Nitridergic platelet pathway activation by hementerin, a metalloprotease from the leech Haementeria depressa

Hementerin (HT) is an 80 kDa fibrino(geno)lytic metalloprotease, purified from saliva of the leech Haementeria depressa. In the present report, the effect of HT on several functional parameters of human platelets was assessed. HT inhibited platelet aggregation and ATP release induced by different agonists such as ADP, adrenaline, collagen, thrombin, and arachidonic acid. HT did neither modify the expression of platelet glycoproteins (Ib, IIb-IIIa, Ia-IIa, IV) nor intraplatelet fibrinogen levels, whereas it markedly decreased CD62P and CD63 levels after the stimulation with thrombin. HT significantly increased thrombin-induced platelet Ca2+ intracellular levels, cGMP content and nitric oxide synthase (NOS) activity. The effect of HT on platelet aggregation was reversed by two NOS inhibitors, N(omega)-Nitro-L-arginine methyl ester and 2 N(G)-Nitro-L-arginine. In summary, these results indicate that HT is an effective inhibitor of human platelet aggregation, presumably through activation of the platelet's nitridergic pathway.     

Effect of plant Kunitz inhibitors from Bauhinia bauhinioides and Bauhinia rufa on pulmonary edema caused by activated neutrophils

Mediators released from polymorphonuclear neutrophils, in particular elastase, are known to induce acute edematous lung injury. In this study we show that the pulmonary edema in isolated perfused rabbit lungs caused by activated neutrophils via release of elastase is significantly decreased by the Kunitz-type Inhibitor BbCI (10(-5) M) from Bauhinia bauhinoides to the same degree as by eglin C (10(-5) M) from Hirudo medicinalis, which was used as a reference. The highly homologous proteinase inhibitor BrPI (10(-5) M) from Bauhinia rufa, however, did not reduce edema formation. The major difference between these inhibitors is the much higher Ki value of BrPI (Ki = 38 nM) for elastase compared to BbCI (Ki = 5.3 nM) and eglin C (Ki = 0.2 nM), respectively. Elastase liberation from activated PMNs was not influenced by the inhibitors. Our results indicate that BbCI can be a useful tool to study the role of neutrophil elastase in pathophysiological processes.     

Mitochondrial COII gene sequences provide new insights into the phylogeny of marmoset species groups (Callitrichidae,Primates)

Mitochondrial cytochrome oxidase II (COII) gene sequences (549 base pairs) were used to investigate the taxonomic relationships among 12 marmoset (Callithrix, Cebuella and Mico) taxa. A large number of substitutions were found in the third base codon positions, providing a strong phylogenetic signal in a gene coding a conserved protein. Despite the significant affinity between the 2 Amazonian genera Cebuella and Mico, found in recent molecular studies, the analysis presented here did not resolve convincingly the phylogenetic relationships between the 3 genera. Mico nevertheless formed 3 distinct clades, reflecting a basic division of species groups based on geographic distribution (east or west of the Rio Tapajós) rather than morphology (presence or absence of auricular hair). This supports the taxonomic distinction of the allopatric emiliae forms. In Callithrix, Callithrix aurita forms a distinct clade, but the remaining morphotypes form a somewhat contradictory cluster, possibly resulting from an extremely rapid radiation.     

Kinetics of T cell-activation molecules in response to Mycobacterium tuberculosis antigens

The phenotypic features acquired subsequent to antigen-specific stimulation in vitro were evaluated by means of the kinetic expressions of CD69 and CD25 activation molecules on T lymphocytes and assayed by flow cytometry in response to PPD, Ag85B, and ferritin in PPD-positive healthy control individuals. In response to PHA, CD69 staining on both CD4+ and CD8+ T cells became initially marked after 4 h, peaked at 24 h, and quickly decreased after 120 h. For CD25, a latter expression was detected around 8 h, having increased after 96 h. As expected, the response rate to the mycobacterial antigens was much lower than that to the mitogen. Positive staining was high after 96 h for CD25 and after 24 h for CD69. CD69 expression was significantly enhanced (p < 0.05) on CD8+ as compared to CD4+ T cells. High levels were also found between 96-120 h. Regarding Ag85B, CD25+ cells were mostly CD4+ instead of CD8+ T cells. Moreover, in response to ferritin, a lower CD25 expression was noted. The present data will allow further characterization of the immune response to new mycobacterial-specific antigens and their evaluation for possible inclusion in developing new diagnostic techniques for tuberculosis as well in a new vaccine to prevent the disease.     

Polyphasic taxonomy of the basidiomycetous yeast genus Rhodotorula: Rh. glutinis sensu stricto and Rh. dairenensis comb. nov

The phenotypic and genetic heterogeneity of the basidiomycetous yeast species Rhodotorula glutinis was investigated in a group of 109 isolates. A polyphasic taxonomic approach was followed which included PCR fingerprinting, determination of sexual compatibility, 26S and ITS rDNA sequence analysis, DNA-DNA reassociation experiments and reassessment of micromorphological and physiological attributes. The relationships with species of the teleomorphic genus Rhodosporidium were studied and isolates previously identified as Rh. glutinis were found to belong to Rhodosporidium babjevae, Rhodosporidium diobovatum and Rhodosporidium sphaerocarpum. Other isolates included in the study were found to belong to Rh. glutinis var. dairenensis, which is elevated to the species level, or to undescribed species. The concept of Rh. glutinis sensu stricto is proposed due to the close phenetic and phylogenetic proximity detected for Rh. glutinis, Rhodotorula graminis and R. babjevae.     

The Complete Amino Acid Sequence of a Trypsin Inhibitor from Bauhinia variegata var. candida Seeds

Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with M _r of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show K _i values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)–Ile (64).     

Induction and inhibition of human lymphocyte transformation by the lectin from the red marine alga Amansia multifida

Lectins are powerful stimulants of quiescent peripheral blood lymphocytes. They can induce blast transformation leading to mitosis of these cells in vitro. We report here the dose-dependent proliferative curve for human peripheral blood monouclear cells (PBMC) stimulated by the lectin amansin, from Amansia multifida. Amansin stimulated proliferation of (PBMC) at relatively low concentrations (3.12 to 12.5 μg mL^-1). We observed also a gradual reduction in mitogenic capacity with progressive increase in the lectin concentration above 12.5 μg mL^-1. This decrease in the mitogenic potential did not result from a toxic effect on the cells, and was predominant at a lectin concentration above 50 μg mL^-1. This decrease in lymphocyte proliferation could be blocked by avidin and could not be overcome by IL-2 or another lectin (Con Br) at stimulatory concentrations. Additionally, we observed that cells incubated at stimulatory concentrations of amansin produced IFN-γ. Analysis of the culture supernatants established a direct correlation between the IFN-γ and the mitogenic and anti-mitogenic capacity of amansin. This revised version was published online in June 2006 with corrections to the Cover Date.     

Meiotic abnormalities in sugarcane (Saccharum spp.) and parental species: Evidence for peri- and paracentric inversions

The modern cultivars of sugarcane (Saccharum spp.) are highly polyploid and accumulate aneuploidies due to their history of domestication, genetic improvement and interspecific hybrid origin involving the domesticated sweet species Saccharum officinarum (‘noble cane’) and the wild Saccharum spontaneum, both with an evolutionary history of polyploidy. The first hybrids were backcrossed with S. officinarum, and selection from progenies in subsequent generations established the genetic basis of modern cultivars. Saccharum genome complexity has inspired several molecular studies that have elucidated aspects of sugarcane genome constitution, architecture and cytogenetics. Herein, we conducted a comparative analysis of the meiotic behaviour of representatives of the parentals S. officinarum and S. spontaneum, and the commercial variety, SP80-3280. S. officinarum, an octoploid species, exhibited regular meiotic behaviour. In contrast, S. spontaneum and SP80-3280 exhibited several abnormalities from metaphase I to the end of division. We reported and typified, for the first time, the occurrence of peri- and paracentric inversions. Using in-situ hybridisation techniques, we were able to determine how pairing association occurred at diakinesis, the origin of lagging chromosomes and, in particular, the mitotic chromosome composition of SP80-3280. Interestingly, S. spontaneum and recombinant chromosomes showed the most marked tendency to produce laggards in both divisions. Future attempts to advance knowledge on sugarcane genetics and genomics should take meiotic chromosome behaviour information into account.     

Protein variation,taxonomy and differentiation in five species of marmosets (genusCallithrix Erxleben, 1777)

The electrophoretic patterns of 15 protein systems codified for 20 genetic loci were investigated using horizontal electrophoresis. A total of 150 blood samples, from five species of the genusCallithrix were analyzed. Polymorphic variation was observed in 10 out 20 loci analyzed. The genotypic distributions are in Hardy-Weinberg equilibrium. The average heterozygosity (H) varied from 1% to 5%, similar to those observed for other Neotropical primates. The genetic distance coefficients revealed a phylogenetic separation of these species into two groups: (1) “argentata” (C. humeralifer andC. emiliae); (2) “jacchus” (C. jacchus, C. penicillata, andC. geoffroyi). This arrangement is according to the taxonomic arrangement proposed byHershkovitz (1977),de Vivo (1988), andMittermeier et al. (1988). The results in each group are compatible with the subspecies values recorded for the Platyrrhini. These values showed that:C. humeralifer andC. emiliae are subspecies ofC. argentata;C. jacchus, C. penicillata, andC. geoffroyi are subspecies ofC. jacchus. These results also suggest thatC. j. geoffroyi is the “jacchus” group taxon, most similar genetically to the “argentata” group.