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Acid Sequence Analysis (acid + sequence_analysis)
Kinds of Acid Sequence Analysis Selected AbstractsIdentification and characterization of Xenopus OMP25DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2004Masafumi Inui This study describes the isolation of mitochondrial outer membrane protein 25 (OMP25) from Xenopus laevis and an analysis of its role in early development. X. laevis OMP25 (xOMP25) is a transmembrane protein of the mitochondrial outer membrane with a PDZ domain in the cytoplasmic tail, and an approximate molecular size of 25 kDa. We isolated xOMP25 from a cDNA library of X. laevis tailbud embryos. Amino acid sequence analysis of xOMP25 showed 57% identity to mouse OMP25, with 73% identity in the PDZ domains. XOMP25 mRNA is expressed maternally, and at a constant level throughout early development. The transcript is localized to eye, otic vesicle, branchial arch and neural tube. Mitochondrial targeting of an EGFP-fusion protein of xOMP25 was visualized using a mitochondria-specific fluorescent dye. Overexpression of xOMP25 in embryos caused curved axes, small eyes and disorganized head structures. Knockdown of xOMP25 protein using antisense morpholino oligonucleotides resulted in slightly shortened axes and decreased neural tissue. Although the mechanism remains unclear, our results implicate xOMP25 protein in the formation of the intact neural tube. [source] Purification of Matrix Gla Protein From a Marine Teleost Fish, Argyrosomus regius: Calcified Cartilage and Not Bone as the Primary Site of MGP Accumulation in Fish,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2003DC Simes Abstract Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas the protein was found to accumulate mainly in bone. However, at that time, it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells because both coexist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. To analyze MGP structure and function in bony fish, MGP was acid-extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius,, separated from the mineral phase by dialysis, and purified by Sephacryl S-100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N-terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription-polymerase chain reaction (RT-PCR) and 5,rapid amplification of cDNA (RACE)-PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was analyzed by Northern blot, in situ hybridization, and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in cartilage from branchial arches, with no expression detected in the different types of bone analyzed, whereas BGP mRNA was located in bone tissue as expected. Accordingly, the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. In soft tissues, MGP mRNA was mainly expressed in heart but in situ hybridization, indicated that cells expressing the MGP gene were located in the bulbus arteriosus and aortic wall, rich in smooth muscle and endothelial cells, whereas no expression was detected in the striated muscle myocardial fibers of the ventricle. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in cartilage, heart, and kidney tissues, but in contrast with results obtained in Xenopus and higher vertebrates, the protein does not accumulate in vertebra of non-osteocytic teleost fish, but only in calcified cartilage. In addition, our results also indicate that the presence of MGP mRNA in heart tissue is due, at least in fish, to the expression of the MGP gene in only two specific cell types, smooth muscle and endothelial cells, whereas no expression was found in the striated muscle fibers of the ventricle. In light of these results and recent information on expression of MGP gene in these same cell types in mammalian aorta, it is likely that the levels of MGP mRNA previously detected in Xenopus, birds, and mammalian heart tissue may be restricted toregions rich in smooth muscle and endothelial cells. Our results also emphasize the need to re-evaluate which cell types are involved in MGP gene expression in other soft tissues and bring further evidence that fish are a valuable model system to study MGP gene expression and regulation. [source] Cloning and Expression of Low Molecular Weight Glutenin Genes from the Chinese Elite Wheat Cultivar "Xiaoyan 54"JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 2 2006Xin-Yu Wang Abstract The low molecular weight (LMW) glutenin subunits account for 40% of wheat gluten protein content by mass and these proteins are considered to significantly affect dough quality characteristics. Five new full-length LMW glutenin genes (designated LMW-5, LMW-7, LMW-42, LMW-58, and LMW-34) were isolated from the Chinese elite wheat cultivar "Xiaoyan 54" by PCR amplification of genomic DNA using a pair of degenerate primers designed from the conserved sequences of the N- and C-terminal regions of published LMW glutenin genes. Deduced amino acid sequence analysis showed that LMW-5 belongs to the LMW-i type genes and that the other four belong to LMW-m type genes. Sequence comparisons revealed that point mutations occasionally occurred in signal peptide and N-terminus domains and often existed in domain III and domain V. Small insertions and deletions are represented in the repetitive domain. There is a stop codon after amino acid position 110 in the repetitive domain of LMW-34, indicating that it is a pseudogene. The other four genes have complete open reading frames and the putative mature regions of these genes were subcloned into pET-30a expression vector and successfully expressed in Escherichia coli. Protein sodium dodecyl sulfate-polyacrylamide gel electro-phoresis analysis showed that all proteins expressed in E. coli by the four genes could be related to B-group LMW glutenin subunits of wheat. (Managing editor: Li-Hui Zhao) [source] Inhibition of a novel sperm gelatinase in prawn sperm by the male reproduction-related kazal-type peptidase inhibitorMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 8 2008Ye Li Abstract Previously, we have identified and characterized a male reproduction-related kazal-type peptidase inhibitor (MRPINK) gene from the prawn, Macrobrachium rosenbergii. In the present study, MRPINK was discovered to have an inhibitory effect on the gelatinolytic activity of M. rosenbergii sperm and immunofluorescence analysis revealed it bound specifically onto the base of sperm. The proteolytic activity of sperm extracts to vitelline coat components was also detected to be interfered by MRPINK. Furthermore, a novel gelatinase on sperm was found to be specifically inhibited by MRPINK and was named M. rosenbergii sperm gelatinase (MSG). MSG was then isolated and purified by reversed-phase high performance liquid chromatography combining with gelatinolytic assay. By amino-terminal amino acid sequence analysis and molecular cloning, the primary structure of MSG was determined. The data presented in this study provided evidence that MRPINK has an inhibitory effect on the gelatinolytic activity as well as proteolytic activity of prawn sperm and specifically blocks the activity of MSG. Mol. Reprod. Dev. 75: 1327,1337, 2008. © 2008 Wiley-Liss, Inc. [source] Vibrio cholerae cytolysin is composed of an ,-hemolysin-like corePROTEIN SCIENCE, Issue 2 2003Rich Olson VCC, Vibrio cholerae cytolysin; ,HL, ,-hemolysin; LukF, leukocidin F component Abstract The enteric pathogen Vibrio cholerae secretes a water-soluble 80-kD cytolysin, Vibrio cholerae cytolysin (VCC) that assembles into pentameric channels following proteolytic activation by exogenous proteases. Until now, VCC has been placed in a unique class of pore-forming toxins, distinct from paradigms such as Staphyloccal ,-hemolysin. However, as reported here, amino acid sequence analysis and three-dimensional structure modeling indicate that the core component of the VCC toxin is related in sequence and structure to a family of hemolysins from Staphylococcus aureus that include leukocidin F and ,-hemolysin. Furthermore, our analysis has identified the channel-forming region of VCC and a potential lipid head-group binding site, and suggests a conserved mechanism of assembly and lysis. An additional domain in the VCC toxin is related to plant lectins, conferring additional target cell specificity to the toxin. [source] Cloning, characterization and expression of two glutathione S -transferase cDNAs in the spruce budworm, Choristoneura fumiferanaARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2009Yufen Huang Abstract Two Choristoneura fumiferana glutathione S -transferase cDNAs (CfGSTs4 and CfGSTd5) were cloned from a cDNA library constructed using mRNA from the midgut cell line, CF-203. These cDNAs encoded two structurally different proteins with a predicted molecular mass of 23 and 24 kDa, respectively. Amino acid sequence analysis indicates that CfGSTs4 and CfGSTd5 contained Sigma and Delta GST domain, respectively. CfGSTs4 cDNA was expressed as a recombinant protein with the same molecular mass as predicted. Semi-quantitative reverse-transcription PCR analyses indicated that both of these genes were expressed in the epidermis, fat body, and midgut of the 6th instar larvae, as well as CF-203 cells. CfGSTs4 was highly and almost constantly expressed in all tissues during the 6th instar stage. There were higher levels of CfGSTs4 protein in the midgut and fat body than in the epidermis. CfGSTd5 was expressed in the fat body when the insects underwent pupal molting and was constantly expressed in the epidermis and midgut during 6th instar development. CfGSTs4 expression was not affected by ecdysone agonist tebufenozide (RH5992), whereas CfGSTd5 expression was slightly suppressed by the compound. Arch Insect Biochem Physiol. 2008. © 2008 Wiley-Liss, Inc. [source] Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus speciesCLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2000W.-L. Lin Background Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species. Objective The object of this study is to generate and characterize monoclonal antibodies against these serine proteinase allergens. Methods BALB/c mice were immunized individually with the Penicillium citrinum culture medium or the crude extract and culture medium preparations of Aspergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies against serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) and two different Aspergillus species (A. fumigatus, and A. flavus) recognized by these monoclonal antibodies were analysed by sodium dodecyl sulphate and two-dimensional polyacrylamide gel electrophoresis immunoblotting and N-terminal amino acid sequence analysis. Results Four (PCM8, PCM10, PCM16 and PCM39) and one (FUM20) monoclonal antibodies against serine proteinase allergens were generated after fusion of NS-1 cells with spleen cells obtained from BALB/c mice immunized with antigens from P. citrinum and A. fumigatus, respectively. Immunoblotting results showed that PCM8 reacted with an alkaline serine proteinase allergen in P. citrinum and P. notatum. PCM10 and PCM39 reacted with the alkaline serine proteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkaline serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus but not with that in P. notatum. MoAb FUM20 reacted with the alkaline serine proteinase allergen in two Aspergillus species (A. fumigatus and A. flavus) but not with that in two different Penicillium species (P. citrinum, P. notatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. notatum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component that reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP. Conclusion Five monoclonal antibodies against different epitopes of the serine proteinase major allergens in prevalent Penicillium and Aspergillus species were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts. [source] | ||||||||||||||||