Gene knockdown of gamma-glutamylcysteine synthetase by RNAi in the parasitic protozoa Trypanosoma brucei demonstrates that it is an essential enzyme

The parasitic protozoa Trypanosoma brucei utilizes a novel cofactor (trypanothione, T(SH)2), which is a conjugate of GSH and spermidine, to maintain cellular redox balance. gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the first step in the biosynthesis of GSH. To evaluate the importance of thiol metabolism to the parasite, RNAi methods were used to knock down gene expression of gamma-GCS in procyclic T. brucei cells. Induction of gamma-GCS RNAi with tetracycline led to cell death within 4-6 days post-induction. Cell death was preceded by the depletion of the gamma-GCS protein and RNA and by the loss of the cellular pools of GSH and T(SH)2. The addition of GSH (80 microM) to cell cultures rescued the RNAi cell death phenotype and restored the intracellular thiol pools to wild-type levels. Treatment of cells with buthionine sulfoximine (BSO), an enzyme-activated inhibitor of gamma-GCS, also resulted in cell death. However, the toxicity of the inhibitor was not reversed by GSH, suggesting that BSO has more than one cellular target. BSO depletes intracellular thiols to a similar extent as gamma-GCS RNAi; however, addition of GSH did not restore the pools of GSH and T(SH)2. These data suggest that BSO also acts to inhibit the transport of GSH or its peptide metabolites into the cell. The ability of BSO to inhibit both synthesis and transport of GSH likely makes it a more effective cytotoxic agent than an inhibitor with a single mode of action. Finally the potential for the T(SH)2 biosynthetic enzymes to be regulated in response to reduced thiol levels was studied. The expression levels of ornithine decarboxylase and of S-adenosylmethionine decarboxylase, two essential enzymes in spermidine biosynthesis, remained constant in induced gamma-GCS RNAi cell lines.     

Gene expression patterns in ovarian carcinomas

We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.     

Socioeconomic Status,Functional Recovery,and Long-Term Mortality among Patients Surviving Acute Myocardial Infarction

Objectives

To examine the relationship between socio-economic status (SES), functional recovery and long-term mortality following acute myocardial infarction (AMI).

Background

The extent to which SES mortality disparities are explained by differences in functional recovery following AMI is unclear.

Methods

We prospectively examined 1368 patients who survived at least one-year following an index AMI between 1999 and 2003 in Ontario, Canada. Each patient was linked to administrative data and followed over 9.6 years to track mortality. All patients underwent medical chart abstraction and telephone interviews following AMI to identify individual-level SES, clinical factors, processes of care (i.e., use of, and adherence, to evidence-based medications, physician visits, invasive cardiac procedures, referrals to cardiac rehabilitation), as well as changes in psychosocial stressors, quality of life, and self-reported functional capacity.

Results

As compared with their lower SES counterparts, higher SES patients experienced greater functional recovery (1.80 ml/kg/min average increase in peak V02, P<0.001) after adjusting for all baseline clinical factors. Post-AMI functional recovery was the strongest modifiable predictor of long-term mortality (Adjusted HR for each ml/kg/min increase in functional capacity: 0.91; 95% CI: 0.87–0.94, P<0.001) irrespective of SES (P = 0.51 for interaction between SES, functional recovery, and mortality). SES-mortality associations were attenuated by 27% after adjustments for functional recovery, rendering the residual SES-mortality association no longer statistically significant (Adjusted HR: 0.84; 95% CI:0.70–1.00, P = 0.05). The effects of functional recovery on SES-mortality associations were not explained by access inequities to physician specialists or cardiac rehabilitation.

Conclusions

Functional recovery may play an important role in explaining SES-mortality gradients following AMI.     

Targeted nitric oxide delivery preferentially induces glioma cell chemosensitivity via altered p53 and O6‐Methylguanine‐DNA Methyltransferase activity

Glioblastoma multiforme is the most common malignant central nervous system tumor, and also among the most difficult to treat due to a lack of response to chemotherapeutics. New methods of countering the mechanisms that confer chemoresistance to malignant gliomas could lead to significant advances in the quest to identify novel drug combinations or targeted drug delivery systems for cancer therapy. In this study, we investigate the use of a targeted nitric oxide (NO) donor as a pretreatment to sensitize glioma cells to chemotherapy. The protein chlorotoxin (CTX) has been shown to preferentially target glioma cells, and we have developed CTX–NO, a glioma‐specific, NO‐donating CTX derivative. Pretreatment of cells with CTX–NO followed by 48‐h exposure to either carmustine (BCNU) or temozolomide (TMZ), both common chemotherapeutics used in glioma treatment, resulted in increased efficacy of both therapeutics. After CTX–NO exposure, both T98G and U‐87MG human malignant glioma cells show increased sensitivity to BCNU and TMZ. Further investigation revealed that the consequences of this combination therapy was a reduction in active levels of the cytoprotective enzyme MGMT and altered p53 activity, both of which are essential in DNA repair and tumor cell resistance to chemotherapy. The combination of CTX–NO and chemotherapeutics also led to decreased cell invasion. These studies indicate that this targeted NO donor could be an invaluable tool in the development of novel approaches to treat cancer. Biotechnol. Bioeng. 2013; 110: 1211–1220. © 2012 Wiley Periodicals, Inc.     

Comparative study of four rice cultivars with different levels of cadmium tolerance

Cadmium (Cd) has been identified as a significant pollutant due to its high solubility in water and soil and high toxicity to plants and animals. Rice, as one of the most important food crops, is grown in soils with variable levels of Cd and therefore, is important to discriminate the Cd tolerance of different rice cultivars to determine their suitability for cultivation in Cd-contaminated soils. This study investigates the primary mechanisms employed by four rice cultivars in attaining Cd tolerance. HA63 cultivar reduces Cd uptake by increasing Fe absorption through activation of phytosiderophores. T3028 cultivar accumulates the highest level of Cd in leaves while also activating its reactive oxygen species (ROS) scavenging system, including antioxidant enzymes and phytochelatins. In some rice cultivars (such as HA63), a cyanide-resistant respiration mechanism, important in Cd detoxification, was also promoted under the Cd stress. In conclusion, different rice cultivars may adopt different biochemical strategies and respond with different efficiency to Cd stress.     

Regulators of G-protein signaling (RGS) 4, insertion into model membranes and inhibition of activity by phosphatidic acid

Regulators of G-protein signaling (RGS) proteins are critical for attenuating G protein-coupled signaling pathways. The membrane association of RGS4 has been reported to be crucial for its regulatory activity in reconstituted vesicles and physiological roles in vivo. In this study, we report that RGS4 initially binds onto the surface of anionic phospholipid vesicles and subsequently inserts into, but not through, the membrane bilayer. Phosphatidic acid, one of anionic phospholipids, could dramatically inhibit the ability of RGS4 to accelerate GTPase activity in vitro. Phosphatidic acid is an effective and potent inhibitor of RGS4 in a G alpha(i1)-[gamma-(32)P]GTP single turnover assay with an IC(50) approximately 4 microm and maximum inhibition of over 90%. Furthermore, phosphatidic acid was the only phospholipid tested that inhibited RGS4 activity in a receptor-mediated, steady-state GTP hydrolysis assay. When phosphatidic acid (10 mol %) was incorporated into m1 acetylcholine receptor-G alpha(q) vesicles, RGS4 GAP activity was markedly inhibited by more than 70% and the EC(50) of RGS4 was increased from 1.5 to 7 nm. Phosphatidic acid also induced a conformational change in the RGS domain of RGS4 measured by acrylamide-quenching experiments. Truncation of the N terminus of RGS4 (residues 1-57) resulted in the loss of both phosphatidic acid binding and lipid-mediated functional inhibition. A single point mutation in RGS4 (Lys(20) to Glu) permitted its binding to phosphatidic acid-containing vesicles but prevented lipid-induced conformational changes in the RGS domain and abolished the inhibition of its GAP activity. We speculate that the activation of phospholipase D or diacylglycerol kinase via G protein-mediated signaling cascades will increase the local concentration of phosphatidic acid, which in turn block RGS4 GAP activity in vivo. Thus, RGS4 may represent a novel effector of phosphatidic acid, and this phospholipid may function as a feedback regulator in G protein-mediated signaling pathways.     

Negligible Immunogenicity of Induced Pluripotent Stem Cells Derived from Human Skin Fibroblasts

Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However, limited information is available regarding the immunologic features of iPSCs. In this study, expression of MHC and T cell co-stimulatory molecules in hiPSCs, and the effects on activation, proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation, hiPSCs failed to induce allogeneic CD45⁺ lymphocyte and CD8⁺ T cell activation and proliferation but could induce a low level of allogeneic CD4⁺ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ, TNF-α and IL-17, hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10, and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity, which may result from their induction of IL-10-secreting Treg.     

Amine fluorescamine compounds inhibit oxidative phosphorylation in rat liver mitochondria

The reaction of fluorescamine with ammonia, benzylamine, o,p-dimethylbenzylamine, 2-phenylethylamine, p-aminobenzoic acid, and the mycosamine-containing macrolide antibiotic, amphotericin B, yield compounds which induce significant effects on mitochondrial activities. From their effects on energy-yielding processes which lead to transmembranous proton movements, the compounds may be divided into three classes. While all modifiers significantly inhibit proton movement induced by both ATP hydrolysis and electron transfer in mitochondria, their influence on the primary energy yielding steps are quite different. Class I modifiers, e.g., the compound made from amphotericin B, inhibit electron transfer but have no effect on the Pi release associated with ATP hydrolysis. Class II modifiers, e.g., the compound made from benzylamine, inhibit respiration but stimulate Pi release. Class III modifiers, e.g., the compound made from p-aminobenzoic acid, on the other hand, only slightly increase Pi release but have no effect on redox reactions. These and other effects of the modifiers are taken to mean that the proton movements and their associated energy-yielding processes are only linked indirectly. The effects of the modifiers on State 3 mitochondrial activities were also investigated. Although all the modifiers decrease the rates of both State 3 respiration and its coupled ATP synthesis, the efficiency of energy conversion measured by the P/O ratio remains unaltered.