Influence of epidermal growth factor on mammalian oocyte maturation via tyrosine-kinase pathway

Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2-5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture with EGF and two specific tyrosine-kinase inhibitors (TKIs, tyrphostins A-23 y A-47). Maturation was determined as the percentage of oocytes at metaphase II stage after culture. Treatments with EGF significantly increased incidences of metaphase II stage compared to controls (86.2% vs. 55% and 70.4% vs. 22.5% in ovine and equine oocytes, respectively). Tyrphostins A-23 and A-47 were effective in suppressing EGF-effect on oocytes. EGF-receptor was localized in follicles, being more prominent in cumulus and granulosa cells. These results confirm that EGF has a physiological role in the regulation of oocyte maturation via tyrosine-kinase pathway.     

Effect of initial glucose concentration and inoculation level of lactic acid bacteria in shrimp waste ensilation

Fermentation conditions and microorganisms were determined, based on acid production, glucose concentration as carbohydrate source. Inoculation levels to obtain a stable shrimp waste silage were also determined. Shrimp waste ensilation was an efficient method of preservation, allowing the recovery of chitin and another added-value products such as pigments, proteins and enzymes. From the various lactic acid bacteria tested, Lactobacillus pentosus and Lactobacillus sp. (B2) were the best lactic acid producers, although small quantities of acetic acid were detected in samples inoculated with Lactobacillus pentosus. Therefore B2 was chosen for the analysis of glucose consumption as well as for the determination of optimum inoculation levels. The best results were obtained at 10% (w/w wet basis) and 5% (v/w wet basis) respectively. Presence of starters and initial glucose concentration were critical factors in the fermentation of shrimp waste. High initial glucose and starter concentrations reduced the time and increased the amount of lactic acid produced. The fermentation pattern changed during ensilation from hetero to homofermentative. Shrimp waste ensilation prevented the growth of spoilage microorganisms keeping their microbial counts steady and pH values within the acid region.     

RAC protein directs the complete removal of the 3' external transcribed spacer by the Pac1 nuclease

In Schizosaccharomyces pombe, interdependency in rRNA processing is mediated by a large protein complex (RAC) which contains independent binding sites for each of the transcribed spacers. The RAC complex exhibits no nuclease activity but dramatically alters the efficiency and specificity of the Pac1 nuclease, leading to the complete removal of the 3' ETS. Furthermore, the affinity of RAC protein for mutant 3' ETS correlates closely with in vivo effects on rRNA processing, and changes which disrupt RAC protein binding also inhibit Pac1 nuclease cleavage at the 3' end of the 25S rRNA sequence. The observations indicate that, in the presence of the RAC protein/3' ETS complex, cleavage by the RNase III-like homolog is not restricted to the known intermediate sites but also is directed at the 3' end of the 25S rRNA.     

Evidence for the involvement of the RNA-dependent protein kinase (PKR) in the induction of human cytotoxic T lymphocytes against a synthetic peptide of HIV-1 by regulatory RNA

The studies on the inhibitory effect exerted by Cholera Toxin (CT) on cell growth and proliferation indicate a remarkable heterogeneity of cell response suggesting that the inhibition represents the final event of many different ways or mechanisms . After CT binding, cAMP accumulation may not occur (as in L1210 leukemia cells) or, when occurring (as in SR-4987 stromal cells), may not be coupled with the antiproliferative effect of CT. In WEHI-3B cells CT binds a Gal-GalNac-GM1b receptor and the anticlonogenic effect of CT seems correlated with cAMP accumulation.To demonstrate the central role of cAMP in WEHI-3B cells, starting from the sensitive cell strain we selected and established a clone of WEHI-3B resistant to CT. This revertant clone (WEHI-3B/CT/REV) is currently cultured in the absence of CT and in the proliferation assay shows a dramatic resistance (>46,000 than the parental cells). Stimulation of WEHI-3B/CT/REV cells by cholera toxin failed to enhance cAMP and the ganglioside-CT binding studied on Thin Layer Chromatography (TLC) blots showed that the resistant cells lost the spot correspondent to the migration of Gal-GalNac-GM1b ganglioside. Both the lines respond at the same level to the adenylate cyclase stimulation by forskolin and the incorporation of GM1a did not decrease the resistance of WEHI-3B/CT/REV. These data confirm that Gal-GalNac-GM1b is the most important functional receptor for CT in WEHI-3B cells able to transduce the signal by enhancing cAMP which in turn inhibits cell proliferation (probably by cAMP dependent protein kinase activation). Our study describes the first cell line resistant to CT originated from a susceptible parental strain and provides a new interesting cell model for studying the cAMP dependent mechanisms involved in cell growth regulation.     

Pituitary luteinizing hormone microheterogeneity in the afternoon of proestrus in rats: some new insights

OBJECTIVE : The aim of the present report was to determine the possible modifications in rat pituitary LH isoforms induced by the spontaneous increase in GnRH at the time of the preovulatory gonadotropin surge. DESIGN: The changes in the quantitative pattern and relative proportions of pituitary LH isoforms in rats on the afternoon of proestrus [INT-P(PM)] were evaluated by comparison with other stages of the estrous cycle (diestrus-1, diestrus-2 and estrus) and ovariectomized (7 and 30 days earlier) animals killed in the morning and in the afternoon of the corresponding day. METHODS: The chromatofocusing technique (pH gradient 11.00-7.00) was used to analyze the different molecular species of intrapituitary LH. RESULTS: Pituitary LH from diestrus-1 animals, considered as a baseline pattern in the cycling rat, eluted as 11 isoforms distributed in pH 9.62-8.82, with greater percentages in pH 9.50-9.01. Except for INT-P(PM) pituitaries, there were no major differences in the pattern of LH heterogeneity in the pituitaries of rats from various stages of the cycle. In contrast, significant changes in the charge distribution and relative abundance of LH isoforms were found in the pituitaries from INT-P(PM) rats. INT-P(PM) pituitaries resolved in 16 LH isoforms with a significant shift to less alkaline pIs (pH 9.62-8.11), the more abundant being focused within pH 9.00-8.51. Conversely, a shift to more basic isoforms resulted after ovariectomy, leading to the accumulation of less mature isoforms in the gonadotrope. CONCLUSIONS: Presumably, the use of animals on INT-P(PM), at the time of the preovulatory LH surge, made it possible to discriminate such changes in LH isoform distribution. That GnRH, released in association with the rising phase of the LH surge, induces these changes in pituitary LH polymorphism appears to be the most likely possibility. In a previous study we demonstrated that GnRH stimulated galactose incorporation into LH in vitro. In the case of pituitaries from INT-P(PM) rats, the shift toward less alkaline isoforms could potentially result from sialylation of increased terminal galactose.     

Massive apoptosis of colonocytes induced by butyrate deprivation overloads resident macrophages and promotes the recruitment of circulating monocytes

Our previous investigations demonstrated a rapid, massive apoptosis of colonocytes after butyrate deprivation. However, while in vitro apoptotic bodies and cells were sludged at the epithelial surface, in vivo they were phagocytosed by the resident macrophages. In the present study the guinea pig colon was perfused in vivo in the presence or absence of butyrate with the aim of identifying the cells involved in the removal of apoptotic material and the method of clearance. Morphological, immunohistochemical and DNA fragmentation analyses were applied. The results demonstrated massive apoptosis of colonocytes in the absence of butyrate. The resident macrophages were tightly clustered below the surface epithelium. Aided by cytoplasmatic projections they phagocytosed and transported apoptotic material from the epithelial intercellular spaces into their bodies. Apparently, the macrophages could not cope with the great amount of apoptotic material they had to eliminate: the recruitment of circulating monocytes occurred. This was revealed by the application of antibodies directed against MAC 387, CD68 (PG-M1), and S-100, which detected distinct monocyte/macrophage populations in the lamina propria. The recruited cells were phenotypically different from resident macrophages, their occurrence being typical in inflamed tissues. In conclusion, butyrate deprivation in vivo led to untimely death of colonocytes and triggered changes in the lamina propria indicative of an inflammatory response.     

Dermatophyte infection and colonization in dogs from South Santiago, Chile

Our main aim was to determine the dermatophyte infection and colonization prevalence in canines from South Santiago, Chile. We studied 241 dogs, 121 of them presented cutaneous lesions suggestive of dermatophytosis and the other 120 were free from lesions and were considered clinically healthy. Dermatophytes were isolated from the lesions of 48.8% of animals with cutaneous diseases and from 5% of healthy dogs. Microsporum canis was the principal species isolated with a frequency of 98.3% from dermatophytosis and from all healthy carriers. The direct microscopic test showed a sensibility of 85% with a positive and negative predictive value of 74 and 79%, respectively. The highest prevalence of dermatophytosis were detected in animals up to one year old (p <= 0.05) and in dogs with high level of irritation, scaly skin, itching and alopecia (p < or = 0.05). The lesions were detected more frequently in head-neck and anterior members (p < or = 0.05). This study shows some clinical characteristics and a frequency of dermatophytes in canines that can be interesting for laboratory professionals and veterinarians.     

TIMP-2 mediated inhibition of angiogenesis: an MMP-independent mechanism

Tissue inhibitors of metalloproteinases (TIMPs) suppress matrix metalloproteinase (MMP) activity critical for extracellular matrix turnover associated with both physiologic and pathologic tissue remodeling. We demonstrate here that TIMP-2 abrogates angiogenic factor-induced endothelial cell proliferation in vitro and angiogenesis in vivo independent of MMP inhibition. These effects require alpha 3 beta 1 integrin-mediated binding of TIMP-2 to endothelial cells. Further, TIMP-2 induces a decrease in total protein tyrosine phosphatase (PTP) activity associated with beta1 integrin subunits as well as dissociation of the phosphatase SHP-1 from beta1. TIMP-2 treatment also results in a concomitant increase in PTP activity associated with tyrosine kinase receptors FGFR-1 and KDR. Our findings establish an unexpected, MMP-independent mechanism for TIMP-2 inhibition of endothelial cell proliferation in vitro and reveal an important component of the antiangiogenic effect of TIMP2 in vivo.     

Histone hyperacetylation in mitosis prevents sister chromatid separation and produces chromosome segregation defects

Posttranslational modifications of core histones contribute to driving changes in chromatin conformation and compaction. Herein, we investigated the role of histone deacetylation on the mitotic process by inhibiting histone deacetylases shortly before mitosis in human primary fibroblasts. Cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with decreased reactivity to the anti-Ser 10 phospho H3 antibody, increased recruitment of protein phosphatase 1-delta on mitotic chromosomes, and depletion of heterochromatin protein 1 from the centromeric heterochromatin. Inhibition of histone deacetylation before mitosis produced defective chromosome condensation and impaired mitotic progression in living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation. In situ hybridization analysis on anaphase cells demonstrated the presence of chromatin bridges, which were caused by persisting cohesion along sister chromatid arms after centromere separation. Thus, the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles.