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Primary Structure (primary + structure)
Selected AbstractsApplication of Electrospray Ionization Mass Spectrometry to the Elucidation of the Primary Structure of LigninMACROMOLECULAR BIOSCIENCE, Issue 7 2003Dmitry V. Evtuguin Abstract Electrospray ionization mass spectrometry (ESI-MS) was successfully applied to the structural analysis of lignin. The structure of oligomers fractionated from Eucalyptus globulus dioxane lignin was elucidated using tandem mass spectrometry, and the information on fragmentation patterns was provided by experiments on dimeric model compounds. Data obtained revealed a significant abundance in the lignin macromolecules of linear fragments that were composed of 8- O -4,-linked syringyl/guaiacyl units and syringaresinol. The proposed linear fragment of the E. globulus lignin molecule. [source] Primary structure of a novel subunit in ba3 -cytochrome oxidase from thermus thermophilusPROTEIN SCIENCE, Issue 11 2000Tewfik Soulimane Abstract The ba3 -type cytochrome c oxidase from Thermus thermophilus is known as a two subunit enzyme. Deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix "subunit IIa" spanning the membrane. The hydrophobic N-terminally blocked protein was isolated in high yield using high-performance liquid chromatography. Its complete amino acid sequence was determined by a combination of automated Edman degradation of both the deformylated and the cyanogen bromide cleaved protein and automated C-terminal sequencing of the native protein. The molecular mass of 3,794 Da as determined by MALDI-MS and by ESI requires the N-terminal methionine to be formylated and is in good agreement with the value calculated from the formylmethionine containing sequence (3,766.5 Da + 28 Da = 3,794.5 Da). This subunit consits of 34 residues forming one helix across the membrane (Lys5-Ala34), which corresponds in space to the first transmembrane helix of subunit II of the cytochrome c oxidases from Paracoccus denitrificans and bovine heart, however, with opposite polarity. It is 35% identical to subunit IV of the ba3 -cytochrome oxidase from Natronobacterium pharaonis. The open reading frame encoding this new subunit IIa (cbaD) is located upstream of cbaB in the same operon as the genes for subunit I (cbaA) and subunit II (cbaB). [source] Earthquake behavior of structures with copper energy dissipatorsEARTHQUAKE ENGINEERING AND STRUCTURAL DYNAMICS, Issue 3 2004Juan C. De la Llera Abstract The earthquake behavior of structures with supplemental copper dampers is evaluated in this study. The investigation is divided into two parts: (i) an experimental work with seven pairs of hourglass copper dampers of different aspect ratios and side profiles; and (ii) a parametric study of 6-, 12-, and 25-story planar structures with elastic as well as inelastic behavior in the primary structure and copper dampers. The copper used in this study is electrolytic tough pitch (ETP) copper C11000; probably the most commonly used of all coppers; ductile, with a low-yield, and highly resistant to corrosion. Experimental results demonstrate that all copper plates reached stable angular distortions of the order of ,=25%, which implies transverse distortions in the devices larger than 40mm. The behavior of the devices is highly dependent on the aspect ratio of the plate, h/t, and a recommendation is made to use plates in the range 11 h/t,18. Plates beyond this range exhibit either large stress and strain concentrations in the neck of the device or a strong influence of axial deformations in their cyclic behavior. The inelastic earthquake response of structures with such devices shows that drift reduction factors of the order of 30 to 40% can be achieved with reasonably economic designs. It is also shown that the efficiency of these devices depends on the soil conditions and flexibility of the primary structure. Finally, it is concluded that supplemental copper dampers are a good alternative for drift reduction in a wide range of structural layouts, ranging from coupled shear-wall systems to moment-resisting frames, and for impulsive as well as non-impulsive ground motions. Copyright © 2003 John Wiley & Sons, Ltd. [source] Protein-Based Capacitive Biosensors: a New Tool for Structure-Activity Relationship StudiesELECTROANALYSIS, Issue 24 2008Alessia Mortari Abstract The present work reports a new application of a protein-based capacitive biosensor as an in vitro assay for the selectivity study of the bacterial periplasmic protein MerP and four MerP variants. The modified MerP proteins were produced by site-directed mutagenesis of the heavy metal associated motif (HMA). The MerP and modified MerPs selectivity for copper, zinc, cadmium and mercury bivalent ions were investigated and compared. The variations in the proteins affinity were related to the primary structure of the HMA motifs. Key amino acids for copper coordination of metalloproteins that contain the metal binding sequence Gly-Met-Thr-Cys-xxx-xxx-Cys were identified. The results brought insights valid for Menkes and Wilson ATPases. The protein-based capacitive biosensors were a simple and useful tool for studying structure-activity relationships of proteins. [source] Development of Biomimetic Chitosan-Based Hydrogels Using an Elastin-Like Polymer,ADVANCED ENGINEERING MATERIALS, Issue 1-2 2010Joaquim S. Barbosa Chitosan and an elastin-like polymer, containing a specific osteoconductive sequence in the primary structure, have been combined to obtain bioactive injectable systems with enhanced mechanical properties and hydrogels. Obtained results indicate that the combination of such polymers may be very promising in the development of biomaterials for minimal invasive orthopaedic reconstructive applications or in bone tissue engineering. The figure shows a thermo-sensitive hydrogel, with a gelation point under physiological temperature. [source] Structural Determination of a Novel O-Chain Polysaccharide of the Lipopolysaccharide from the Bacterium Xanthomonas campestris pv. pruniEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 12 2003Antonio Molinaro Abstract In this paper, we report the structure of the O-specific polysaccharide of the LPS fraction of the strain type NCPPB416 of X. campestris pv. pruni. It is built up of three different monosaccharides , glucose, rhamnose and xylose , in an intricate block-wise polymer. Herein, the primary structure is elucidated by means of chemical degradation and 2D NMR spectroscopy. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003) [source] Purification of three aminotransferases from Hydrogenobacter thermophilus TK-6 , novel types of alanine or glycine aminotransferaseFEBS JOURNAL, Issue 8 2010Enzymes, catalysis Aminotransferases catalyse synthetic and degradative reactions of amino acids, and serve as a key linkage between central carbon and nitrogen metabolism in most organisms. In this study, three aminotransferases (AT1, AT2 and AT3) were purified and characterized from Hydrogenobacter thermophilus, a hydrogen-oxidizing chemolithoautotrophic bacterium, which has been reported to possess unique features in its carbon and nitrogen anabolism. AT1, AT2 and AT3 exhibited glutamate:oxaloacetate aminotransferase, glutamate:pyruvate aminotransferase and alanine:glyoxylate aminotransferase activities, respectively. In addition, both AT1 and AT2 catalysed a glutamate:glyoxylate aminotransferase reaction. Interestingly, phylogenetic analysis showed that AT2 belongs to aminotransferase family IV, whereas known glutamate:pyruvate aminotransferases and glutamate:glyoxylate aminotransferases are members of family I,. In contrast, AT3 was classified into family I, distant from eukaryotic alanine:glyoxylate aminotransferases which belong to family IV. Although Thermococcus litoralis alanine:glyoxylate aminotransferase is the sole known example of family I alanine:glyoxylate aminotransferases, it is indicated that this alanine:glyoxylate aminotransferase and AT3 are derived from distinct lineages within family I, because neither high sequence similarity nor putative substrate-binding residues are shared by these two enzymes. To our knowledge, this study is the first report of the primary structure of bacterial glutamate:glyoxylate aminotransferase and alanine:glyoxylate aminotransferase, and demonstrates the presence of novel types of aminotransferase phylogenetically distinct from known eukaryotic and archaeal isozymes. [source] Identification of substrates for transglutaminase in Physarum polycephalum, an acellular slime mold, upon cellular mechanical damageFEBS JOURNAL, Issue 11 2007Fumitaka Wada Transglutaminases are Ca2+ -dependent enzymes that post-translationally modify proteins by crosslinking or polyamination at specific polypeptide-bound glutamine residues. Physarum polycephalum, an acellular slime mold, is the evolutionarily lowest organism expressing a transglutimase whose primary structure is similar to that of mammalian transglutimases. We observed transglutimase reaction products at injured sites in Physarum macroplasmodia upon mechanical damage. With use of a biotin-labeled primary amine, three major proteins constituting possible transglutimase substrates were affinity-purified from the damaged slime mold. The purified proteins were Physarum actin, a 40 kDa Ca2+ -binding protein with four EF-hand motifs (CBP40), and a novel 33 kDa protein highly homologous to the eukaryotic adenine nucleotide translocator, which is expressed in mitochondria. Immunochemical analysis of extracts from the damaged macroplasmodia indicated that CBP40 is partly dimerized, whereas the other proteins migrated as monomers on SDS/PAGE. Of the three proteins, CBP40 accumulated most significantly around injured areas, as observed by immunofluoresence. These results suggested that transglutimase reactions function in the response to mechanical injury. [source] Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthodermaFEBS JOURNAL, Issue 3 2007Magdalena Kujawa We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose,methanol,choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9- S -cysteinyl, 8-,- N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are d -glucose, d -galactose, l -arabinose, and d -xylose. As shown by in situ NMR analysis, d -glucose and d -galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (kcat/Km). The enzyme may play a role in lignocellulose degradation. [source] Identification of a cowpea ,-thionin with bactericidal activityFEBS JOURNAL, Issue 15 2006Octávio L. Franco Antimicrobial peptides are an abundant group of proteinaceous compounds widely produced in the plant kingdom. Among them, the ,-thionin family, also known as plant defensins, represents one typical family and comprises low molecular mass cysteine-rich proteins, usually cationic and distributed in different plant tissues. Here, we report the purification and characterization of a novel ,-thionin from cowpea seeds (Vigna unguiculata), named Cp-thionin II, with bactericidal activity against Gram-positive and Gram-negative bacteria. Once the primary structure was elucidated, molecular modelling experiments were used to investigate the multimerization and mechanism of action of plant ,-thionins. Furthermore, Cp-thionin II was also localized in different tissues in cowpea seedlings during germination in contrasting conditions, to better understand the plant protection processes. The use of plant defensins in the construction of transgenic plants and also in the production of novel drugs with activity against human pathogens is discussed. [source] Fish and molluscan metallothioneinsFEBS JOURNAL, Issue 23 2005A structural, functional comparison Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals. They exhibit an unusual primary sequence and unique 3D arrangement. Whereas vertebrate MTs are characterized by the well-known dumbbell shape, with a ,,domain that binds three bivalent metal ions and an ,,domain that binds four ions, molluscan MT structure is still poorly understood. For this reason we compared two MTs from aquatic organisms that differ markedly in primary structure: MT 10 from the invertebrate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both proteins were overexpressed in Escherichia coli as glutathione S -transferase fusion proteins, and the MT moiety was recovered after protease cleavage. The MTs were analyzed by gel electrophoresis and tested for their differential reactivity with alkylating and reducing agents. Although they show an identical cadmium content and a similar metal-binding ability, spectropolarimetric analysis disclosed significant differences in the Cd7 -MT secondary conformation. These structural differences reflect the thermal stability and metal transport of the two proteins. When metal transfer from Cd7 -MT to 4-(2-pyridylazo)resorcinol was measured, the mussel MT was more reactive than the fish protein. This confirms that the differences in the primary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability. The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms. [source] Identification of a 250 kDa putative microtubule-associated protein as bovine ferritinFEBS JOURNAL, Issue 3 2005Evidence for a ferritin, microtubule interaction We reported previously on the purification and partial characterization of a putative microtubule-associated protein (MAP) from bovine adrenal cortex with an approximate molecular mass of 250 kDa. The protein was expressed ubiquitously in mammalian tissues, and bound to microtubules in vitro and in vivo, but failed to promote tubulin polymerization into microtubules. In the present study, partial amino acid sequencing revealed that the protein shares an identical primary structure with the widely distributed iron storage protein, ferritin. We also found that the putative MAP and ferritin are indistinguishable from each other by electrophoretic mobility, immunological properties and morphological appearance. Moreover, the putative MAP conserves the iron storage and incorporation properties of ferritin, confirming that the two are structurally and functionally the same protein. This fact led us to investigate the interaction of ferritin with microtubules by direct electron microscopic observations. Ferritin was bound to microtubules either singly or in the form of large intermolecular aggregates. We suggest that the formation of intermolecular aggregates contributes to the intracellular stability of ferritin. The interactions between ferritin and microtubules observed in this study, in conjunction with the previous report that the administration of microtubule depolymerizing drugs increases the serum release of ferritin in rats [Ramm GA, Powell LW & Halliday JW (1996) J Gastroenterol Hepatol11, 1072,1078], support the probable role of microtubules in regulating the intracellular concentration and release of ferritin under different physiological circumstances. [source] Structural determination of the O-chain polysaccharide from Agrobacterium tumefaciens, strain DSM 30205FEBS JOURNAL, Issue 12 2002Cristina De Castro Agrobacterium tumefaciens is a Gram-negative, phytopathogenic bacterium and is characterized by an unique mode of action on dicotyledonous plants: it is able to genetically modify the host, and because of this feature, it is used as a tool for transgenic plants. Many experiments have demonstrated that lipopolysaccharides (LPSs) play an important role for the disease development, as they are involved in the adhesion process of the bacterium on the plant cell wall. Despite the wealth of information on the role of LPS on phytopathogenesis, the present paper appears as the first report on the molecular primary structure of the O-chain produced from Agrobacterium. Its repeating unit was determined by means of chemical and spectroscopical analysis, and has the following structure: (3)-,- d -Araf -(1,3)-,- l -Fucp -(1,. [source] Purification and cDNA cloning of nitric oxide reductase cytochrome P450nor (CYP55A4) from Trichosporon cutaneumFEBS JOURNAL, Issue 11 2001Li Zhang Cytochrome P450nor is involved in fungal denitrification as nitric oxide (NO) reductase. Although the heme protein has been known to occur in restricted species of fungi that belong to ascomycotina, we have previously suggested that it would also occur in the yeast Trichosporon cutaneum, which is phylogenetically far from those P450nor-producing ascomycetous fungi. Here we isolated and characterized the heme protein from the basidiomycetous yeast T. cutaneum. P450nor of the yeast (TcP450nor) exhibited properties in terms of catalysis, absorption spectrum and molecular mass that are almost identical to those of its counterparts in ascomycetous fungi. We also isolated and sequenced its cDNA. The predicted primary structure of TcP450nor showed high sequence identities (around 65%) to those of other P450nors, indicating that they belong to the same family. TcP450nor protein cofractionated with cytochrome c oxidase by subcellular fractionation and its predicted primary structure contained an extension on its amino terminus that is characteristic of a mitochondrial-targeting signal, indicating that it is a mitochondrial protein like some of the isoforms of other fungi. On the other hand, TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells. The occurrence of P450nor across the subdivisions of eumycota suggests that P450nor and denitrification are distributed more universally among fungi than was previously thought. [source] Complete primary structure of rainbow trout type I collagen consisting of ,1(I),2(I),3(I) heterotrimersFEBS JOURNAL, Issue 10 2001Masataka Saito The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be ,1(I),2(I),3(I) and [,1(I)]2,2(I), respectively. The occurrence of ,3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 °C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of pro,(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each pro,(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific ,(I) chain, pro,3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for pro,1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in pro,3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that pro,3(I) had diverged from pro,1(I). This study is the first report of the complete primary structure of fish type I procollagen. [source] A putative lipoprotein of Sphingomonas sp. strain A1 binds alginate rather than a lipid moietyFEMS MICROBIOLOGY LETTERS, Issue 2 2008Jinshan He Abstract Gram-negative Sphingomonas sp. strain A1 accumulates alginate in the cell surface pit and directly incorporates the polysaccharide into its cytoplasm through a ,superchannel'. A cell surface protein Algp7 (27 kDa) is inducibly expressed in the presence of alginate. Although the protein Algp7 was initially classified as a lipoprotein based on its primary structure, Algp7 purified from strain A1 cells did not possess a lipid moiety. Algp7 bound alginate efficiently at a neutral pH with a Kd of 3.6 × 10,8 M, suggesting that the cell surface protein contributed to accumulation of alginate in the pit. [source] Genes for an alkaline d -stereospecific endopeptidase and its homolog are located in tandem on Bacillus cereus genomeFEMS MICROBIOLOGY LETTERS, Issue 1 2003Hidenobu Komeda Abstract Alkaline d -peptidase (Adp) from Bacillus cereus DF4-B is a d -stereospecific endopeptidase acting on oligopeptides composed of d -phenylalanine and the primary structure deduced from its gene, adp, shows a similarity with d -stereospecific hydrolases from Ochrobactrum anthropi strains. We have isolated DNA fragments covering the flanking region of adp from DF4-B genome and found an additional gene, adp2, located upstream of adp. The deduced amino acid sequence of Adp2 showed 96% and 85% identity with those of Adp from B. cereus strains AH559 and DF4-B, respectively. The recombinant Adp2 expressed in Escherichia coli was purified to homogeneity and characterized. It had hydrolyzing activity toward (d -Phe)3, (d -Phe)4, and (d -Phe)6 but did not act on (l -Phe)4, d -Phe-NH2, and l -Phe-NH2, some characteristics that are closely related to those of Adp from strain DF4-B. These results indicate that highly homologous genes encoding d -stereospecific endopeptidases are arranged in a tandem manner on the genomic DNA of B. cereus DF4-B. [source] Application of proton nuclear magnetic resonance spectroscopy to the study of Cryptococcus and cryptococcosisFEMS YEAST RESEARCH, Issue 4 2006Tania C. Sorrell Abstract Proton nuclear magnetic resonance spectroscopy is a nondestructive technique that identifies chemicals in solution and in living cells. It has been used in cryptococcal research to identify the primary structure of capsular glucuronoxylomannans, link cellular apoptosis susceptibility (CAS) genes to positioning of residues on the mannose backbone of glucuronoxylomannan, and verify that the cryptococcal virulence determinant, phospholipase B, is elaborated in vivo. Promising clinical applications include speciation (Cryptococcus neoformans and Cryptococcus gattii), with preliminary evidence that varieties neoformans and grubii can also be distinguished, non-invasive diagnosis of cerebral cryptococcomas, and, in cases of meningitis, monitoring therapeutic response by analysis of cerebrospinal fluid. [source] ALys amyloidosis caused by compound heterozygosity in Exon 2 (Thr70Asn) and Exon 4 (Trp112Arg) of the lysozyme gene,,HUMAN MUTATION, Issue 1 2006Christoph Röcken Abstract Hereditary amyloidoses are caused by germline mutations, which increase the propensity of a protein to form cross-, aggregates and deposit as amyloid. Hereditary amyloidoses are particularly interesting as they help to understand how changes in the primary structure of an otherwise non-amyloidogenic protein contribute to amyloidogenesis. Here we report on a novel form of systemic ALys amyloidosis, caused by compound heterozygosity in exon 2 (p.T70N) and exon 4 (p.W112R) of the lysozyme gene (LYZ), with both mutations being present on the same allele. This type of hereditary ALys amyloidosis is characterized by extended amyloid deposits in the upper gastrointestinal tract, entire colon, and kidney, leading to gastrointestinal bleeding. Both mutations are probably effective in disease manifestation. The novel mutation at position 112 in the mature protein is located within the ,-helical domain of the protein and therefore outside the cluster of residues that has so far been implicated in ALys amyloidosis. Taken together with the p.T70N mutation, this results in a lysozyme species where the correct folding of various protein domains is probably impaired and increases the propensity of amyloid fibril formation. Interestingly, this form of ALys amyloidosis is also characterized by the occurrence of proteolytic fragments of lysozyme in the amyloid deposits. © 2005 Wiley-Liss, Inc. [source] Identification of Modified Proteins by Mass SpectrometryIUBMB LIFE, Issue 2 2002Albert Sickmann Abstract Because it is obvious that high-throughput genomics do not lead to a molecular description or even a prediction of protein function, modern techniques for protein analysis become increasingly more important. Sequence analysis of proteins and peptides is not limited to the elucidation of the primary structure of a protein. The analysis of posttranslational modifications is an important task of protein chemistry in proteome research. Increased sensitivity in mass spectrometry as a result of more efficient ionization techniques and better detection systems has allowed the stepwise reduction of protein quantity for analysis. Protein spots of 2D-PAGE separated samples are now sufficient for an unequivocal identification of a protein by mass spectrometry. In addition to protein identification, a closer look at posttranslational modifications is now also possible. It is assumed that modifications such as phosphorylation or glycosylation exist on every second protein and that they are important for the protein function. [source] Isolation and Identification of Bitter Peptides of Tryptic Hydrolysate of Soybean 11S Glycinin by Reverse-phase High-performance Liquid ChromatographyJOURNAL OF FOOD SCIENCE, Issue 8 2003I M.-R. ABSTRACT: The 21 peptides purified from the bitter fraction of tryptic hydrolysates of soybean 11S glycinin by using gel-permeation high-performance liquid chromatography (HPLC) and a series of 3 C18 reverse phase (RP)-HPLC were in the molecular weight range of 200-1400 Da and showed mostly the hydrophobicity of less than 1400 cal/mol. Although the primary structures of the bitter peptides from 11S glycinin were not exactly the same as those of the proglycinin, many bitter peptides were basic mimics of the common structure, indicating the significance of the primary structure of a peptide playing a role in the bitter taste perception. [source] Purification and initial characterization of a novel protein with factor Xa activity from Lonomia obliqua caterpillar spiculesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2005S. Lilla Abstract A novel protein with factor Xa-like activity was isolated from Lonomia obliqua caterpillar spicules by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The protein had a mass of 20745.7 Da, as determined by mass spectrometry, and contained four Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of the protein and the cysteine residues linked by disulfide bridges. The positions of 24 sequenced tryptic peptides, including the N-terminal, were deduced by comparison with a homologous protein from the superfamily Bombycoidea. Approximately 90% of the primary structure of the active protein was determined. Copyright © 2005 John Wiley & Sons, Ltd. [source] Neudesin, a novel secreted protein with a unique primary structure and neurotrophic activityJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2005Ikuo Kimura Abstract We identified a novel secreted protein and named it neudesin. Mouse neudesin of 171 amino acids is unique with no primary structural similarity to any known proteins. The neudesin protein produced in cultured cells was secreted efficiently into the culture medium. Mouse neudesin mRNA was expressed abundantly in the developing brain and spinal cord in embryos, but was expressed widely in postnatal tissues including brain, heart, lung, and kidney. Mouse neudesin mRNA was expressed in neurons but not glial cells of the brain. The protein exhibited significant neurotrophic activity in primary cultured mouse neurons but not mitogenic activity in primary cultured mouse astrocytes. Neudesin activated the mitogen-activated protein (MAP) and phosphatidylinositol-3 (PI-3) kinase pathways. The activity of neudesin was inhibited by the inhibitor pertussis toxin for Gi/Go-protein but not by inhibitors for receptor tyrosine kinases. These results indicated that the activity was mediated via the activation of the MAP and PI-3 kinase pathways, potentially by the activation of a Gi/Go-protein-coupled receptor. Human neudesin of 172 amino acids with high similarity (,91% identity) to mouse neudesin was also identified. The human neudesin gene was mapped to chromosome 1p33. The identification of neudesin, a novel secreted protein with a unique primary structure and neurotrophic activity, will provide new insights into the development and maintenance of neurons. © 2004 Wiley-Liss, Inc. [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] cDNA cloning of the housefly pigment-dispersing factor (PDF) precursor protein and its peptide comparison among the insect circadian neuropeptidesJOURNAL OF PEPTIDE SCIENCE, Issue 2 2004Ayami Matsushima Abstract Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a principal circadian neurotransmitter for the circadian rhythms of the locomotor activity in flies. Recently, two completely different types of PDF precursor were clarified; that of the cricket Gryllus bimaculatus and that of the last-summer cicada Meimuna opalifera. The G. bimaculatus PDF precursor is extraordinarily short and comprises a nuclear localization signal (NLS), while the M. opalifera PDF precursor is of ordinary length, comparable to that seen for the precursors of crustacean ,-PDH homologues. Although their PDF peptide regions were exactly the same, the regions containing a signal peptide combined with a PDF-associated peptide (PAP) were remarkably different from each other. Such a grouping suggested a fundamental role for the PAP peptide in the circadian clock, perhaps associated with PDF function. In the present study, the cDNA cloning of PDF from the adult brains of the housefly Musca domestica was carried out and it was found that an isolated clone (527 bp) encodes a PDF precursor protein of ordinary length. The PDF peptide shows a high sequence identity (78%,94%) and similarity (89%,100%) to insect PDFs and also to the crustacean ,-PDH peptides. In particular, there is only a single amino acid difference between the PDFs of Musca and Drosophila; at position 14 Ser for Musca PDF and Asn for Drosophila PDF. A characteristic Ser10 in Drosophila was retained in Musca, indicating the presence of a structural profile unique to these PDFs. The results of sequence analyses suggest that Musca and Drosophila PDFs are to be considered members of a single group that has evolved structurally. When the primary structure of the PAP regions was compared, the Musca PDF precursor also belonged to the same group as that to which the Drosophila PDF precursor belongs. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd. [source] Structure elucidation and 3D solution conformation of the antibiotic enduracidin determined by NMR spectroscopy and molecular dynamicsMAGNETIC RESONANCE IN CHEMISTRY, Issue 8 2005F. Castiglione Abstract Enduracidin and ramoplanin belong to the large family of cyclodepsipeptide antibiotics, highly effective against Gram-positive bacteria. The primary and 3D solution structure of ramoplanin is already well known, and the primary structure of enduracidin has been determined by a combination of chemical and NMR spectroscopic methods. Both antibiotics share a similar peptide core of 17 amino acids and differ mainly in the length of the acyl chain and the presence of two D -mannose moieties in ramoplanin. Based on the high sequence homology with ramoplanin, the structure in solution of enduracidin is modeled as a cyclic peptide. The tertiary structure thus obtained was refined through molecular dynamics (MD) simulation, in which the interatomic NOE-derived distance restraints were imposed. MD simulations yielded a family of representative 3D structures (RMSD = 0.89), which highlighted a backbone geometry similar to that of ramoplanin in its ,-hairpin arrangement. In contrast, enduracidin displays a different arrangement of the side-chain and of the residues forming the hydrophobic core. Copyright © 2005 John Wiley & Sons, Ltd. [source] Mass spectrometry analysis of the influenza virus,MASS SPECTROMETRY REVIEWS, Issue 1 2009Kevin M. Downard Abstract The role of mass spectrometry to probe characteristics of the influenza virus, and vaccine and antiviral drugs that target the virus, are reviewed. Genetic and proteomic approaches have been applied which incorporate high resolution mass spectrometry and mass mapping to genotype the virus and establish its evolution in terms of the primary structure of the surface protein antigens. A mass spectrometric immunoassay has been developed and applied to assess the structure and antigenicity of the virus in terms of the hemagglutinin antigen. The quantitation of the hemagglutinin antigen in vaccine preparations has also been conducted that is of importance to their efficacy. Finally, the characterization and quantitation of antiviral drugs against the virus, and their metabolites, have been monitored in blood, serum, and urine. The combined approaches demonstrate the strengths of modern mass spectrometric methods for the characterization of this killer virus. © 2008 Wiley Periodicals, Inc., Mass Spec Rev 28:35,49, 2009 [This article was published online 10 September 2008. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 7 November 2008.] [source] Application of microscopy in authentication of traditional Tibetan medicinal plant Halenia ellipticaMICROSCOPY RESEARCH AND TECHNIQUE, Issue 1 2008Jie Li Abstract Halenia elliptica D. Don, a popularly used ethnodrug from Qinghai-Tibetan plateau, was studied to reveal the indispensable morphoanatomic details. The fixed, sectioned, and stained plant materials as well as the epidermis, powder, and maceration materials were studied using light microscope according to the usual microscopic techniques. The results of the microscopic features were systematic described and illustrated. In the root, an endodermal cell was divided into 8-16-22 and 38-50-62 daughter cells in transverse section and in face view, respectively, and 9-11-13 phloem strands were present in primary structure; in the stem, stone cells were observed in the cortex, pericycle, and external phloem while 17-19-21 internal phloem strands were present in an incontinuous ring; in the pedicel, 8-10-12 internal phloem strands were observed to form an incontinuous ring; anisocytic and anomocytic stomata were present in leaf and sepal epidermis; pollen grain was with three germinal apertures and furrows; a few tracheids, a large number of spiral vessels, and various fibers were observed. Also, semiquantitative and quantitative micrographic parameter tables were simultaneously presented. Further, the key authentication parameters were concluded. The study indicated that light microscopy and related techniques could be unambiguously applied to the authentication of Halenia elliptica. Microsc. Res. Tech., 2008. © 2007 Wiley-Liss, Inc. [source] A novel lysis system in PM2, a lipid-containing marine double-stranded DNA bacteriophageMOLECULAR MICROBIOLOGY, Issue 6 2007Mart Krupovi Summary In this study we investigated the lysis system of the lipid-containing double-stranded DNA bacteriophage PM2 infecting Gram-negative marine Pseudoalteromonas species. We analysed wt and lysis-deficient phage-induced changes in the host physiology and ascribed functions to two PM2 gene products (gp) involved in lysis. We show that bacteriophage PM2 uses a novel system to disrupt the infected cell. The novelty is based on the following findings: (i) gp k is needed for the permeabilization of the cytoplasmic membrane and appears to play the role of a typical holin. However, its unique primary structure [53 aa, 1 transmembrane domain (TMD)] places it into a new class of holins. (ii) We have proposed that, unlike other bacteriophages studied, PM2 relies on lytic factors of the cellular origin for digestion of the peptidoglycan. (iii) gp l (51 aa, no TMDs) is needed for disruption of the outer membrane, which is highly rigidified by the divalent cations abundant in the marine environment. The gp l has no precedent in other phage lytic systems studied so far. However, the presence of open reading frame l-like genes in genomes of other bacterial viruses suggests that the same system might be used by other phages and is not unique to PM2. [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. 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