Exogenous prostaglandin E2 inhibits TPA induced matrix metalloproteinase-9 production in MCF-7 cells

Elevated levels of prostaglandin E2 (PGE2) have been reported in many high metastatic human breast cancers, but no relationship between exogenous PGE2 activity, expression of matrix metalloproteinases (MMPs) and metastasis in human tumor cells has been reported. The poorly invasive human breast cancer cell line MCF-7 was cultured for 24h in the presence of both phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 nM) and PGE2 (1 microM) and the activity of MMP-9, one of the MMPs involved in metastasis, was measured, in growth medium by gelatin substrate zymography. TPA induced a strong production of MMP-9 while exogenous PGE2 had no effect on the basal MMP-9 level, but inhibited the TPA induced enzyme expression and matrigel invasiveness. We showed that MCF-7 cells expressed EP2, EP3 and EP4 receptors for PGE2 and that its action was probably mediated by EP4 receptor and adenylyl cyclase activation while cAMP dependent PKA was not involved in the process of inhibition of MMP-9 production. These findings suggest a possible inhibitory role for exogenous PGE2 in the metastatic process development.     

Analysis of kavalactones from Piper methysticum (kava-kava)

The chemical analysis and quality control of both Piper methysticum G. Forster (kava-kava) and extracts obtained by aqueous acetone or aqueous methanol as well as supercritical fluid extraction are reviewed. In the last two decades various procedures concerning the separation and detection of kavalactones have been routinely carried out by gas chromatography (without previous derivatization of kavalactones) and high performance liquid chromatography but most of them are not validated or only partially validated. Recently, analyses by supercritical fluid chromatography and micellar electrokinetic chromatography have also been reported. Both gas chromatography and high performance liquid chromatography can be used for the analysis of kavalactones with some advantages and disadvantages for each method. Using gas chromatography analysis, methysticin and yangonin, which are two of the major components, are generally not separated. In addition, the high temperature of the injection port caused the decomposition of methysticin. Concerning high performance liquid chromatography analyses, the reversed-phase is generally better because highly reproducible with a very low detection limit for all compounds even if the quantitative analysis of the kavalactones by liquid chromatography needs to be carried out in the absence of light to prevent the cis/trans isomerisation of yangonin.     

Abl-dependent tyrosine phosphorylation of Sos-1 mediates growth-factor-induced Rac activation

The non-receptor tyrosine kinase Abl participates in receptor tyrosine kinase (RTK)-induced actin cytoskeleton remodelling, a signalling pathway in which the function of Rac is pivotal. More importantly, the activity of Rac is indispensable for the leukaemogenic ability of the BCR-Abl oncoprotein. Thus, Rac might function downstream of Abl and be activated by it. Here, we elucidate the molecular mechanisms through which Abl signals to Rac in RTK-activated pathways. We show that Sos-1, a dual guanine nucleotide-exchange factor (GEF), is phosphorylated on tyrosine, after activation of RTKs, in an Abl-dependent manner. Sos-1 and Abl interact in vivo, and Abl-induced tyrosine phosphorylation of Sos-1 is sufficient to elicit its Rac-GEF activity in vitro. Genetic or pharmacological interference with Abl (and the related kinase Arg) resulted in a marked decrease in Rac activation induced by physiological doses of growth factors. Thus, our data identify the molecular connections of a pathway RTKs-Abl-Sos-1-Rac that is involved in signal transduction and actin remodelling.     

Identification and tissue-specific distribution of sulfated glycosaminoglycans in the blood-sucking bug Rhodnius prolixus (Linnaeus)

We have previously characterized heparan sulfate (HS) as the major ovarian sulfated glycosaminoglycan (GAG) in females of Rhodnius prolixus, while chondroitin sulfate (CS) was the minor component. Using histochemical procedures we found that GAGs were concentrated in the ovarian tissue but not found inside the oocytes. Here, we extend our initial observations of GAG expression in R. prolixus by characterizing these molecules in other organs: the fat body, intestinal tract, and the reproductive tracts. Only HS and CS were found in the three organs analyzed, however CS was the major GAG species in these tissues. We also determined the compartmental distribution of GAGs in these organs by histochemical analysis using 1,9-dimethylmethylene blue, and evaluated the specific distribution of CS within both male and female reproductive tracts by immunohistochemistry using an anti-CS antibody. We also determined the GAG composition in eggs at days 0 and 6 of embryonic development. Only HS and CS were found in eggs at day 6, while no sulfated GAGs were detected at day 0. Our results demonstrate that HS and CS are the only sulfated GAG species expressed in the fat body and in the intestinal and reproductive tracts of Rhodnius male and female adults. Both sulfated GAGs were also identified in Rhodnius embryos. Altogether, these results show no qualitative differences in the sulfated GAG composition regarding tissue-specific or development-specific distribution.     

Fishing new proteins in the twilight zone of genomes: the test case of outer membrane proteins in Escherichia coli K12, Escherichia coli O157:H7, and other Gram-negative bacteria

We address the problem of clustering the whole protein content of genomes into three different categories-globular, all-alpha, and all-beta membrane proteins-with the aim of fishing new membrane proteins in the pool of nonannotated proteins (twilight zone). The focus is then mainly on outer membrane proteins. This is performed by using an integrated suite of programs (Hunter) specifically developed for predicting the occurrence of signal peptides in proteins of Gram-negative bacteria and the topography of all-alpha and all-beta membrane proteins. Hunter is tested on the well and partially annotated proteins (2160 and 760, respectively) of Escherichia coli K 12 scoring as high as 95.6% in the correct assignment of each chain to the category. Of the remaining 1253 nonannotated sequences, 1099 are predicted globular, 136 are all-alpha, and 18 are all-beta membrane proteins. In Escherichia coli 0157:H7 we filtered 1901 nonannotated proteins. Our analysis classifies 1564 globular chains, 327 inner membrane proteins, and 10 outer membrane proteins. With Hunter, new membrane proteins are added to the list of putative membrane proteins of Gram-negative bacteria. The content of outer membrane proteins per genome (nine are analyzed) ranges from 1.5% to 2.4%, and it is one order of magnitude lower than that of inner membrane proteins. The finding is particularly relevant when it is considered that this is the first large-scale analysis based on validated tools that can predict the content of outer membrane proteins in a genome and can allow cross-comparison of the same protein type between different species.     

Oocyte age and nuclear donor cell type affect the technical efficiency of somatic cloning in rabbits

The present study in rabbits compared, in the first experiment, the effect of two commonly used oocyte ages, 13 h and 17 h after ovulation induction treatment, on the technical efficiency of somatic nuclear transfer steps, using fresh cumulus cells as nuclear donors. Recently ovulated metaphase II oocytes (13 h) showed higher fusion (13 h: 83% vs 17 h: 67%, p < 0.05) and in vitro development rates than in vivo slightly aged metaphase II oocytes (morula, 13 h: 74% vs 17 h: 25%, p < 0.05; blastocyst, 13 h: 16% vs 17 h: 8%; p < 0.05). In contrast, activation rate was higher in the 17 h group (13 h: 45% vs 17 h: 67%; p < 0.05). In a second experiment, using recently ovulated oocytes (13 h) as recipients, two donor cell types (from primary cultures of either cumulus cells or fetal fibroblasts) were tested to evaluate their effects on the efficiencies of the different technical steps of somatic nuclear transfer procedure. A better fusion ratewas obtained when fetal fibroblasts were used as nuclear donors (cumulus cells: 45% vs fetal fibroblasts: 67%, p < 0.05). No statistically significant differences were detected in cleavage rate regardless of the cell type used (cumulus cells: 44% vs fetal fibroblasts: 60%, p > 0.05). However, in vitro development to morula (cumulus cells: 41% vs fetal fibroblasts: 14%, p < 0.05) and to blastocyst stage (cumulus cells: 27% vs fetal fibroblasts: 3%, p < 0.05) were different between cell types.     

Abnormal splicing of ABCA1 pre-mRNA in Tangier disease due to a IVS2 +5G>C mutation in ABCA1 gene

Two point mutations of ABCA1 gene were found in a patient with Tangier disease (TD): i) G>C in intron 2 (IVS2 +5G>C) and ii) c.844 C>T in exon 9 (R282X). The IVS2 +5G>C mutation was also found in the brother of another deceased TD patient, but not in 78 controls and 33 subjects with low HDL. The IVS2 +5G>C mutation disrupts ABCA1 pre-mRNA splicing in fibroblasts, leading to three abnormal mRNAs: devoid of exon 2 (Ex2-/mRNA), exon 4 (Ex4-/mRNA), or both these exons (Ex2-/Ex4-/mRNA), each containing a translation initiation site. These mRNAs are expected either not to be translated or generate short peptides. To investigate the in vitro effect of IVS2 +5G>C mutation, we constructed two ABCA1 minigenes encompassing Ex1-Ex3 region, one with wild-type (WTgene) and the other with mutant (MTgene) intron 2. These minigenes were transfected into COS1 and NIH3T3, two cell lines with a different ABCA1 gene expression. In COS1 cells, WTgene pre-mRNA was spliced correctly, while the splicing of MTgene pre-mRNA resulted in Ex2-/mRNA. In NIH3T3, no splicing of MTgene pre-mRNA was observed, whereas WTgene pre-mRNA was spliced correctly. These results stress the complexity of ABCA1 pre-mRNA splicing in the presence of splice site mutations.