排序方式: 共有47条查询结果,搜索用时 0 毫秒
1.
2.
3.
Structural and functional characterization of myotoxin, Cr-IV 1, a phospholipase A2 D49 from the venom of the snake Calloselasma rhodostoma 总被引:1,自引:0,他引:1
V.L. Bonfim L.A. Ponce-Soto D. Martins de Souza G.H.M.F. Souza P.A. Baldasso M.N. Eberlin S. Marangoni 《Biologicals》2008,36(3):168-176
A new D49 PLA(2) was purified from the venom of Calloselasma rhodostoma after two chromatographic steps. Molecular exclusion chromatography was done through a Protein-Pack 300 SW column (0.78 cm x 30 cm), eluting with 0.25 M ammonium bicarbonate, pH 7.9, at a flow rate of 0.3 ml/min. Reverse-phase HPLC was then performed on mu-Bondapack C-18. The sample was determined to have a molecular mass of 13,870.94 Da MALDI-TOF by mass spectrometry, and the amino acid composition showed that Cr-IV 1 presented a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). Cr-IV 1 presented a sequence of 122 amino acid residues: DLWEFGQMILKETGSLPFPY YTTYGCYCGV GGRGGKPKDA TDRCCFVHDC CYGKLTGCPK TNDRYSYSRL DYTIVCGEGG PCKQICECDK AAAVCFRENL RTYNKKYRYHLKPFCKEPAE TC and a calculated pI value of 8.0. Cr-IV 1 had PLA(2) activity in the presence of a synthetic chromogenic substrate (4-nitro-3-(octanoyloxy)benzoic acid) and showed a rapid cytolytic effect on mouse skeletal muscle myoblasts and myotubes in culture. In mice, Cr-IV 1 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD(50) of Cr-IV 1 was determined to be 0.07 mg/k body weight by intracerebroventricular (i.c.v.) injection. The combination of structural and functional information obtained herein classifies Cr-IV 1 as a new member of the D49 PLA(2) family, as it presents the typical behavior of a phospholipase A(2) from this family. 相似文献
4.
Ponce-Soto LA Bonfim VL Novello JC Navarro Oviedo R Yarlequé Chocas A Marangoni S 《The protein journal》2007,26(6):387-394
A serine protease from Bothrops
atrox (Peruvian specimen’s venom) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. This
protein was denominated Ba III-4 (33,080.265 Da determinated by MALDI-TOF mass spectrometry) and showed pI of 5.06, Km 0.2 × 10−1
M and the V
máx 4.1 × 10−1 nmoles p-NA/lt/min on the synthetic substrate BapNA. Ba III-4 also showed ability to coagulate bovine fibrinogen. The serine
protease was inhibited by soyben trypsin inhibitor and DA2II, which is an anti-hemorrhagic factor isolated from the opossum
specie Didelphis albiventris. The primary structure of Ba III-4 showed the presence of His(44), Asp(94) and Ser(193) residues in the corresponding positions
to the catalytic triad established in the serine proteases and Ser(193) are inhibited by phenylmethylsulfonylfluoride (PMSF).
Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro, as well as 12 half-cysteine residues. Ba III-4
contained 293 amino acid residues and the primary structure of VIGGDECDIN EHPFLAFMYY SPRYFCGMTL INQEWVLTAA HCRYFCGMTL IHLGVHRESE
KANYDEVRRF PKEKYFIFCD NNFTDDEVDK DIMLIRLDKP VSNSEHIAPL SLPSNPPSVG SVCRIMGWGQ TTTSPIDVLS PDEPHCANIN LFDNTVCHTA HPQVANTRTS TDTLCAGDLQ
GGRDTCNGDS GGPLICNEQL HGILSWGGDP CAQPNKPAFY TKVYYFDHPW IKSIIAGNKK TVNFTCPPLR SDAKDDSTTY INQEWDWVLT AEHCDRTHMR NSFYDYSSIN SDS.
Titration experiments did not show the presence of free sulfhydryl groups after 4 h incubation, nor were differences found
in relation to titration kinetics in the presence of nondenaturating buffer. The isolation of this protein, Ba III-4, is of
potential interest for the understanding of the pathomechanism of the snake venom action and for the identification of new
blood coagulation enzymes of natural sources. 相似文献
5.
J. A. Florêncio C. R. Soccol L. F. Furlanetto T. M. B. Bonfim N. Krieger M. Baron J. D. Fontana 《Bioprocess and biosystems engineering》1998,19(3):161-164
A sugarcane juice-based low cost culture medium was previously explored to produce the carotenoid pigment astaxanthin in liquid culture by the red yeast Phaffia rhodozyma (1300?μg astaxanthin/g of dry yeast and 6500?μg/l whole culture medium). Two peculiar limitations in Phaffia are growth temperature (<26?°C) and lack of sugar osmotolerance. Two advantages are the wide biochemical ability for the assimilation and metabolization of disaccharides and the prompt utilization of simple nitrogen sources. For instance, the sucrolytic/ureolytic enzymatic activities deserves exploration. In order to improve the culture medium composition and the conditions of fermentation for highly oxygenated carotenoids (e.g., astaxanthin) a study was carried out with a factorial design in two steps. As a first step, the production of astaxanthin was studied as a function of the nutrient concentration levels and their interactions. The production increase (μg/l) obtained was 23.0% but at the expense of 16.0% pigment content decrease (μg/g). In the second step, the variables pH and agitation level (OTR, oxygen transfer rate) were optimized and then, both goals were attained: the increase of pigment content (418?μg astaxanthin/g of yeast) as well as the absolute pigment production enhancement (1987?μg/l). 相似文献
6.
Bonfim VL Toyama MH Novello JC Hyslop S Oliveira CR Rodrigues-Simioni L Marangoni S 《Journal of Protein Chemistry》2001,20(3):239-245
A novel basic phospholipase A2 (PLA2) isoform was isolated from Bothrops jararacussu snake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLPSYTTY . . .) showed a high degree of homology with basic D49 PLA2 myotoxins from other Bothrops venoms. Bj IV had high PLA2 activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45°C. Full PLA2 activity required Ca2+ but was inhibited by Cu2+ and Zn2+, and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. Crotapotins from Crotalus durissus terrificus rattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA2 was similar to that in the basic PLA2 of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA2 could partly explain the low PLA2 activity of Bothrops venoms. 相似文献
7.
8.
Costa VV Fagundes CT Valadão DF Cisalpino D Dias AC Silveira KD Kangussu LM Ávila TV Bonfim MR Bonaventura D Silva TA Sousa LP Rachid MA Vieira LQ Menezes GB de Paula AM Atrasheuskaya A Ignatyev G Teixeira MM Souza DG 《PLoS neglected tropical diseases》2012,6(5):e1663
There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ(-/-) mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2(-/-) mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection. 相似文献
9.
Vivian V. Costa Caio T. Fagundes Deborah F. Valad?o Daniel Cisalpino Ana Carolina F. Dias K��tia D. Silveira Lucas M. Kangussu Thiago V. ��vila Maria Rosa Q. Bonfim Daniela Bonaventura Tarc��lia A. Silva Lirlandia P. Sousa Milene A. Rachid Leda Q. Vieira Gustavo B. Menezes Ana Maria de Paula Alena Atrasheuskaya George Ignatyev Mauro M. Teixeira Danielle G. Souza 《PLoS neglected tropical diseases》2012,6(5)
10.
Bruna Carla Agustini Gildo Almeida da Silva Tania Maria Bordin Bonfim 《Folia microbiologica》2018,63(6):685-693
The study of grape microflora is of interest when autochthonous yeasts, which are related to typical wine characteristics, are intended to be used in winemaking. The election of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) as the first method for yeast identification was based on its accuracy and rapidity compared to alternative laboratory protocols for identification. The aims of this study are to consolidate the MALDI-TOF MS Supplementary database for environmental yeasts already constructed, to expand it through the addition of standard spectra of not included yeast species, and to discuss the grape microflora encountered in Southern Brazil. A total of 358 strains, isolated from grape berries, were submitted to protein profiling employing Biotyper and Supplementary database. Molecular biology techniques were used as alternatives to identify 6.4% of strains not promptly designated by protein profiling. These strains corresponded to the species Candida californica, Zygoascus meyerae, Candida akabanensis, Candida azyma, and Hanseniaspora vineae. The MALDI-TOF MS spectra of the identified species were added to Supplementary database. The presented results strengthen the need for further expansion of the mass spectra database to broaden its microbiological application. 相似文献