A Recombination Directionality Factor Controls the Cell Type-Specific Activation of σK and the Fidelity of Spore Development in Clostridium difficile

The strict anaerobe Clostridium difficile is the most common cause of nosocomial diarrhea, and the oxygen-resistant spores that it forms have a central role in the infectious cycle. The late stages of sporulation require the mother cell regulatory protein σ^K. In Bacillus subtilis, the onset of σ^K activity requires both excision of a prophage-like element (skin^Bs) inserted in the sigK gene and proteolytical removal of an inhibitory pro-sequence. Importantly, the rearrangement is restricted to the mother cell because the skin^Bs recombinase is produced specifically in this cell. In C. difficile, σ^K lacks a pro-sequence but a skin^Cd element is present. The product of the skin^Cd gene CD1231 shares similarity with large serine recombinases. We show that CD1231 is necessary for sporulation and skin^Cd excision. However, contrary to B. subtilis, expression of CD1231 is observed in vegetative cells and in both sporangial compartments. Nevertheless, we show that skin^Cd excision is under the control of mother cell regulatory proteins σ^E and SpoIIID. We then demonstrate that σ^E and SpoIIID control the expression of the skin^Cd gene CD1234, and that this gene is required for sporulation and skin^Cd excision. CD1231 and CD1234 appear to interact and both proteins are required for skin^Cd excision while only CD1231 is necessary for skin^Cd integration. Thus, CD1234 is a recombination directionality factor that delays and restricts skin^Cd excision to the terminal mother cell. Finally, while the skin^Cd element is not essential for sporulation, deletion of skin^Cd results in premature activity of σ^K and in spores with altered surface layers. Thus, skin^Cd excision is a key element controlling the onset of σ^K activity and the fidelity of spore development.     

P-I Snake Venom Metalloproteinase Is Able to Activate the Complement System by Direct Cleavage of Central Components of the Cascade

Background

Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom.

Results

Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity.

Conclusion

We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation.     

K121Q PC‐1 Gene Polymorphism Is Not Associated with Insulin Resistance in a Spanish Population

Objective : To investigate the effect of the K121Q plasma cell membrane glycoprotein (PC‐1) polymorphism on the components of the insulin resistance syndrome in a population‐based nationwide multicenter study in Spain. Research Methods and Procedures : The subjects of the study were 293 nonrelated adults (44.7% men and 55.3% women) ages 35 to 64 years randomly chosen from a nationwide population‐based survey on obesity and related conditions, including insulin resistance and cardiovascular risk factors. Obesity‐related anthropometric measurements included blood pressure, oral glucose tolerance test, lipid profile (total cholesterol, high‐density lipoprotein‐ and low‐density lipoprotein‐cholesterol, and triglycerides), plasma leptin, insulin levels by radioimmunoassay, and insulin resistance (homeostasis model assessment). K121Q PC‐1 genotypes were determined by restriction fragment‐length polymorphism‐polymerase chain reaction. Results : Overall Q allele frequency was 0.14, with no differences between obese and nonobese individuals (0.15 vs. 0.13). After adjustment for sex, age, BMI, and degree of glucose tolerance, the Q allele was associated with high plasma leptin and triglyceride levels, but not with insulin resistance. Discussion : The results showed that the K121Q PC‐1 polymorphism in the Spanish population has no significant impact on insulin sensitivity.     

Anti-Tumor Effects of Ganoderma lucidum (Reishi) in Inflammatory Breast Cancer in In Vivo and In Vitro Models

The medicinal mushroom Ganoderma lucidum (Reishi) was tested as a potential therapeutic for Inflammatory Breast Cancer (IBC) using in vivo and in vitro IBC models. IBC is a lethal and aggressive form of breast cancer that manifests itself without a typical tumor mass. Studies show that IBC tissue biopsies overexpress E-cadherin and the eukaryotic initiation factor 4GI (eIF4GI), two proteins that are partially responsible for the unique pathological properties of this disease. IBC is treated with a multimodal approach that includes non-targeted systemic chemotherapy, surgery, and radiation. Because of its non-toxic and selective anti-cancer activity, medicinal mushroom extracts have received attention for their use in cancer therapy. Our previous studies demonstrate these selective anti-cancer effects of Reishi, where IBC cell viability and invasion, as well as the expression of key IBC molecules, including eIF4G is compromised. Thus, herein we define the mechanistic effects of Reishi focusing on the phosphoinositide-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, a regulator of cell survival and growth. The present study demonstrates that Reishi treated IBC SUM-149 cells have reduced expression of mTOR downstream effectors at early treatment times, as we observe reduced eIF4G levels coupled with increased levels of eIF4E bound to 4E-BP, with consequential protein synthesis reduction. Severe combined immunodeficient mice injected with IBC cells treated with Reishi for 13 weeks show reduced tumor growth and weight by ∼50%, and Reishi treated tumors showed reduced expression of E-cadherin, mTOR, eIF4G, and p70S6K, and activity of extracellular regulated kinase (ERK1/2). Our results provide evidence that Reishi suppresses protein synthesis and tumor growth by affecting survival and proliferative signaling pathways that act on translation, suggesting that Reishi is a potential natural therapeutic for breast and other cancers.     

Oleylethanolamide impairs glucose tolerance and inhibits insulin-stimulated glucose uptake in rat adipocytes through p38 and JNK MAPK pathways

Oleylethanolamide (OEA) is a lipid mediator that inhibits food intake and body weight gain and also exhibits hypolipemiant actions. OEA exerts its anorectic effects peripherally through the stimulation of C-fibers. OEA is synthesized in the intestine in response to feeding, increasing its levels in portal blood after the meal. Moreover, OEA is produced by adipose tissue, and a lipolytic effect has been found. In this work, we have examined the effect of OEA on glucose metabolism in rats in vivo and in isolated adipocytes. In vivo studies showed that acute administration (30 min and 6 h) of OEA produced glucose intolerance without decreasing insulin levels. Ex vivo, we found that 10 min of preincubation with OEA inhibited 30% insulin-stimulated glucose uptake in isolated adipocytes. Maximal effect was achieved at 1 microM OEA. The related compounds palmitylethanolamide and oleic acid had no effect, suggesting a specific mechanism. Insulin-stimulated GLUT4 translocation was not affected, but OEA promoted Ser/Thr phosphorylation of GLUT4, which may impair transport activity. This phosphorylation may be partly mediated by p38 and JNK kinases, since specific inhibitors (SB-203580 and SP-600125) partly reverted the inhibitory effect of OEA on insulin-stimulated glucose uptake. These results suggest that the lipid mediator OEA inhibits insulin action in the adipocyte, impairing glucose uptake via p38 and JNK kinases, and these effects may at least in part explain the glucose intolerance produced in rats in vivo. These effects of OEA may contribute to the anorectic effects induced by this mediator, and they might be also relevant for insulin resistance in adipose tissue.     

The Arabidopsis HAL2-like gene family includes a novel sodium-sensitive phosphatase

The yeast HAL2 gene encodes a lithium- and sodium-sensitive phosphatase that hydrolyses 3'-phosphoadenosine-5'-phosphate (PAP). Salt toxicity in yeast results from Hal2 inhibition and accumulation of PAP, which inhibits sulphate assimilation and RNA processing. We have investigated whether the model plant Arabidopsis thaliana contains sodium-sensitive PAP phosphatases. The Arabidopsis HAL2-like gene family is composed of three members: AtAHL and AtSAL2, characterized in the present work, and the previously identified AtSAL1. The AtAHL and AtSAL2 cDNAs complement the auxotrophy for methionine of the yeast hal2 mutant and the recombinant proteins catalyse the conversion of PAP to AMP in a Mg(2+)-dependent reaction sensitive to inhibition by Ca2+ and Li+. The PAP phosphatase activity of AtAHL is sensitive to physiological concentrations of Na+, whereas the activities of AtSAL1 and AtSAL2 are not. Another important difference is that AtAHL is very specific for PAP while AtSAL1 and AtSAL2 also act as inositol polyphosphate 1-phosphatases. AtAHL constitutes a novel type of sodium-sensitive PAP phosphatase which could act co-ordinately with plant sulphotransferases and serve as target of salt toxicity in plants.