Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis

The performances of ELISA assays with different antigen preparations, such as Leishmania amazonensis or L. chagasi lysates and the recombinant antigens rK-39 and rK-26, were compared using sera or eluates from dried blood collected on filter paper to detect anti-Leishmania antibodies in dogs from a visceral leishmaniasis-endemic area in Brazil. Of 115 IFAT-reactive dogs at 1:40 titre, 106 (92.2%) were positive in parasitological exams (skin and/or spleen). These animals were compared to healthy animals (n = 25), negative for IFAT at a titre of 1:40 and parasitological exams. The sensitivities of crude and recombinant antigens were similar and remarkably high for both sera and eluates (97-100%). Specificity was higher than 96% for sera and eluates for different antigens, except for L. chagasi antigen using eluates (88%). Concordance values among the tests were higher either for sera or eluates (J = 0.95-1.00). High concordances were observed between sera and eluates tested with different antigens (kappa = 0.93-0.97). Crude and recombinant antigens identified different clinical phases of canine leishmaniasis. These results show that eluates could be used in canine surveys to identify L. chagasi infection. Recombinant antigens added little when compared to crude antigen in identifying positive dogs. Cross-reactivity with other diseases whose distribution often overlaps VL-endemic areas is a limitation of crude antigen use however.     

Expansion of CD22lo B cells in the spleen of autoimmune-prone flaky skin mice

Similar to murine models with compromised CD22/SHP-1 function, flaky skin (fsn) mutant mice exhibit lymphocyte hyperactivation and an autoimmune phenotype characterized by circulating autoantibodies to dsDNA and glomerulonephritis. Immunophenotyping of fsn/fsn splenic B cells was performed to determine if abnormalities in CD22 expression contributed to the phenotype. We identified an expansion of an IgM(bright) CD22lo population consistent with immature B-lymphocytes. While normal B-lymphocytes require IL-4 to achieve down-modulation of CD22 expression in response to BCR cross-linking, culture with anti-IgM alone led to reduced CD22 expression in fsn/fsn mice. Furthermore, when IL-4 was added to fsn/fsn cultures, no further reduction in CD22 expression was observed. This suggested that fsn/fsn B cells were pre-activated in vivo by chronic IL-4 exposure. A portion of these CD22lo cells expressed the B-1 surface marker CD11b. We contend that decreased activation thresholds among CD22lo B-lymphocytes contributes to the expansion of immature and B-1 B cell populations and to the development of autoimmune pathology in fsn/fsn mice.     

Sucrose hydrolases from the midgut of the sugarcane stalk borer Diatraea saccharalis

A beta-fructosidase (EC 3.2.1.26) was isolated from the midgut of larval sugar cane stalk borer Diatraea saccharalis by mild-denaturing electrophoresis and further purified to near homogeneity by gel filtration. beta-Fructosidase hydrolysed sucrose, raffinose and the fructosyl-trisaccharide isokestose, but it had no activity against maltose, melibiose and synthetic substrates for alpha-glucosidases. Two other sucrose hydrolases, one resembling a alpha-glucosidase (EC 3.2.1.20) and the other one active specifically against sucrose (sucrase) were detected in the larval midgut of D. saccharalis. All three sucrose hydrolases were associated with the midgut epithelium of larval D. saccharalis. Relative molecular mass (M(r)) of the beta-fructosidase was estimated around 45,000 (by gel filtration). The other two sucrose hydrolases had M(r) of 54,000 (alpha-glucosidase) and 59,000 (sucrase). The pH optima of the sucrose hydrolases were 5-10 for both alpha-glucosidase and sucrase and 7-8 for beta-fructosidase. Considering V(max)/K(m) ratios, beta-fructosidase preferentially cleaves isokestose rather than raffinose and sucrose. In order to evaluate the possible contribution of microorganisms isolated from the midgut to the pool of sucrose hydrolases, washed midgut epithelia were homogenised and plated onto appropriate media. Seven bacterial and one yeast species were isolated. None of the sucrose hydrolases extracted from the microorganisms corresponded to the enzymes isolated from midgut tissue homogenates. This result suggests that the major sucrose hydrolases found in the midgut of larval D. saccharalis were probably produced by the insect themselves not by the gut microflora.     

Variation in ostracod (Crustacea,Ostracoda) communities in the alluvial valley of the upper Paraná River (Brazil) in relation to substrate

Large river floodplains are convenient model systems to test for variation in animal and plant community structure, as they have a variety of habitats and substrates and are generally dynamic systems through the occurrence of flood pulses with varying intensity. South American floodplain systems furthermore have unique types of substrates, in the form of root systems of floating macrophytes. Here, we investigate the variation in ostracod (small, bivalved crustaceans) communities in relation to substrates and related environmental variables. Sampling was effected in 2004 in the alluvial valley of the upper Paraná River, Brazil, in the wet and dry seasons. Five different substrates, including littoral sediment and four macrophyte species root and leaf systems, in four hydrological systems and a variety of habitat types, were sampled. Fifty-four species of Ostracoda were found. Variation partitioning analysis (RDA) showed that ostracod communities significantly differed between different substrates, mainly between the littoral and plants with small root systems (Eichhornia azurea) on the one hand, and plants with large and complex root systems on the other hand (Eichhornia crassipes and Pistia stratiotes). RDA analyses indicated that the pleuston (biotic communities associated with root systems of floating plants) of E. crassipes comprised more non-swimming species than the pleuston of the smaller roots of P. stratiotes, but species-level Kruskal–Wallis analyses could not detect significant differences between both macrophyte species. Also habitat type and hydrological systems contributed to variation amongst ostracod communities, but less so than the factor substrate. Abiotic factors also contributed to variation, but the ranges of all measured water chemistry variables were narrow. This uniformity in abiotic factors, which might be owing to the occurrence of large flooding events, unites all water bodies, even those that are generally separated.     

Regulation of mitochondrial morphology and cell survival by Mitogenin I and mitochondrial single-stranded DNA binding protein

We found that a mouse homolog of human DNA polymerase delta interacting protein 38, referred to as Mitogenin I in this paper, and mitochondrial single-stranded DNA-binding protein (mtSSB), identified as upregulated genes in the heart of mice with juvenile visceral steatosis, play a role in the regulation of mitochondrial morphology. We demonstrated that overexpression of Mitogenin I or mtSSB increased elongated or fragmented mitochondria in mouse C2C12 myoblast cells, respectively. On the other hand, the silencing of Mitogenin I or mtSSB by RNA interference led to an increase in fragmented or elongated mitochondria in the cells, respectively, suggesting that Mitogenin I and mtSSB are involved in the processes of mitochondrial fusion and fission, respectively. In addition, we showed that the silencing of Mitogenin I resulted in an increase in the number of trypan blue-positive cells and the silencing of mtSSB resulted in an enhancement of the sensitivity of the cells to apoptotic stimulation by etoposide. The present results demonstrated that these proteins play a role in cell survival.     

The Conformational State of Actin Filaments Regulates Branching by Actin-related Protein 2/3 (Arp2/3) Complex

Actin is a highly ubiquitous protein in eukaryotic cells that plays a crucial role in cell mechanics and motility. Cell motility is driven by assembling actin as polymerizing actin drives cell protrusions in a process closely involving a host of other actin-binding proteins, notably the actin-related protein 2/3 (Arp2/3) complex, which nucleates actin and forms branched filamentous structures. The Arp2/3 complex preferentially binds specific actin networks at the cell leading edge and forms branched filamentous structures, which drive cell protrusions, but the exact regulatory mechanism behind this process is not well understood. Here we show using in vitro imaging and binding assays that a fragment of the actin-binding protein caldesmon added to polymerizing actin increases the Arp2/3-mediated branching activity, whereas it has no effect on branch formation when binding to aged actin filaments. Because this caldesmon effect is shown to be independent of nucleotide hydrolysis and phosphate release from actin, our results suggest a mechanism by which caldesmon maintains newly polymerized actin in a distinct state that has a higher affinity for the Arp2/3 complex. Our data show that this new state does not affect the level of cooperativity of binding by Arp2/3 complex or its distribution on actin. This presents a novel regulatory mechanism by which caldesmon, and potentially other actin-binding proteins, regulates the interactions of actin with its binding partners.     

Susceptibility to antimicrobial agents and genotypic relatedness of enterococcal strains isolated from patients and hospital environment

The aim of this study was to evaluate antibiotic susceptibility of Enterococcus sp. strains isolated from two hospitals in ?ód?, during 2005-2006. The second goal was to determine possible transmission of these strains within hospital wards by using melting profile PCR. Enterococcal strains were identified to species according standard microbiological methods. There was isolated 159 strains of E. faecalis, 51 strains of E. faecium and two E. avium, 1 E. durans. Susceptibility to antimicrobial agents was tested by disc diffusion method. None of these strains was resistant to vancomycin, teicoplanin or linezolid. There was high percentage of strains resistant to aminoglicosides, 22% of E. faecalis strains, and 54.9% of E. faecium strains, respectively. Additionally it was shown that 11.7% of E. faecium is resistant to chinuprisin-dalfopristin. The strains with similar pattern of resistance to antibiotics and fenotypic characteristics were genotyped by mpPCR. This technique was useful to confirm relatedness of bacterial strains suspected of being spread within hospital wards.     

Antioxidant activity of extracts from leaves and roots of Salvia miltiorrhiza Bunge, S. przewalskii Maxim., and S. verticillata L

Three Salvia species have been studied for antioxidant activity in methanol extracts from roots and leaves. The presence of the polyphenols and tanshinones was screened by HPLC and spectrophotometric assays and related to the antioxidant potential. The antioxidant capacity of the studied species is high, but differences between species and organs have been also revealed. Salvia przewalskii leaf extract was the strongest one in all tests, followed by Salvia miltiorrhiza root and Salvia verticillata leaf. Among the roots, the most active was S. miltiorrhiza extract, followed by S. verticillata. The antioxidant activity correlates to the total polyphenol and, depending on the assay, to the hydroxycinnamic acids content. The high content of tanshinones in both S. miltiorrhiza and S. przewalskii roots is unlikely to contribute to the antioxidant activity.     

Pioglitazone, a PPAR-gamma ligand, exerts cytostatic/cytotoxic effects against cancer cells, that do not result from inhibition of proteasome

Thiazolidinediones are oral antidiabetic agents that activate peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and exert potent antioxidant and anti-inflammatory properties. It has also been shown that PPAR-gamma agonists induce G0/G1 arrest and apoptosis of malignant cells. Some of these effects have been suggested to result from inhibition of proteasome activity in target cells. The aim of our studies was to critically evaluate the cytostatic/cytotoxic effects of one of thiazolidinediones (pioglitazone) and its influence on proteasome activity. Pioglitazone exerted dose-dependent cytostatic/cytotoxic effects in MIA PaCa-2 cells. Incubation of tumor cells with pioglitazone resulted in increased levels of p53 and p27 and decreased levels of cyclin D1. Accumulation of polyubiquitinated proteins within cells incubated with pioglitazone suggested dysfunction of proteasome activity. However, we did not observe any influence of pioglitazone on the activity of isolated proteasome and on the proteolytic activity in lysates of pioglitazone-treated MIA PaCa-2 cells. Further, treatment with pioglitazone did not cause an accumulation of fluorescent proteasome substrates in transfected HeLa cells expressing unstable GFP variants. Our results indicate that pioglitazone does not act as a direct or indirect proteasome inhibitor.     

Sesquiterpenoids from the fruits of Ferula kuhistanica and antibacterial activity of the constituents of F. kuhistanica.

Ethyl acetate extracts of the air-dried fruits of Ferula kuhistanica afforded three daucane esters: kuhistanicaol H, I and J, together with nine other known compounds. Their structures were established on the basis of spectroscopic evidence. Isolated compounds in this paper and previously reported compounds from the roots and stems of F. kuhistanica were tested for antibacterial activity. Some of them were selectively toxic against Gram-positive bacteria, including methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA).