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Jayasekera Sharmini Arseculeratne S. N. Atapattu D. N. Kumarasiri R. Tilakaratne W. M. 《Mycopathologia》2001,152(2):69-79
There is no published data on Cell Mediated Immune Responses in experimental animals to Rhinosporidium seeberi the causative agent of human and animal rhinosporidiosis. The quantitative mouse foot-pad model was used to assay the Delayed-Type
Hypersensitivity (DTH) cell-mediated immune response to extracts of purified endospores and sporangia of R. seeberi. Histological examination was used to confirm that the foot-pad reactions were compatible with DTH reactions in the mouse.
We report that sonically disintegrated rhinosporidial endospores/sporangia induced DTH responses in the foot-pads of sensitized
mice which were comparable in intensity and histological profile to that induced by sheep red blood cells in SRBC sensitized
mice. Anti-rhinosporidial antibody was also induced. Filtrates of the soluble antigens in sonicated suspensions failed to
evoke a DTH-foot-pad (DTH-FP) response in sensitized mice although an anti-rhinosporidial antibody response to this preparation
was detected. Prolonged pre-treatment with sonicated suspensions of endospores and sporangia resulted in a decrease of DTH
reactivity as compared with reactions following pre-treatment of a shorter duration.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
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Bishara NB Dunlop ME Murphy TV Darby IA Sharmini Rajanayagam MA Hill MA 《Journal of cellular physiology》2002,193(1):80-92
Studies have shown diabetes to be associated with alterations in composition of extracellular matrix and that such proteins modulate signal transduction. The present studies examined if non-enzymatic glycation of fibronectin or a mixed matrix preparation (EHS) alters endothelial cell Ca(2+) signaling following agonist stimulation. Endothelial cells were cultured from bovine aorta and rat heart. To glycate proteins, fibronectin (10 microg/ml), or EHS (2.5 mg/ml) were incubated (37 degrees C, 30 days) with 0.5 M glucose-6-phosphate. Matrix proteins were coated onto cover slips after which cells (10(5) cells/ml) were plated and allowed to adhere for 16 h. For measurement of intracellular Ca(2+), cells were loaded with fura 2 (2 microM) and fluorescence intensity monitored. Bovine cells on glycated EHS showed decreased ability for either ATP (10(-6) M) or bradykinin (10(-7) M) to increase Ca(2+) (i). In contrast, glycated fibronectin did not impair agonist-induced increases in Ca(2+) (i). In the absence of extracellular Ca(2+), ATP elicited a transient increase in Ca(2+) (i) consistent with intracellular release. Re-addition of Ca(2+) resulted in a secondary rise in Ca(2+) (i) indicative of store depletion-mediated Ca(2+) entry. Both phases of Ca(2+) mobilization were reduced in cells on glycated mixed matrix; however, as the ratio of the two components was similar in all cells, glycation appeared to selectively impair Ca(2+) release from intracellular stores. Thapsigargin treatment demonstrated an impaired ability of cells on glycated EHS to increase cytoplasmic Ca(2+) consistent with decreased endoplasmic reticulum Ca(2+) stores. Further support for Ca(2+) mobilization was provided by increased baseline IP(3) levels in cells plated on glycated EHS. Impaired ATP-induced Ca(2+) release could be induced by treating native EHS with laminin antibody or exposing cells to H(2)O(2) (20-200 microM). Glycated EHS impaired Ca(2+) signaling was attenuated by treatment with aminoguanidine or the antioxidant alpha-lipoic acid. The results demonstrate that matrix glycation impairs agonist-induced Ca(2+) (i) increases which may impact on regulatory functions of the endothelium and implicate possible involvement of oxidative stress. 相似文献
3.
A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: structural determinants of redox potential 下载免费PDF全文
Alagaratnam S van Pouderoyen G Pijning T Dijkstra BW Cavazzini D Rossi GL Van Dongen WM van Mierlo CP van Berkel WJ Canters GW 《Protein science : a publication of the Protein Society》2005,14(9):2284-2295
Flavodoxin II from Azotobacter vinelandii is a "long-chain" flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 A by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five-stranded parallel beta-sheet flanked on either side by alpha-helices. An eight-residue insertion, compared with other long-chain flavodoxins, forms a short 3(10) helix preceding the start of the alpha3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent-accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular. 相似文献
4.
Background
Risk stratification in ST-elevation myocardial infarction (STEMI) is important, such that the most resource intensive strategy is used to achieve the greatest clinical benefit. This is essential in developing countries with wide variation in health care facilities, scarce resources and increasing burden of cardiovascular diseases. This study sought to validate the Thrombolysis In Myocardial Infarction (TIMI) risk score for STEMI in a multi-ethnic developing country.Methods
Data from a national, prospective, observational registry of acute coronary syndromes was used. The TIMI risk score was evaluated in 4701 patients who presented with STEMI. Model discrimination and calibration was tested in the overall population and in subgroups of patients that were at higher risk of mortality; i.e., diabetics and those with renal impairment.Results
Compared to the TIMI population, this study population was younger, had more chronic conditions, more severe index events and received treatment later. The TIMI risk score was strongly associated with 30-day mortality. Discrimination was good for the overall study population (c statistic 0.785) and in the high risk subgroups; diabetics (c statistic 0.764) and renal impairment (c statistic 0.761). Calibration was good for the overall study population and diabetics, with χ2 goodness of fit test p value of 0.936 and 0.983 respectively, but poor for those with renal impairment, χ2 goodness of fit test p value of 0.006.Conclusions
The TIMI risk score is valid and can be used for risk stratification of STEMI patients for better targeted treatment. 相似文献5.
Pamela Orjuela-Sánchez Nadira D Karunaweera Mônica da Silva-Nunes Natal S da Silva Kézia KG Scopel Raquel M Gonçalves Chanaki Amaratunga Juliana M Sá Duong Socheat Rick M Fairhust Sharmini Gunawardena Thuraisamy Thavakodirasah Gawrie LN Galapaththy Rabindra Abeysinghe Fumihiko Kawamoto Dyann F Wirth Marcelo U Ferreira 《BMC genetics》2010,11(1):65
Background
The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite P. vivax remain little characterized.Results
We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of P. vivax in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains (Salvador-I, from El Salvador, and Belem, from Brazil). We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for ~40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of P. vivax. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the pvmdr-1 locus, putatively associated with drug resistance.Conclusion
These findings support the feasibility of genome-wide association studies in carefully selected populations of P. vivax, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.See commentary: http://www.biomedcentral.com/1741-7007/8/906.
Alagaratnam S Mertens BJ Dalebout JC Deelder AM van Ommen GJ den Dunnen JT 't Hoen PA 《Proteomics》2008,8(8):1552-1563
Protein profiling in blood serum by fractionation and MS analysis has been applied in mice to assess its applicability as a fast, economical alternative to current DNA and RNA analyses for diagnosis of neuromuscular disorders. Mass spectra of peptides and proteins were generated using serum from dystrophin-deficient mdx and control mice by WCX ClinProt bead fractionation, followed by MALDI-MS. Double cross-validatory linear discriminant and logistic regression data analysis methods were compared with a new Bayesian logistic regression method. These were evaluated on their ability to discriminate between healthy and dystrophic samples, and to identify the discriminatory peaks in the mass spectra. All three approaches classified the spectra with comparable misclassification rates (between 18.4 and 20.6%), with much overlap between the differential peaks identified between the methods. The differential peak pattern from the Bayesian method was sparser and easier to interpret than from the other two methods, without compromising classifying strength. One of the two main differentiating peaks at m/z 3908 was identified as an N-terminal peptide of coagulation Factor XIIIa, previously identified in human serum. This work underlines the translational aspect of serum protein profiling in mice and supports a further study with serum from patients with neuromuscular disorders. 相似文献
7.
Ralf Schmauder Sharmini Alagaratnam Chris Chan Thomas Schmidt Gerard W. Canters Thijs J. Aartsma 《Journal of biological inorganic chemistry》2005,10(6):683-687
The blue copper protein azurin from Pseudomonas aeruginosa has been covalently labelled with the fluorescing dye Cy5. The optical spectrum of the azurin changes markedly with its redox
state. These changes are reflected in the fluorescence intensity of the dye through fluorescence resonance energy transfer
(FRET). This provides a sensitive way to monitor biological redox events. The method shown to work in the nanomolar range
of protein concentrations, can be easily extended into the sub-nanomolar regime and holds promise for single-molecule detection. 相似文献
8.
Alagaratnam S Meeuwenoord NJ Navarro JA Hervás M De la Rosa MA Hoffmann M Einsle O Ubbink M Canters GW 《The FEBS journal》2011,278(9):1506-1521
The reactivity of a variant of the blue copper protein, azurin from Pseudomonas aeruginosa, was investigated with laser flash photolysis and compared with the reactivity of the wild-type (WT) protein. The variant was obtained by changing the Cu ligating His117 for a glycine. The mutation creates a gap in the ligand shell of the Cu that can be filled with external ligands or water molecules. The crystal structure of the H117G variant is reported. It shows that the immediate surrounding of the Cu site in the variant exhibits less rigidity than in the WT protein and that the loop containing the Cu ligands Cys112, His117 and Met121 in the WT protein has gained flexibility in the H117G variant. Flash photolysis experiments were performed with 5-deazariboflavin and 8α-imidazolyl-(N-propylyl)-amino riboflavin as electron donors to probe the reactivity of WT and H117G azurin, and of H117G azurin for which the gap in the Cu co-ordination shell was filled with imidazole. 8α-Imidazolyl-(N-propylyl)-amino riboflavin appears one to two orders less efficient as a photo-flash reductant than 5-deazariboflavin. The reactivity of the H117G variant in the absence of external ligands appears to be 2.5-fold lower than the WT reactivity (second-order rate constants of 51 ± 2 × 10(7) m(-1) ·s(-1) versus 21 ± 1 × 10(7) m(-1) ·s(-1) ), whereas the addition of imidazole restores reactivity to above the WT level (71 ± 4 × 10(7) m(-1) ·s(-1) ). The differences are discussed in terms of structural modifications and changes in reorganizational energy and electronic coupling. Database Structural data are available in the Protein Data Bank under the accession number 3N2J. 相似文献
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