The Bacillus subtilis genome encodes two multidrug efflux transporters sharing 51% sequence identity: Bmr, described previously, and Blt, described here. Overexpression of either transporter in B. subtilis leads to a similar increase in resistance to ethidium bromide, rhodamine and acridine dyes, tetraphenylphosphonium, doxorubicin, and fluoroquinolone antibiotics. However, Blt differs widely from Bmr in its expression pattern. Under standard cultivation conditions, B. subtilis expresses Bmr but Blt expression is undetectable. We have previously shown that Bmr expression is regulated by BmrR, a member of the family of MerR-like transcriptional activators. Here we show that blt transcription is regulated by another member of the same family, BltR. The DNA-binding domains of BmrR and BltR are related, but their putative inducer-binding domains are dissimilar, suggesting that Bmr and Blt are expressed in response to different inducers. Indeed, rhodamine, a substrate of Bmr and Blt and a known inducer of Bmr expression, does not induce Blt expression. Blt expression has been observed only in B. subtilis carrying mutation acfA, which, as we show here, alters the sequence of the blt gene promoter. Unlike bmr, which is transcribed as a monocistronic mRNA, blt is cotranscribed with a downstream gene encoding a putative acetyltransferase. Overall, the differences in transcriptional control and operon organization between bmr and blt suggest that the transporters encoded by these genes have independent functions involving the transport of distinct physiological compounds.

Children with neurofibromatosis (NF1) typically develop central nervous system (CNS) abnormalities, including aberrant proliferation of astrocytes and formation of benign astrocytomas. The NF1 gene encodes neurofibromin, a Ras-GAP, highly expressed in developing neural cells; the mechanism of regulation of neurofibromin as a Ras-GAP, remains however unknown. We now show that, in response to EGF, neurofibromin is in vivo phosphorylated on serine residues by PKC-a, in human, rat, and avian CNS cells and cell lines. EGF-induced PKC phosphorylation was prominent in the cysteine/serine-rich domain (CSRD) of neurofibromin, which lies in the Nterminus and upstream of the Ras-GAP domain (GRD), and this modification significantly increased the association of neurofibromin with actin in co-immunoprecipitations. In addition, we show that Ras activation in response to EGF was significantly lowered when C62B cells overexpressed a construct encoding both CSRD þ GRD. Moreover, when PKC-a was downregulated, the Ras-GAP activity of CSRD þ GRD was significantly diminished, whereas overexpressed GRD alone acted as a weaker GAP and in a PKC-independent manner. Most importantly, functional Ras inhibition and EGF signaling shifts were established at the single cell level in C6-derived cell lines stably overexpressing CSRD þ GRD, when transient co-overexpression of Ras and PKC-depletion prior to stimulation with EGF-induced mitosis. Taken together, these data provide the first evidence of a functional, allosteric regulation of GRD by CSRD, which requires neurofibromin phosphorylation by PKC and association with the actin cytoskeleton. Our data may suggest a novel mechanism for regulating biological responses to EGF and provide a new aspect for the understanding of the aberrant proliferation seen in the CNS of children with NF1.

Background: Recent epidemiological studies have suggested that obesity is associated with ovarian cancer. Obesity hormone leptin and its receptor (Ob-R) contribute to tumor development by enhancing cell growth and survival. This study was design to investigate the prevalence of leptin and Ob-R in Middle Eastern epithelial ovarian cancer (EOC) and to analyze the role of leptin and the mechanisms under its action in EOC tissue sample and cell lines.

Background A number of constitutively activated signaling pathways play critical roles in the survival and growth of primary effusion lymphoma cells (PELs) including NFkB and PI3/AKT kinase cascades. NFkBis constitutively activated in a number of malignancies, including multiple myeloma, Burkitt’s lymphoma and diffuse large cell B-cell lymphoma. However, its role in primary effusion lymphoma has not been fully explored. Methodology/Principal Findings We used pharmacological inhibition and gene silencing to define the role of NFkB in growth and survival of PEL cells. Inhibition of NFkB activity by Bay11-7085 resulted in decreased expression of p65 in the nuclear compartment as detected by EMSA assays. In addition, Bay11-7085 treatment caused de-phosphorylation of AKT and its downstream targets suggesting a cross-talk between NFkB and the PI3-kinase/AKT pathway. Importantly, treatment of PEL cells with Bay11-7085 led to inhibition of cell viability and induced apoptosis in a dose dependent manner. Similar apoptotic effects were found when p65 was knocked down using specific small interference RNA. Finally, co-treatment of PEL cells with suboptimal doses of Bay11-7085 and LY294002 led to synergistic apoptotic responses in PEL cells. Conclusion/Significance These data support a strong biological-link between NFkB and the PI3-kinase/AKT pathway in the modulation of anti-apoptotic effects in PEL cells. Synergistic targeting of these pathways using NFKB- and PI3-kinase/AKT- inhibitors may have a therapeutic potential for the treatment of PEL and possibly other malignancies with constitutive activation of these pathways.

To identify genes potentially playing an important role in the progression of colorectal carcinoma (CRC), we screened global gene expression using cDNA expression array on 41 CRC tissue samples and 25 noncancerous colorectal tissue samples. Among the up-regulated genes, forkhead box M1 (FOXM1) has been shown to play a critical role in pathogenesis of various malignancies. Using immunohistochemistry on 448 Saudi CRC samples in tissue microarray format, FoxM1 protein overexpression was seen in 66% of CRC tissues and was significantly associated with poorly differentiated and highly proliferative tumors (P = 0.0200 and 0.0018, respectively). FoxM1 expression was also significantly associated with MMP-9 protein expression (P = 0.0002). In vitro data using CRC cell lines showed that inhibition of FoxM1 by thiostrepton resulted in inhibition of proliferation and induction of apoptosis in a dose-dependent manner. Overexpression of FoxM1 potentiated cell proliferation, cell transformation, and migration/invasion of CRC cells via up-regulation of FoxM1 target genes MMP2 and MMP9 and protected these cells from thiostrepton-mediated antiproliferative effects. Finally, in vivo, overexpression of FoxM1 promoted growth of CRC-cell line xenograft tumors in nude mice. Altogether, our data indicate that FoxM1 signaling contributes to aggressiveness in a subset of CRC and that the FOXM1 gene may serve as a useful molecular biomarker and potential therapeutic target.