Molecular cell biology research communications : MCBRC最新文献

Vascular TTB cells derive from murine Kaposi's sarcoma-like dermal lesions and share several phenotypic features with AIDS-associated KS spindle cells. We have recently reported that fibroblast growth factor-2 (FGF-2) promotes dramatic cytoskeletal and morphological alterations in TTB cells, concomitant with the induction of an autocrine loop for hepatocyte growth factor and a relocalization of the urokinase receptor. Since all these alterations are hallmarks of cell transformation. we attempted to verify whether FGF-2 induces a transformed phenotype in TTB cells. Our results show that FGF-2-treated TTB cells acquire the ability to grow under anchorage-independent conditions. In addition, FGF-2 markedly reduced the levels of thrombospondin-1, an antiangiogenic and tumor suppressor protein, in TTB cells. Therefore, FGF-2 induces KS-like spindle cells to acquire properties characteristic of transformed cells. This suggests that FGF-2 plays a pathogenetic role in KS not only by promoting angiogenesis, but also by conferring a transformed phenotype upon KS cells. In light of previous reports on Tat-induced release of FGF-2 into the extracellular space, our findings may provide an additional mechanism for the observed synergism between Tat and FGF-2 in the pathogenesis of KS.

In order to elucidate the mechanisms involved in the downregulation of mdr 1 gene expression reported in experimentally-induced inflammation, we examined the effects of experimentally-induced inflammation and interleukin-(IL-) 6 on transcriptional control of the mdr1 genes in rats. RNA, nuclear extracts, and nuclear protein fractions were isolated from livers harvested from saline or turpentine-treated male Sprague–Dawley rats or from IL-6 treated or nontreated (controls) cultured rat hepatocytes. mdr gene expression and regulation was examined by RT-PCR, mRNA stability studies, nuclear run-on analysis of transcription, and gel shift analysis of promoter-transcription factor interaction. As compared to controls, significantly lower levels of mdr1a and mdr1b mRNA and significantly decreased mdr1a and mdr1b transcription rates were observed in livers isolated from the turpentine-treated rats. In vitro treatments of cultured hepatocytes with IL-6 also suppressed mdr1a and mdr1b mRNA expression and imposed similar reductions in mdr1a and mdr1b transcriptional activity. Significant effects of IL-6 on mdr1 mRNA stability were not seen. Our results indicate that reductions in mdr1 expression in experimental models of inflammation likely occurs through altered gene transcription. Furthermore, as IL-6 was found to decrease mdr1 expression and gene transcription rates in vitro, this cytokine is likely involved in the reduction of mdr1 expression that is seen in vivo during an acute inflammatory response.

In Alzheimer's disease (AD) and Down's syndrome (DS) patients, posttranscriptional alterations of sequences encoded by exon 9 and exon 10 of the β-amyloid precursor protein (βAPP) mRNA result in mutant proteins (βAPP⁺) that colocalize with neurofibrillary tangles and senile plaques. These aberrant messages may contribute to the development of sporadic or late-onset Alzheimer's disease; thus, eliminating them or attenuating their expression could significantly benefit AD patients. In the present work, self-cleaving hammerhead ribozymes targeted to βAPP exon 9 (Rz9) and βAPP⁺ mutant exon 10 (Rz10) were examined for their ability to distinguish between βAPP and βAPP⁺ mRNA. In transiently transfected A-204 cells, quantitative confocal fluorescence microscopy showed that Rz9 preferentially lowered endogenous βAPP. In contrast, in transient cotransfection experiments with βAPP⁺ mRNAs containing a wild-type exon 9 and mutant exon 10 (βAPP-9/βAPP-10+1), or a mutant exon 9 and wild-type exon 10 (βAPP-9+1/βAPP-10) we found that Rz9 and Rz10 preferentially reduced βAPP⁺-mutant exon 10 mRNA in a concentration and a ribozyme-dependent manner.

We have identified two cellular proteins that are specifically immunoprecipitated by an anti-SV40 T antigen monoclonal antibody. This antibody, PAb419, recognizes an epitope contained within a region of T antigen which we have recently demonstrated is required for the initiation of immortalization by SV40 T antigen, but is not essential for maintenance of the immortal state. The two proteins were identified as BAP37 and Prohibitin. Recent results suggest Prohibitin may enhance the transcriptional inactivation of E2F by the retinoblastoma family of pocket proteins (pRb, p107, p130). BAP37 and Prohibitin are specifically recognized by PAb419 and PAb210, another anti-SV40 T antigen monoclonal antibody, which has an overlapping epitope, but not by other anti-SV40 T antigen monoclonal antibodies, demonstrating the specificity of the interaction.

A number of DNaseI-hypersensitive (DH) sites have been mapped within a regulatory region situated upstream of the human apolipoprotein B (apoB) promoter (−5262 to −899) that is required for high level expression of human apoB transgenes in the livers of mice. These DH sites were observed in nuclei from transcriptionally active liver-derived HepG2 cells, but were absent from transcriptionally inactive HeLa cell nuclei. Several nuclear protein binding sites were detected in the DNaseI-hypersensitive region by DNaseI footprinting with HepG2 nuclear extracts, representing putative binding sites for the liver-specific activators. The locations of binding sites for these transcription factors were revealed via computer analysis of the DNA sequence of this region against a transcription factor database. Many micrococcal nuclease hypersensitive (MH) sites were also observed in nuclei from HepG2 cells but not in HeLa cell nuclei, implying that in hepatic cells, nucleosomes are either absent or have been displaced from this region by the liver-specific transcriptional activators, as inferred by the correspondence between the DH sites, the MH sites and the footprints.

The current study characterizes a mediated transport for GSH uptake in murine fibroblasts NIH3T3. The presence of GSH mediated transport is indicated by the behaviour of GSH uptake time-course, as well as by kinetic saturation and the specific inhibition of the initial rate of GSH transport. Moreover, a concentrative GSH uptake has been measured, whose driving force may be due to a change of membrane potential and the direct involvement of ATP. Hyperbolic kinetic saturation shows a single mediated transport with high affinity for GSH (K_m = 0.209 ± 0.03 mM). High specificity of this GSH transporter for the entire structure of GSH is also demonstrated. To summarize, GSH uptake into NIH3T3 cells occurs by an active transport system and shows characteristics similar to ATP-dependent mechanisms previously identified in hepatocyte membranes. Moreover, a possible physiological role of this GSH transporter related to NIH3T3 cell proliferation has been hypothesized.