of 1060 amino acids. Dsg1-, protein shares 94% and 76% identity with mouse Dsg1-, and human DSG1, respectively. RT-PCR using a multitissue cDNA panel demonstrated that while Dsg1-, mRNA was expressed in 15- to 17-day-old embryos and adult spleen and testis, Dsg1-, mRNA was detected in 17-day-old embryos only. To assess subcellular localization, a FLAG-tagged expression construct of Dsg1-, was transiently expressed in epithelial HaCaT cells. Dsg1-,-FLAG was found at the cell,cell border and was recognized by the anti-Dsg1/Dsg2 antibody DG3.10. In summary, we have cloned and characterized a novel member of the mouse desmoglein gene family, Dsg1-,. [source] Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle, Psacothea hilarisFEBS JOURNAL, Issue 16 2003Masahiro Sugimura A cellulase (endo-,-1,4-glucanase, EC 3.2.1.4) was purified from the gut of larvae of the yellow-spotted longicorn beetle Psacothea hilaris by acetone precipitation and elution from gels after native PAGE and SDS/PAGE with activity staining. The purified protein formed a single band, and the molecular mass was estimated to be 47 kDa. The purified cellulase degraded carboxymethylcellulose (CMC), insoluble cello-oligosaccharide (average degree of polymerization 34) and soluble cello-oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. The specific activity of the cellulase against CMC was 150 µmol·min,1·(mg protein),1. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cello-oligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 µmol·min,1·(mg protein),1. The optimal pH of P. hilaris cellulase was 5.5, close to the pH in the midgut of P. hilaris larvae. The N-terminal amino-acid sequence of the purified P. hilaris cellulase was determined and a degenerate primer designed, which enabled a 975-bp cDNA clone containing a typical polyadenylation signal to be obtained by PCR and sequencing. The deduced amino-acid sequence of P. hilaris cellulase showed high homology to members of glycosyl hydrolase family 5 subfamily 2, and, in addition, a signature sequence for family 5 was found. Thus, this is the first report of a family 5 cellulase from arthropods. [source] Homo-oligomer formation by basigin, an immunoglobulin superfamily member, via its N-terminal immunoglobulin domainFEBS JOURNAL, Issue 14 2000Seiya Yoshida Basigin (Bsg) is a highly glycosylated transmembrane protein with two immunoglobulin (Ig)-like domains. A number of studies, including gene targeting, have demonstrated that Bsg plays pivotal roles in spermatogenesis, implantation, neural network formation and tumor progression. In the present study, to understand the mechanism of action of Bsg, we determined its expression status on the plasma membrane. Cotransfection of Bsg expression vectors with two different tags clarified that Bsg forms homo-oligomers in a cis -dependent manner on the plasma membrane. If the disulfide bond of the more N-terminally located Ig-like domain was destroyed by mutations, Bsg could not form oligomers. In contrast, the mutations of the C-terminal Ig-like domain or N-glycosylation sites did not affect the association. The association of mouse and human Bsgs, which exhibit high homology in the transmembrane and intracellular domains but low homology in the extracellular domain, was very weak as compared with that within the same species, suggesting the importance of the extracellular domain in the association. If the extracellular domain of the human Ret protein was replaced with the N-terminal Ig-like domain of Bsg, the resulting chimera protein was associated with intact wild-type Bsg, but not if the C-terminal Ig-like domain, instead of the N-terminal one, of Bsg was used. No oligomer formation took place between the intact wild-type Ret and Bsg proteins. In conclusion, these data indicate that the N-terminal Ig-like domain is necessary and sufficient for oligomer formation by Bsg on the plasma membrane. [source] The HOG pathway in the halophilic black yeast Hortaea werneckii: isolation of the HOG1 homolog gene and activation of HwHog1pFEMS MICROBIOLOGY LETTERS, Issue 2 2002Martina Turk Abstract The mitogen-activated protein kinase (MAPK) Hog1p plays an essential role in the yeast hyperosmotic response. A homolog of the HOG1 gene was isolated from the halophilic black yeast Hortaea werneckii encoding a putative 359 amino acid protein, HwHog1p, with high homology to Saccharomyces cerevisiae Hog1p and to other eukaryotic Hog1p homologs. HwHog1p contains a TGY motif within a protein kinase catalytic domain and a C-terminal common docking (CD) motif. Its activation by increased salinity is regulated at the posttranscriptional level. HwHog1p is located on the plasma membrane under nonstress conditions. Upon increased external salinity it is translocated from the membrane, presumably to the nucleus. [source] Cloning and characterization of CmGPD1, the Candida magnoliae homologue of glycerol-3-phosphate dehydrogenaseFEMS YEAST RESEARCH, Issue 8 2008Dae-Hee Lee Abstract Glycerol-3-phosphate dehydrogenase (GPDH) plays a central role in glycerol metabolism. A genomic CmGPD1 gene encoding NADH-dependent GPDH was isolated from Candida magnoliae producing a significant amount of glycerol. The gene encodes a polypeptide of 360 amino acids, which shows high homology with known NADH-dependent GPDHs of other species. The CmGPD1 gene was expressed in recombinant Escherichia coli with the maltose-binding protein (MBP) fusion system and purified to homogeneity using simple affinity chromatography. The purified CmGpd1p without the MBP fusion displayed an apparent molecular mass of 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The CmGpd1p enzyme exhibited a Kcat/Km value of 195 min,1 mM,1 for dihydroxyacetone phosphate whereas Kcat/Km for glycerol-3-phosphate is 0.385 min,1 mM,1. In a complementation study, CmGpd1p rescued the ability of glycerol synthesis and salt tolerance in a Saccharomyces cerevisiae GPD1,GPD2, mutant strain. The overall results indicated that CmGPD1 encodes a functional homologue of S. cerevisiae GPDH. [source] Cloning and characterization of genes encoding trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from Zygosaccharomyces rouxiiFEMS YEAST RESEARCH, Issue 4 2003Hawk-Bin Kwon Abstract In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively. [source] Genes involved in the determination of the rate of inversions at short inverted repeatsGENES TO CELLS, Issue 6 2000Malgorzata M. Slupska Background Not all of the enzymatic pathways involved in genetic rearrangements have been elucidated. While some rearrangements occur by recombination at areas of high homology, others are mediated by short, often interrupted homologies. We have previously constructed an Escherichia coli strain that allows us to examine inversions at microhomologies, and have shown that inversions can occur at short inverted repeats in a recB,C -dependent fashion. Results Here, we report on the use of this strain to define genetic loci involved in limiting rearrangements on an F, plasmid carrying the lac genes. Employing mini-Tn10 derivatives to generate insertions near or into genes of interest, we detected three loci (rmuA,B,C) that, when mutated, increase inversions. We have mapped, cloned and sequenced these mutator loci. In one case, inactivation of the sbcC gene leads to an increase in rearrangements, and in another, insertions near the recE gene lead to an even larger increase. The third gene involved in limiting inversions, rmuC, has been mapped at 86 min on the E. coli chromosome and encodes a protein of unknown function with a limited homology to myosins, and some of the SMC (structural maintenance of chromosomes) proteins. Conclusions This work presents the first example of an anti-mutator role of the sbcC,D genes, and defines a new gene (rmuC) involved in DNA recombination. [source] Molecular characterization of the amplified carboxylesterase gene associated with organophosphorus insecticide resistance in the brown planthopper, Nilaparvata lugensINSECT MOLECULAR BIOLOGY, Issue 6 2000Graham J. Small Abstract Widespread resistance to organophosphorus insecticides (OPs) in Nilaparvata lugens is associated with elevation of carboxylesterase activity. A cDNA encoding a carboxylesterase, Nl-EST1, has been isolated from an OP-resistant Sri Lankan strain of N. lugens. The full-length cDNA codes for a 547-amino acid protein with high homology to other esterases/lipases. Nl-EST1 has an N-terminal hydrophobic signal peptide sequence of 24 amino acids which suggests that the mature protein is secreted from cells expressing it. The nucleotide sequence of the homologue of Nl-EST1 in an OP-susceptible, low esterase Sri Lankan strain of N. lugens is identical to Nl-EST1. Southern analysis of genomic DNA from the Sri Lankan OP-resistant and susceptible strains suggests that Nl-EST1 is amplified in the resistant strain. Therefore, resistance to OPs in the Sri Lankan strain is through amplification of a gene identical to that found in the susceptible strain. [source] Porcine ESTs detected by differential display representing possible candidates for the trait ,eye muscle area'JOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 1 2000By S. Ponsuksili In order to identify ESTs which represent possible candidates for carcass traits in pigs, the differential display approach was used. F2 animals of a resource population and pure-bred German Landrace (DL) pigs were selected for the trait ,eye muscle area' in order to build up groups of three high and three low performing individuals within each population. To increase the probability that differentially expressed DNA fragments were not found due to the genetic background but due to differences in a few genes affecting the trait of interest, siblings were included in the high and in the low performing groups. RNA was isolated from M. longissimus dorsi and four ,intra-litter constrasting pools' were prepared: high performing F2, low performing F2, high performing DL and low performing DL. Differential display banding patterns were produced using (d)T11VA (V:A,C,G) and 20 arbitrary primers. Comparing the banding patterns of the four RNA pools revealed 27 nonshared bands. Here we report on the analysis of seven of these bands, including sequencing, search for homology and mapping using a somatic cell hybrid panel. Two clones showed high homology to known genes, two were homologous to an EST and a SINE sequence. Three clones did not show any homology. Differential expression was tested by semiquantitative reverse transcription,polymerase chain reaction (RT,PCR) and could be confirmed for six clones. [source] Bacillus pumilus SG2 isolated from saline conditions produces and secretes two chitinasesJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2007G. Ahmadian Abstract Aims: Isolation and characterization of chitinases from a halotolerant Bacillus pumilus. Methods and Results: Bacillus pumilus strain SG2 was isolated from saline conditions. It is able to produce chitinase activity at high salt concentration. SDS-PAGE analysis of the B. pumilus SG2 culture supernatant showed two major bands that were induced by chitin. The amino acid sequence of the two proteins, designated ChiS and ChiL, showed a high homology with the chitinase of B. subtilis CHU26, and chitinase A of B. licheniformis, respectively. N -terminal signal peptide of both proteins was also determined. The molecular weight and isoelectric point of the chitinases were determined to be 63 and 74 kDa, and 4·5 and 5·1, for ChiS and ChiL respectively. The genes encoding for both chitinases were isolated and their sequence determined. The regulation of the chitinase genes is under the control of the catabolite repression system. Conclusions: Secreted chitinase genes and their flanking region on the genome of B. pumilus SG2 have been identified and sequenced. Significance and Impact of the Study: This is the first report of a multiple chitinases-producing B. pumilus halotolerant strain. We have identified two chitinases by using a reverse genetics approach. The chitinases show resistance to salt. [source] Use of a Phage Display Technique to Identify Potential Osteoblast Binding Sites Within Osteoclast Lacunae,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2002Tzong-Jen Sheu Abstract There is a temporal coupling between the processes of bone resorption and bone formation in normal skeletal remodeling. That is, osteoblastic activity usually follows episodes of osteoclastic activity. However, what has not been universally appreciated is that there also is a spatial coupling between these processes. Bone formation only occurs in the immediate vicinity of the resorptive event. In this study, we describe a phage display technique that has been used to identify the mechanisms by which osteoblasts recognize components of the prior resorbed lacunar surface. Using a type V tartrate-resistant acid phosphatase (TRAP) as the bait and a random peptide M13 phage display library as the probe, we have identified specific sequences that show a very high affinity for TRAP. One of these peptides, designated clone 5, has a subnanomolar Kd for TRAP, interacts with TRAP in a Far-Western assay, binds exclusively to TRAP within osteoclast lacunae, is present in osteoblasts, and can effectively block osteoblast binding to resorption surfaces. The clone 5 peptide shows a high homology to glypican 4 (GPC4), a proteoglycan attachment receptor found in a number of cell types. [source] Amino Acid Absorption in Portal Blood After Duodenal Infusions of a Soy Protein Hydrolysate Prepared by a Novel Soybean Protease D3JOURNAL OF FOOD SCIENCE, Issue 7 2006Tomohiro Kodera ABSTRACT:, The intestinal absorption of amino acids from decapeptide was investigated in rats under unrestrained conditions. The soy protein hydrolysate utilized in the experiment was produced by a novel soybean protease D3. The enzymatic features of protease D3 showed high homology with cathepsin L and cathepsin K and the average molecular weight of D3 hydrolysate is approximately 1200. We compared the intestinal absorption of D3 hydrolysate in portal blood with that of an amino acids mixture and soy protein with the same amino acid composition by determining the concentration of individual amino acids after a single administration of a nitrogen source. The absorptive velocity and intensity of each amino acid were calculated from its rate of elevation in the portal blood. And in most cases, these were higher in the D3 hydrolysate than in amino acids mixture and protein. The proportion of the amount of each amino acid absorbed in portal blood from D3 hydrolysate was much more like the composition of the administrated amino acids than like that from the amino acids mixture. The result of in vitro digestion assay indicated that D3 hydrolysate was hydrolyzed easier than the hydrolysates produced by microbial proteases. This is the first report to demonstrate that the D3 hydrolysate, which contains decapeptide as a dominant fraction, was more rapidly utilized than the amino acids mixture and protein as is the case with di-, tripeptides. This suggested that this hydrolysate could be available for nutraceutical use as well as use in nutritious foods for athletes and patients. [source] Cloning and expression profile of FLT3 gene during progenitor cell-dependent liver regenerationJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 12 2007Iraz T Aydin Abstract Background and Aim:, The liver has a unique capacity to regenerate upon exposure to viral infections, toxic reactions and cancer formation. Liver regeneration is a complex phenomenon in which several factors participate during its onset. Cellular proliferation is an important component of this process and the factors that regulate this proliferation have a vital role. FLT3, a well-known hematopoietic stem cell and hepatic lineage surface marker, is involved in proliferative events of hematopoietic stem cells. However, its contribution to liver regeneration is not known. Therefore, the aim of this study was to clone and examine the role of FLT3 during liver regeneration in rats. Methods:, Partial cDNA of rat homolog of FLT3 gene was cloned from thymus and the tissue specific expression of this gene at mRNA and protein levels was examined by RT-PCR and Western blot. After treating with 2-AAF and performing hepatectomy in rats to induce progenitor-dependent liver regeneration, the mRNA and protein expression profile of FLT3 was investigated by real-time PCR and Western blot during liver regeneration. In addition, cellular localization of FLT3 protein was determined by immunohistochemistry. Results:, The results indicated that rat FLT3 cDNA has high homology with mouse and human FLT3 cDNA. It was also found that FLT3 is expressed in most of the rat tissues and during liver regeneration. In addition, its intracellular localization is altered during the late stages of liver regeneration. Conclusion:, The FLT3 receptor is activated at the late stages of liver regeneration and participates in the proliferation response that is observed during progenitor-dependent liver regeneration. [source] Influence of methylated p15 INK4b and p16 INK4a genes on clinicopathological features in colorectal cancerJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 8 2006Atsushi Ishiguro Abstract Background and Aim:, Genetic silencing by promoter methylation has attracted attention in the carcinogenesis of colorectal cancer. Methylation of the p16INK4a gene has been found in primary colorectal cancer. Although the p15INK4b gene displays high homology to the p16INK4a gene in the amino acid sequence, methylation of p15INK4b has not been fully studied. We investigated p15INK4b methylation status in patients with colorectal cancer to verify the association between the methylation of p15INK4b and clinicopathological features compared with p16INK4a. Methods:, DNA samples from the tissues of primary colorectal cancer and corresponding adjacent normal colon mucosa were obtained from surgical resections of 88 patients (47 males and 41 females, aged 29,83 years). Methylation-specific polymerase chain reaction was used to analyze p15INK4b and p16INK4a methylation status after bisulfite modification. Cumulative survival rates (mean follow-up period: 53.2 months) were calculated by the Kaplan-Meier analysis. Results:, Methylations of p15INK4b and p16INK4a genes were detected in 23 (26.1%) and 20 (22.7%) colorectal cancers, respectively. Methylation of p15INK4b was not associated with any clinicopathological features. Compared with normal mucosa, the methylation of p15INK4b was more prominent in tumor tissue (P < 0.001). Reverse transcription-polymerase chain reaction (RT-PCR) revealed that p15INK4b methylaton decreased mRNA expression. Kaplan-Meier analysis showed that patients with stage I-II had a significant difference in survival rate between those with and without methylated p15INK4b (P = 0.018). Conclusions:, Our results suggest that methylation of the p15INK4b gene contributes to the process of carcinogenesis in colorectal cancer as well as p16INK4a and is useful as a prognostic factor in the early stage. [source] Multiple promoter elements required for leukemia inhibitory factor-stimulated M2 muscarinic acetylcholine receptor promoter activityJOURNAL OF NEUROCHEMISTRY, Issue 4 2006George S. Laszlo Abstract Treatment of neuronal cells with leukemia inhibitory factor (LIF) results in increased M2 muscarinic acetylcholine receptor promoter activity. We demonstrate here that multiple promoter elements mediate LIF stimulation of M2 gene transcription. We identify a LIF inducible element (LIE) in the M2 promoter with high homology to a cytokine-inducible ACTG-containing sequence in the vasoactive intestinal peptide promoter. Mutagenesis of both a STAT (signal transducers and activators of transcription) element and the LIE in the M2 promoter is required to attenuate stimulation of M2 promoter activity by LIF completely. Mobility shift assays indicate that a LIF-stimulated complex binds to a 70 base pair M2 promoter fragment. Furthermore, a STAT element within this fragment can bind to LIF-stimulated nuclear STAT1 homodimers in vitro. Mutagenesis experiments show that cytokine-stimulated activation of M2 promoter activity requires tyrosine residues on glycoprotein 130 (gp130) that are also required for both STAT1 and STAT3 activation. Dominant negative STAT1 or STAT3 can block LIF-stimulated M2 promoter activity. Real-time RT-PCR analysis indicates that LIF-stimulated induction of M2 mRNA is partially dependent on protein synthesis. These results show that regulation of M2 gene transcription in neuronal cells by LIF occurs through a complex novel mechanism that is dependent on LIE, STAT and de novo protein synthesis. [source] Complete structure determination of the A chain of mistletoe lectin III from Viscum album L. ssp. albumJOURNAL OF PEPTIDE SCIENCE, Issue 3 2004Roland Wacker Abstract The complete primary structure of the A chain of mistletoe lectin III (ML3A), a type II ribosome-inactivating protein, was determined using proteolytic digests of ML3A, HPLC separation of the peptides, Edman degration and MALDI-MS. Based on our results, ML3A consists of 254 amino acid residues, showing a high homology to the A chain of isolectin ML1 with only 24 amino acid residue exchanges. A striking important structural difference compared with ML1A is the lack of the single N-glycosylation site in ML3A due to an amino acid exchange at position 112 (ML1A: N112GS,ML3A: T112GS). The alignment of ML3A with the A chains of ML1, isoabrins, ricin D, Ricinus communis agglutinin and three lectins, identified from the Korean mistletoe Viscum album ssp. coloratum, demonstrates the rigid conservation of all amino acid residues, responsible for the RNA-N-glycosidase activity as reported for ricin D. In addition, the fully determined primary structure of ML3A will give further information about the biological mechanism of mistletoe lectin therapy. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd. [source] Cloning, Expression and Characterization of Protein Elicitors from the Soyabean Pathogenic Fungus Phytophthora sojaeJOURNAL OF PHYTOPATHOLOGY, Issue 3 2000J. Becker The oomycete Phytophthora sojae is a severe pathogen of soybean. Several resistance genes against races of P. sojae exist in soybean but the nature of corresponding avirulence genes is unknown. Clones encoding four different isoforms of a protein elicitor from P. sojae (sojein 1,4) belonging to the class of acidic ,-elicitins have been isolated. These 98 amino acid proteins show high homology to elicitins from other Phytophthora species. The different sojein isoforms were expressed in Escherichia coli as His-tagged fusion proteins. Purified sojein as well as recombinant sojein isoforms induce hypersensitive reaction (HR)-like lesions in tobacco but are not active as race-specific elicitors in soybean. However all sojein isoforms induce defence-related genes like those encoding phenylalanine ammonia lyase, glutathione-S-transferase and chalcone synthase in tobacco and soybean plants and cell cultures. It is concluded that sojeins contribute to the induction of defence responses but that they are not involved in race specific recognition of the P. sojae races by soybean plants. Zusammenfassung Klonierung, Expression und Charactier von Proteinelictoren aus dem Soyabohnenpathogen Phytophthora sojae Der Oomycet Phytophthora sojae ist ein ernstes Pathogen der Sojabohne. In der Sojabohne gibt es mehrere Resistenzgene gegen verschiedene Rassen von P. sojae, jedoch ist die Natur der korrespondierenden Avirulenzgene unbekannt. Wir haben 4 verschiedene Isoformen eines Protein-Elicitors aus P. sojae (Sojein 1,4) kloniert, die zur Klasse der sauren ,-Elicitine gehören. Sie kodieren für Proteine mit 98 Aminosäuren und zeigen hohe Homologie zu Elicitinen aus anderen Phytophthora Spezies. Aus genomischer DNA und aus revers-transkribierter mRNA wurden die gleichen 4 Isoformen erhalten. Die verschiedenen Sojeine wurden in Escherichia coli als His-markierte Fusionproteine exprimiert. Sowohl gereinigtes als auch rekombinantes Sojein induziert HR-ähnliche Läsionen in Tabak. In der Sojabohne sind sie allerdings nicht als rassenspezifische Elicitoren aktiv. Dagegen induzieren alle Sojein-Isoformen Abwehrgene wie die Phenylalanin Ammonium-Lyase, Glutathion-S-Transferase und Chalkonsynthase in Tabak-und Sojabohnenpflanzen und Zellkulturen. Die Sojeine tragen also zur Induktion von Abwehrreaktionen bei, sind aber nicht in die rassenspezifische Erkennung von P. sojae durch Sojabohnenpflanzen involviert. [source] Cloning and some properties of Japanese pear (Pyrus pyrifolia) polyphenol oxidase, and changes in browning potential during fruit maturation,JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 11 2003Makiyo Nishimura Abstract A PCR-amplified genomic DNA fragment encoding Japanese pear (Pyrus pyrifolia) polyphenol oxidase (PPO) was cloned and sequenced. The DNA appears to encode a 66 kDa precursor protein consisting of a 56 kDa mature protein and a 9.5 kDa N-terminal transit peptide. The amino acid sequence showed high homology with apple PPO. The PPO mainly existed as a soluble fraction in cells and was limitedly proteolysed, while the mature form (56 kDa) was detected in plastids. Immature fruits showing high browning potential had high PPO activity and a high level of phenolics, while mature fruits showing little browning had high PPO activity but a low level of phenolics. Copyright © 2003 Society of Chemical Industry [source] Molecular phenotype of Fragile X syndrome: FMRP, FXRPs, and protein targetsMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2002Walter E. Kaufmann Abstract Fragile X syndrome (FraX) is one of the most prevalent genetic causes of mental retardation. FraX is associated with an unstable expansion of a polymorphism within the 5, untranslated region of the FMR1 gene. The main consequence of this mutation is a reduction in the levels of the gene product (FMRP). FMRP is an RNA-binding protein with multiple spliced variants (isoforms) and high levels of expression in a variety of tissues, including neurons. In the latter cells, it is localized not only to the perikaryon but also to dendrites and dendritic spines. FMRP belongs to a family of proteins that includes the Fragile X Related Proteins or FXRPs. FXRPs share high homology in their functional domains with FMRP, and also associate with mRNA and components of the protein synthesis apparatus. However, FXRPs do not have the same temporo-spatial pattern of distribution (and other properties) of FMRP. Immunochemical assays have confirmed that a functionally uncompensated FMRP deficit is the essence of the FraX molecular phenotype. Here, we report our preliminary study on FXRPs levels in leukocytes from FraX males. By immunoblotting, we found that a marked reduction in FMRP levels is associated with a modest increase in FXR1P and no changes in FXR2P levels. The consequences of this reduced FMRP expression on protein synthesis, in other words, the identification of FMRP targets, can be studied by different molecular approaches including protein interaction and proteomics methods. By two-dimensional gel electrophoresis, we showed that in FraX leukocytes there is a defect in acetylation that involves prominently the regulatory protein annexin-1. Extension of current studies of the molecular phenotype to more brain-relevant tissue samples, a wider range of proteomics-based methods, and correlative analyses of FMRP homologues and FMRP targets with multiple behavioral measures, will greatly expand our understanding of FraX pathogenesis and it will help to develop and monitor new therapeutic strategies. Microsc. Res. Tech. 57:135,144, 2002. © 2002 Wiley-Liss, Inc. [source] Identification of spore allergens from the indoor mould Aspergillus versicolorALLERGY, Issue 4 2008D. Benndorf Background:, Indoor mould growth and dampness are associated with respiratory health effects and allergies and several studies demonstrated that mainly Aspergillus versicolor and Penicillium expansum are responsible for indoor mould exposure. In contrast, commercialized test systems to diagnose allergic reactions to this mould species are not available. In this study, allergenic proteins from spores of the indoor relevant species A. versicolor and P. expansum should get detected and identified. Methods:, We used two-dimensional (2D)-gel electrophoresis of spore proteins and immunoblotting with sera from patients participating in an epidemiologic study about indoor exposure of moulds and their influence on the development of allergies (ESTERSPEGA). Sera were screened for IgE antibodies specific for proteins from A. versicolor, A. fumigatus and P. expansum in one-dimensional blots and in 2D immunoblots. From the 2D gels, the corresponding spots were picked and identified by mass spectrometry. Results:, More than 20 allergens from A. versicolor were identified; in particular, seven major allergens were selected, which were detected by more than 90% of the positive sera. The most abundant allergen was glyceraldehyde-3-phosphate dehydrogenase, followed by an unnamed protein, which displays a high homology to sobitol/xylose reductase. The other allergens were identified as catalase A, hypothetical protein AN6918.2, enolase, hypothetical protein AN0297.2 and a protein with homology to a fungal malate dehydrogenase. Conclusions:, The results indicate an important role of spore proteins from A. versicolor for sensitization against indoor moulds and identification of the major allergens might enable species-specific diagnosis of allergic reactions. [source] Acquisition of double-stranded DNA-binding ability in a hybrid protein between Escherichia coli CspA and the cold shock domain of human YB-1MOLECULAR MICROBIOLOGY, Issue 3 2000Nan Wang Escherichia coli CspA, a major cold shock protein, is dramatically induced upon temperature downshift. As it binds co-operatively to single-stranded DNA (ssDNA) and RNA without apparent sequence specificity, it has been proposed that CspA acts as an RNA chaperone to facilitate transcription and translation at low temperature. CspA consists of a five-stranded ,-barrel structure containing two RNA-binding motifs, RNP1 and RNP2. Eukaryotic Y-box proteins, such as human YB-1, are a family of nucleic acid-binding proteins that share a region of high homology with CspA (43% identity), termed the cold shock domain (CSD). Their cellular functions are very diverse and are associated with growth-related processes. Here, we replaced the six-residue loop region of CspA between the ,3 and ,4 strands with the corresponding region of the CSD of human YB-1 protein. The resulting hybrid protein became capable of binding to double-stranded DNA (dsDNA) in addition to ssDNA and RNA. The dsDNA-binding ability of an RNP1 point mutant (F20L) of the hybrid was almost unchanged. On the other hand, the dsDNA-binding ability of the hybrid protein was abolished in high salt concentrations in contrast to its ssDNA-binding ability. These results indicate that the loop region between the ,3 and ,4 strands of Y-box proteins, which is a little longer and more basic than that of CspA, plays an important role in their binding to dsDNA. [source] Molecular and physiological analysis of an OxyR-regulated ahpC promoter in Xanthomonas campestris pv. phaseoliMOLECULAR MICROBIOLOGY, Issue 6 2000Suvit Loprasert In Xanthomonas campestris pv. phaseoli, a gene for the alkyl hydroperoxide reductase subunit C (ahpC) had unique patterns of regulation by various forms of OxyR. Reduced OxyR repressed expression of the gene, whereas oxidized OxyR activated its expression. This dual regulation of ahpC is unique and unlike all other OxyR-regulated genes. The ahpC transcription start site was determined. Analysis of the region upstream of the site revealed promoter sequences that had high homology to the Xanthomonas consensus promoter sequence. Data from gel shift experiments indicated that both reduced and oxidized OxyR could bind to the ahpC regulatory region. Moreover, the reduced and the oxidized forms of OxyR gave different DNase I footprint patterns, indicating that they bound to different sites. The oxidized OxyR binding site overlapped the ,35 region of the ahpC promoter by a few bases. This position is consistent with the role of the protein in activating transcription of the gene. Binding of reduced OxyR to the ahpC promoter showed an extended DNase I footprint and DNase I hypersensitive sites, suggesting that binding of the protein caused a shift in the binding site and bending of the target DNA. In addition, binding of reduced OxyR completely blocked the ,35 region of the ahpC promoter and prevented binding of RNA polymerase, leading to repression of the gene. Monitoring of the ahpC promoter activity in vivo confirmed the location of the oxidized OxyR binding site required for activation of the promoter. A mutant that separated OxyR regulation from basal ahpC promoter activity was constructed. The mutant was unable to respond to oxidants by increasing ahpC expression. Physiologically, it had a slower aerobic growth rate and was more sensitive to organic peroxide killing. This indicated that oxidant induction of ahpC has important physiological roles in normal growth and during oxidative stress. [source] Expression of a transcription factor (FsERF1) involved in ethylene signalling during the breaking of dormancy in Fagus sylvatica seedsPHYSIOLOGIA PLANTARUM, Issue 3 2005Jesús Angel Jiménez By means of reverse transcriptase-polymerase chain reaction, using degenerate oligonucleotides conserved among ethylene-responsive transcription factors, we have isolated and characterized a cDNA clone encoding a protein involved in ethylene signalling during the breaking of dormancy in Fagus sylvatica L. seeds. This clone, named FsERF1, exhibits high homology to ethylene-responsive factors (ERFs) from several plant species. The expression of FsERF1 as a fusion protein in Escherichia coli confirmed that it was able to bind to the GCC box, a cis element present in the promoters of several ethylene-responsive genes, corroborating its role as a DNA-binding protein. Northern analysis showed that the transcript levels increased when dormancy was broken by ethephon (an ethylene-releasing compound), or by moist prechilling pretreatment at restricted water content, and were almost undetectable when seeds remained dormant by the addition of abscisic acid, aminooxyacetic acid (an ethylene biosynthesis inhibitor) or warm pretreatment, and when seeds were artificially dried, suggesting that FsERF1 function may be more closely related with the transition from seed dormancy to germination than with responses to drought stress mediated by ethylene. [source] Characterization of two members of the cryptochrome/photolyase family from Ostreococcus tauri provides insights into the origin and evolution of cryptochromesPLANT CELL & ENVIRONMENT, Issue 10 2010MARC HEIJDE ABSTRACT Cryptochromes (Crys) are blue light receptors believed to have evolved from the DNA photolyase protein family, implying that light control and light protection share a common ancient origin. In this paper, we report the identification of five genes of the Cry/photolyase family (CPF) in two green algae of the Ostreococcus genus. Phylogenetic analyses were used to confidently assign three of these sequences to cyclobutane pyrimidine dimer (CPD) photolyases, one of them to a DASH-type Cry, and a third CPF gene has high homology with the recently described diatom CPF1 that displays a bifunctional activity. Both purified OtCPF1 and OtCPF2 proteins show non-covalent binding to flavin adenine dinucleotide (FAD), and additionally to 5,10-methenyl-tetrahydrofolate (MTHF) for OtCPF2. Expression analyses revealed that all five CPF members of Ostreococcus tauri are regulated by light. Furthermore, we show that OtCPF1 and OtCPF2 display photolyase activity and that OtCPF1 is able to interact with the CLOCK:BMAL heterodimer, transcription factors regulating circadian clock function in other organisms. Finally, we provide evidence for the involvement of OtCPF1 in the maintenance of the Ostreococcus circadian clock. This work improves our understanding of the evolutionary transition between photolyases and Crys. [source] Phosphoenolpyruvate carboxylase genes in C3, crassulacean acid metabolism (CAM) and C3/CAM intermediate species of the genus Clusia: rapid reversible C3/CAM switches are based on the C3 housekeeping genePLANT CELL & ENVIRONMENT, Issue 12 2006ANJA VAASEN ABSTRACT The genus Clusia includes species that exhibit either the C3 or crassulacean acid metabolism (CAM) mode of photosynthesis, or those that are able to switch between both modes according to water availability. In order to screen for species-specific genetic variability, we investigated the key carboxylase for CAM, phosphoenolpyruvate carboxylase (PEPC). Sequence analysis of DNA isolated from the obligate CAM species, Clusia hilariana, the obligate C3 species, Clusia multiflora, and an intermediate species that can switch between C3 and CAM photosynthesis, Clusia minor, revealed three different isoforms for C. hilariana and one each for the other two species. Sequence alignments indicated that PEPC from the intermediate species had high homology with the C3 protein and with one of CAM plant proteins. These were assumed to constitute ,housekeeping' proteins, which can also support CAM in intermediate species. The other two isoforms of the CAM plant C. hilariana were either CAM-specific or showed homologies with PEPC from roots. Phylogenetic trees derived from neighbour-joining analysis of amino acid sequences from 13 different Clusia species resulted in two distinct groups of plants with either ,housekeeping' PEPC only, or additionally CAM-related isoforms. Only C. hilariana showed the third, probably root-specific isoform. The high homology of the PEPC from the intermediate species with the C3 protein indicates that for the reversible transition from the C3 to CAM mode of photosynthesis, the C3 type of PEPC is sufficient. Its expression, however, is strongly increased under CAM-inducing conditions. The use of the C3 isoform could have facilitated the evolution of CAM within the genus, which occurred independently for several times. [source] Crystal structure of human coactosin-like protein at 1.9 Å resolutionPROTEIN SCIENCE, Issue 11 2004Xuemei Li Abstract Human coactosin-like protein (CLP) shares high homology with coactosin, a filamentous (F)-actin binding protein, and interacts with 5LO and F-actin. As a tumor antigen, CLP is overexpressed in tumor tissue cells or cell lines, and the encoded epitopes can be recognized by cellular and humoral immune systems. To gain a better understanding of its various functions and interactions with related proteins, the crystal structure of CLP expressed in Escherichia coli has been determined to 1.9 Å resolution. The structure features a central ,-sheet surrounded by helices, with two very tight hydrophobic cores on each side of the sheet. CLP belongs to the actin depolymerizing protein superfamily, and is similar to yeast cofilin and actophilin. Based on our structural analysis, we observed that CLP forms a polymer along the crystallographic b axis with the exact same repeat distance as F-actin. A model for the CLP polymer and F-actin binding has therefore been proposed. [source] | |||||||||||||||||||||||||||||||