Category: clinically Small Molecule

In developed countries, the prevalence of asthma has increased considerably over the past three decades. Asthma is a complex inflammatory disorder that results from interactions between more than 100 susceptibility genes and multiple environmental factors. It is, therefore, important to identify the gene variants contributing to asthma pathogenesis. Numerous studies have focused on this field, and the cytotoxic T-lymphocyte associated antigen 4 gene has been extensively studied. CTLA-4, a B7-binding protein, was initially described as a classical type I glycoprotein on the surface of activated T cells. Cumulative evidence suggested that CTLA-4 may play an important role in the pathogenesis of asthma. CTLA-4 is a powerful negative regulator of T cell activation and is associated with Th cell differentiation. Oosterwegel et al. demonstrated that CTLA-4 was a potent and critical inhibitor of Th2 cell differentiation. Expression of CTLA-4 in Th2 cells was much higher than in Th1 cells. CTLA-4 was also demonstrated to suppress the production of cytokines produced by Th2 cells. A number of studies showed that administration of CTLA-4-Ig significantly ameliorated airway hyperresponsiveness, reduced the level of eosinophils in average bronchoalveolar lavage fluid and serum IgE, as well as cytokine production in murine asthma model. Recently, Choi et al. reported that intranasal administration of Hph-1-ctCTLA-4 could significantly reduce infiltration of inflammatory cells, secretion of Th2 cytokines, serum IgE levels and AHR in a mouse model of allergic airway inflammation. Lin and co-workers demonstrated that decreased allergic inflammation by NVP-BEZ235 surfactant protein D was mediated by an increased expression of CTLA-4 in T cells. The human CTLA-4 gene is located on chromosome 2q33.2. Several single nucleotide polymorphisms of the CTLA-4 gene have been identified. Some of these studies have demonstrated a significant association of CTLA-4 polymorphisms with atopy or asthma. However, the results were not consistent in other studies. Considering a single study may lack the power of providing a reliable conclusion, we performed a meta-analysis to investigate the relationship between CTLA-4 gene variants and asthma. To our knowledge, this is the first meta-analysis of the association between CTLA-4 polymorphisms and asthma susceptibility. A positive association between these polymorphisms and asthma could not be ruled out because studies with small sample size may have insufficient statistical power to detect a slight effect. Publication bias and heterogeneity may influence the results of meta-analyses. In our meta-analysis, only studies indexed by the selected databases were included. Negative studies were less likely to be published in journals and be available in computerized database, resulting in potential overestimation of effect sizes. In this meta-analysis, Begg’s test and Egger’s test showed significant publication bias, thus the current results should be interpreted cautiously. Therefore, heterogeneity did not seem to have influenced the results, suggesting the reliability of our results. Some limitations of this meta-analysis should be considered. First, the number of available studies that could be included was moderate. Therefore, the results could be influenced by factors like random error. Second, only 7 of the 17 studies were conducted in non-Asian population. Third, the overall outcomes were based on individual unadjusted ORs, while a more precise evaluation should be adjusted by other potentially suspected factors including age, sex and lifestyle.

In our study, stable BHK-21 cell lines expressing targeting shRNAs were selected via FACS sorting for eGFP, which is co-expressed in H1 Lenti-virus vector. Thus, to evaluate efficiency of any given candidate RNAi, the stable clones should be BAY 43-9006 established in order to achieve a high level of anti-virus activity. In this study, we transfected RNAi-VP4 into bovine fetal fibroblasts cells, followed by transfering the transgenic cells into enucleated oocyte cytoplasts, selection of reconstructed embryos were selected based on their expression of eGFP and finally transferring the reconstructed embryos to synchronized recipient cows. Since the major focus of our study is to evaluate the efficiency of FMDV shRNA targeting after transgenic delivery, we used 4-month-old fetuses, insteading of adult animals, for the sake of saving time and money. We confirmed shRNA integration into chromosome of cloned fetuses by Southern Blotting and the expression of shRNA by Northern. Since four-month-old transgenic fetuses could not survive in vitro, for FMDV challenge assay, we used primary tongue epithelium cells established from small pieces of the mucosa collected from the tip of bovine tongue. Since the targeting sequence of RNAi-VP4 was conserved among O, A, and Aisa1 serotypes of FMDV, and ASIA1/YS/CHA/05 strain is able to grow well in BHK-21 cells and in primary tongue epithelium cells, we used ASIA1/YS/CHA/05 strain as challenge virus in this study. We found that shRNA expressed in transgenic fetuses could significantly degrade viral RNA after inoculation of FMDV at a titer of 100 TCID50, and inhibited viral replication. Thus, primary transgenic bovine fetus tongue epithelium cells became much more resistant to FMDV challenge. The most important threat caused by FMDV is the high speed of viral replication, short incubation time, and high contagiousness. Thus, although protective immune responses against FMDV can be efficacious, the rapidity of virus replication and spread can outpace the development of immune defenses and overrun the immune system. Our observation that shRNA inhibited over 91% of viral replication at 48 h after challenge suggest that RNAi-based virus targeting is useful for transgenic cows to get more time to develop immune defense. Needless to say, whether transgenic cows indeed become resistant to FMDV infection will wait for the future study using adult transgenic cows upon FMDV challenge. In fact, we have so far obtained on six-month old male transgenic dairy cattle. Due to their high degree of sequence specificity, shRNAs become ineffective in the presence of escape mutations within and outside the targeted regions, and effective silencing of a single viral gene does not always translate into antiviral effect due to genetic compensation or redundancy. Furthermore, variations within multiple regions of the quasispecies of FMDV were retrospectively revealed by sequencing of FMDV genes, strategies to inhibit RNA virus multiplication based on the use of siRNAs have to consider the high genetic polymorphism exhibited by this group of virus. Thus, it may be important to use multiplex shRNAs if RNAi is to be developed for therapeutic use. In this study, we used shRNAs targeting of viral genes VP2, VP3, and VP4, and observed a significant inhibition of FMDV. Combination of these shRNAs may be necessary to avoid the evolution of escape variants. In conclusion, we obtained three transgenic fetuses expressing RNAi-VP4 against FMDV. Study using primary tongue epithelium cells derived from these fetuses reveal that RNAi-VP4 degraded viral RNA and inhibited viral replication.

LS2 was unable to grow at 42uC since gene could not replicate. It demonstrated that the MSMEG_1556 gene is essential for the growth of M. smegmatis. Furthermore, we found that GlmM protein was not expressed in M. smegmatis LS2 strain. To observe morphological change of LS2 strain, the LS2 cells were harvested after shifting temperature from 30uC to 42uC. The morphological analysis of LS2 by SEM revealed that the LS2 cells were longer and had rougher surface compared to the wild type cells. With the increased incubation time at 42uC, many cells fused and lysed eventually. These results suggested that lacking GlmM could block the PG synthesis and make the structure of cell wall changed, resulting in cell death. Therefore, GlmM is a potential target for development of anti-tuberculosis drugs. HSP90 is a molecular chaperone that functions in association with a cohort of co-chaperones to guide the stabilization and activation of an array of signaling proteins, including oncogenic kinases, transcription factors and hormone SAR131675 receptors. Cdc37 is considered as a key component of this multimeric chaperone machinery, playing a specialized and indispensable role in the maturation and/or stabilization of a large subset of protein kinases, implicated in signal transduction, proliferation and survival. By blocking the closure of the Nterminal HSP90 ATP-binding site, Cdc37 inhibits the ATPase activity of HSP90 and assists loading of kinase client proteins onto the chaperone machinery. In particular, Cdc37 acts as an adaptor or scaffold, facilitating client kinase interaction with HSP90 and subsequently by recruiting these client kinases into the HSP90 complex, it stabilizes and/or maintains them in a folding-competent conformation. In addition, Cdc37 promotes the assembly of HSP90-protein kinase complexes and expression of a dominant form that lacks the HSP90 binding domain inhibits kinase activation in mammalian cells. Many client proteins interact directly with both Cdc37 and HSP90 and their folding, maturation and stability depend on the activity of both chaperones. Hence Cdc37 mediates the formation of HSP90-Raf1 and HSP90-Cdk4 complexes. The complex relationship between Cdc37 and HSP90 is illustrated by the finding that their interaction is stabilized by the client protein. Over the past years there has been increasing evidence demonstrating that intracellular HSP90 plays a pivotal role in the acquisition and maintenance of the malignant phenotype. Accordingly, there is growing interest in Cdc37 in the context of malignancy since Cdc37 also regulates multiple oncogenic kinase clients. Indeed Cdc37 levels are found increased in many clinical cancers. In particular, Cdc37, is increased in proliferating tissues, and is heavily expressed in certain cancers including anaplastic large cell lymphoma acute myelocytic leukaemia, hepatocellular carcinoma and multiple myeloma. Furthermore, data have been presented indicating that Cdc37 can function as an oncogene, as mice engineered to over-express Cdc37 develop tumors at a high frequency, suggesting that the establishment of protein kinase pathways mediated by HSP90/Cdc37 can be a rate-limiting event in epithelial cell transformation. More recently it has been shown that Cdc37 is essential for maintaining prostate tumor cell growth. Additionally, the platelet-derived growth factor receptor alpha which is up-regulated and activated in several malignancies forms a complex with HSP90 and the cochaperone Cdc37 in ovarian, glioblastoma, and lung cancer cells. Together, these results support the targeting of Cdc37 for cancer therapy.

Furthermore, and by exploiting the function blocking properties of a cell-impermeable monoclonal antibody named mAb 4C5, specifically targeted against HSP90 we have shown that extracellular HSP90 interacts with HER-2 on the cell surface as well as metalloproteinases MMP-2 and MMP-9. Although a Torin 1 growing number of HSP90’s co-chaperones such as HSP70, Hop and p23 were also found on the cell surface, their action and underlying mechanisms have not been elucidated yet. Taking the above into consideration, in the present work we explore the cell surface localization of Cdc37 and we examine its possible involvement in cancer cell invasion processes as well as its potential interacting partners during this process, using the MDA-MB-453 and MDA-MB-231 human breast cancer cell lines and a commercially available polyclonal antibody against Cdc37. Furthermore and taking into account previously reported data showing that mAb 4C5 inhibits cancer cell invasion by disrupting association of extracellular HSP90 with HER2 and metalloproteinases MMP2 and MMP9 in this work we investigate the possible effect of this cell impermeable anti-HSP90 antibody on the interactions of surface Cdc37 with HSP90 and the ErbB receptors. In the present work we demonstrate that the co-chaperone Cdc37 is localized on the surface of MDA-MB-453 and MDAMB-231 breast cancer cells, where it is necessary for the motility of these cells and similarly to its intracellular counterpart it specifically interacts with the molecular chaperone HSP90. Moreover our findings show that this surface pool of Cdc37 directly interacts with members of the ErbB family of growth factor receptors possibly acting as a co-factor in HSP90 extracellular chaperoning activities implicated in cancer cell invasion processes and that the anti-HSP90 antibody mAb 4C5 disrupts these interactions. Cell surface localization of Cdc37 was demonstrated by both immunocytochemistry on live MDA-MB-453 and MDA-MB-231 breast cancer cells and western blot using membrane fraction lysates derived from these cell lines. The inhibition of MDA-MB-453 and MDA-MB-231 breast cancer cell motility, by the anti-Cdc37 antibody was shown using the wound healing assay. Presence of this antibody in the culture medium significantly reduced the motility rate of both cell lines studied. At this point it is interesting to note that when the antiHSP90 antibody mAb 4C5 and the anti-Cdc37 antibody were included either separately or combined in the culture medium of the above cells no statistically significant differences were observed in the wound closures. This is not surprising since mAb 4C5 and anti-Cdc37 are targeted against two different molecules involved in the cell motility process. To assess the participation of cell surface and not intracellular Cdc37 in the motility process of the cancer cells studied, internalization of the anti-Cdc37 antibody was examined. Interestingly, the antibody remained bound to the cell surface of both MDA-MB-453 and MDA-MB-231 cells, even after 16 h in culture and was not internalized indicating that the previously mentioned inhibitory effect of the anti-Cdc37 antibody is due to binding of the antibody to the cell surface pool of Cdc37. It is well established that intracellular Cdc37 acts as cochaperone of HSP90 by targeting protein kinases to the chaperone machinery and thus contributing to their activation. Taking this into account together with the above mentioned results we next examined the possible association of cell surface Cdc37 with the surface pools of HSP90 and the ErbB receptors.

Human self-reports of stress during pregnancy are associated with increased circulating cortisol, but only 10–20% of maternal cortisol passes to the fetus. This barrier function of the placenta is achieved through the actions of the enzyme 11b-hydroxysteroid dehydrogenase type 2, which converts the glucocorticoids cortisol/corticosterone into the inactive metabolites cortisone/11b-dehydrocorticosterone, thus preventing the activation of glucocorticoid receptors. In contrast, the enzyme 11ß–HSD1 converts inactive glucocorticoids to cortisol/corticosterone. Targeted gene deletion and pharmacological studies suggest a functional consequence of 11ß–HSD for the development of the hypothalamicpituitary adrenal response to stress. In mice, mutation of the HSD11B2 gene leads to hypertension, excess mineralocorticoid activity, and increased anxiety-like behavior in adulthood whereas HSD11B1 mutation leads to attenuated negative-feedback of the HPA response to stress and improved cognitive performance in aging. Pharmacological inhibition of 11ß–HSD2 during pregnancy or administration of dexamethasone, a synthetic glucocorticoid that is not metabolized by 11ß–HSD2, leads to molecular and neurobiological changes within the HPA axis associated with increased stress responsivity and anxiety-like behavior in adulthood. These glucocorticoid programming effects may also be evident in humans, consequent to prenatal betamethasone exposure or inhibition of 11ß–HSD2 through elevated glycyrrhizin consumption during pregnancy. These studies suggest that the regulation of placental 11ß–HSD2 levels may be a mechanistic link between the experience of maternal gestational stress and long-term health outcomes in offspring. There is increasing evidence that maternal adversity during pregnancy may lead to a down-regulation of 11ß–HSD2. In rats, chronic restraint stress during gestational days 11–20 was found to decrease placental 11ß–HSD2 enzymatic activity and decrease mRNA levels of this gene. Similarly, rat dams that are food restricted from gestational days 10–20, a manipulation that increases maternal plasma corticosterone and induces similar phenotypes to those observed following prenatal stress, have reduced placental protein levels of 11ß–HSD2. In humans, Nutlin-3 heightened maternal anxiety was found to be negatively correlated with placental HSD11B2 mRNA levels. Reduced placental HSD11B2 mRNA levels have also been found associated with intrauterine growth retardation and pre-term birth, suggesting that the transcriptional activity of this enzyme may be predicted by maternal adversity and predictive of high risk birth outcomes. Regulation of gene expression through epigenetic mechanisms – factors that alter gene transcription without altering DNA sequence – is being increasingly explored within the context of environmentally-induced changes in neurobiology, metabolism, and disease risk.