Influence of co-culture with oviductal epithelial cells on in vitro maturation of canine oocytes

The process of oocyte maturation in the canine species is unique among mammals: oocytes are immature at ovulation and the resumption and progression of meiotic maturation occur in the oviduct. This study was performed to investigate (i) the effect of co-culture with infundibulum and ampullar oviductal epithelial cells on the in vitro maturation of canine oocytes and (ii) the culture time necessary to reach full meiotic maturation. For this purpose the oocytes, collected from the ovaries of bitches undergoing ovariectomies, were divided into three groups and cultured for 48 and 72 h with the following systems: (A) TCM 199 + 10% oestrus bitch serum + FSH (0.1 IU.mL(-1)), LH (0.1 IU.mL(-1)) + progesterone (1 microg.mL(-1)) + oestradiol (1 microg.mL(-1)) + cysteamine (100 microM); (B) medium A plus infundibulum cells; (C) medium A plus ampullar cells. Infundibulum and ampullar cells were recovered from the oviducts of bitches at the oestrus stage of their cycle. The results showed that after 48 h of incubation, a significantly higher meiotic resumption (P < 0.01) was observed in the oocytes cultured with infundibulum (59%) and ampullar cells (60.0%), than in the control group (40.0%). There was also a significantly (P < 0.01) higher meiotic progression to the MII in systems B and C (15.6% and 16.7%) than in system A (4.0%). After 72 h of culture, the percentages of meiotic resumption and progression were unchanged. These results showed that both the infundibulum and the ampullar oviductal epithelial cells positively influence the meiotic resumption and progression of canine oocytes and that 48 h are sufficient for the completion of nuclear maturation.     

Mitochondrial targeting and membrane anchoring of a viral replicase in plant and yeast cells

Replication of the Carnation Italian ringspot virus genomic RNA in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection. ORF1 in the viral genome encodes a 36-kDa protein, while ORF2 codes for the 95-kDa replicase by readthrough of the ORF1 stop codon. We have shown previously that the N-terminal part of ORF1 contains the information leading to vesiculation of mitochondria and that the 36-kDa protein localizes to mitochondria. Using infection, in vivo expression of green fluorescent protein fusions in plant and yeast cells, and in vitro mitochondrial integration assays, we demonstrate here that both the 36-kDa protein and the complete replicase are targeted to mitochondria and anchor to the outer membrane with the N terminus and C terminus on the cytosolic side. Analysis of deletion mutants indicated that the anchor sequence is likely to correspond approximately to amino acids 84 to 196, containing two transmembrane domains. No evidence for a matrix-targeting presequence was found, and the data suggest that membrane insertion of the viral proteins is mediated by an import receptor-independent signal-anchor mechanism relying on the two transmembrane segments and multiple recognition signals present in the N-terminal part of ORF1.     

ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors

ADAMTS1 is a secreted protein that belongs to the recently described ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats) family of proteases. Evaluation of ADAMTS1 catalytic activity on a panel of extracellular matrix proteins showed a restrictive substrate specificity which includes some proteoglycans. Our results demonstrated that human ADAMTS1 cleaves aggrecan at a previously shown site by its mouse homolog, but we have also identified additional cleavage sites that ultimately confirm the classification of this protease as an 'aggrecanase'. Specificity of ADAMTS1 activity was further verified when a point mutation in the zinc-binding domain abolished its catalytic effects, and latency conferred by the prodomain was also demonstrated using a furin cleavage site mutant. Suppression of ADAMTS1 activity was accomplished with a specific monoclonal antibody and some metalloprotease inhibitors, including tissue inhibitor of metalloproteinases 2 and 3. Finally, we developed an activity assay using an artificial peptide substrate based on the interglobular domain cleavage site (E(373)-A) of rat aggrecan.     

Lignans from leaves of Rollinia mucosa

A new furofuranic lignan named (+)-epimembrine together with known (+)-epieudesmine and (+)-epimagnoline were isolated from leaves of R. mucosa. Their structures were determined by spectroscopic data. Palmitone and a mixture of beta-sitosterol and stigmasterol were also isolated.     

Betulaceae and Corylaceae in Trieste (NE-Italy):Aerobiological and clinical data

Airborne pollen produced by Betulaceae and Corylaceaeis present in the Trieste area for a long period fromJanuary to June, but only in April it does representa considerable proportion (436 p/m³) of the totalpollen count (1193 p/m³). In the years considered (1995–1997), there was a gradual increase in thepollen count of Corylaceae and Betulaceae: frommaximum levels of 580 p/m³ in 1995 to 1218 p/m³ in 1997, and the taxon making the mostsignificant contribution to the pollen concentrationcurve was Ostrya carpinifolia. Sensitization to Betulaceae and Corylaceae wasanalyzed in 2213 subjects visiting our clinic betweenJanuary 1st 1995 and December 31th 1997, withallergic symptoms believed to be IgE mediated. Of thegroup, 1292 (58.4%) were atopic by skin prick testand 328 of them (25.4%) were sensitized to Betulaceaeand Corylaceae. Of the 328 subjects, 72.6% werecosensitized to Gramineae, 56.1% to Oleaceae, 42.1%to Compositae, and just over half to house-dust mites(52.1%). Only ten cases were mono-sensitized (3%).Of the subjects sentitized to Betulaceae andCorylaceae, 163 complained of rhinitis (72%) and 110of asthma (33.6%), often in association withrhinitis; 177 (54%) had only seasonal symptoms.Sensitization to Betulaceae and Corylaceae is high,but its role in inducing allergic respiratorysymptoms is difficult to evaluate because almost allpatients were sensitized to other pollen types. Inconclusion, the role played by this family of trees indetermining allergic respiratory symptoms could becomeincreasingly important in this geographical area inthe future, if pollen levels continue to rise at theirpresent rates. For the moment, sensitivity toBetulaceae-Corylaceae among the population is presentalmost exclusively in subjects sensitized also toother pollen types.     

Leptospira spp., rotavirus,norovirus, and hepatitis E virus surveillance in a wild invasive golden‐headed lion tamarin (Leontopithecus chrysomelas; Kuhl, 1820) population from an urban park in Niterói,Rio de Janeiro,Brazil

The world currently faces severe biodiversity losses caused by anthropogenic activities such as deforestation, pollution, the introduction of exotic species, habitat fragmentation, and climate changes. Disease ecology in altered environments is still poorly understood. The golden‐headed lion tamarin (GHLT, Leontopithecus chrysomelas) is an endangered species that became invasive in an urban park in Niterói, Rio de Janeiro, Brazil. The initially few invasive GHLT individuals became hundreds, adapted to living in proximity to humans and domestic animals. These GHLTs were captured as part of a conservation project; some animals were translocated to Bahia and some were kept in captivity. This study tested 593 GHLT for Leptospira serology; 100 and 95 GHLT for polymerase chain reaction (PCR) toLeptospira and hepatitis E virus genotype 3 (HEV‐3), respectively, and 101 familiar groups for PCR to viruses (rotavirus A, norovirus GI and GII, and HEV‐3). One animal had antibodies for Leptospira serovar Shermani and another for serovar Hebdomadis. One saprophyticLeptospira was found by the 16S PCR and sequencing. Viruses were not detected in samples tested. Findings suggest that the epidemiological importance of such pathogens in this GHLT population is either low or nonexistent. These data are important to understand the local disease ecology, as well as monitoring a translocation project, and to contribute data for species conservation.     

Ultrastructural and biochemical studies on Candida albicans after prolonged incubation in sea water

Abstract Candida albicans yeast cells suspended in sterilized sea water and cultivated in Brain Heart Infusion broth were compared. Viability, chemical composition, surface hydrophobicity and ultrastructural characteristics showed variations after incubation in sea water. The yeast cells developed some ultrastructural changes after about a month in sea water. The surface hydrophobicity of the yeast cells was gradually reduced, starting from day 16, and continued to decline throughout the 32 days in sea water. A decrease in total carbohydrate, lipid and protein contents was also observed and corresponded with ultrastructural modifications.     

Dissociation of Human αB-Crystallin Aggregates by Thiocyanate Is Structurally and Functionally Reversible

Conformational modifications and changes in the aggregation state of human αB-crystallin were investigated at different concentrations of SDS, KBr, urea, and NH₄SCN and at different temperatures. Intrinsic fluorescence measurements indicated complete and reversible unfolding of the protein at 2 M NH₄SCN, whereas the concentration of urea required for complete and irreversible unfolding was 6 M. Gel permeation chromatography indicated almost complete dissociation of the micelle-like aggregate of αB-crystallin in 2 M NH₄SCN, but only partial dissociation into large-sized aggregates in 6 M urea. Thiocyanate-treated αB-crystallin recovered its chaperone-like activity upon dilution of the dissociating agent, whereas the urea-treated protein did not.