The Alpha and Omega of Galactosylceramides in T Cell Immune Function

Glycosphingolipids are a subgroup of glycolipids that contain an amino alcohol sphingoid base linked to sugars. They are found in the membranes of cells ranging from bacteria to vertebrates. This group of lipids is known to stimulate the immune system through activation of a type of white blood cell known as natural killer T cell (NKT cell). Here we summarize the extensive research that has been done to identify the structures of natural glycolipids that stimulate NKT cells and to determine how these antigens are recognized. We also review studies designed to understand how glycolipid variants, both natural and synthetic, can alter the responses of NKT cells, leading to dramatic changes in the global immune response.     

Age and anesthetic effects on murine electrocardiography

Murine models offer potential insights regarding human cardiac disease, but efficient and reliable methods for phenotype evaluation are necessary. We employed non-invasive electrocardiography (ECG) in mice, investigating statistical reliability of these parameters with respect to anesthetic and animal age. Mice (C57BL/6, 8 or 48 weeks) were anesthetized by ketamine/xylazine (K/X, 80/10 mg/kg ip) or by inhalation anesthetic (halothane, HAL; sevoflurane, SEV) and 6 lead ECGs were recorded. P wave duration and QT interval was significantly prolonged with K/X compared to HAL and SEV, indicating slowed atrial and ventricular conduction. P-R interval (atrio-ventricular conduction) was significantly increased in aged mice under all anesthetics. Heart rate was inversely correlated to QT interval and P wave duration. We also detected significant age effects with respect to optimal approaches for QT interval corrections. Power analysis showed 4-fold higher number of mice/group, were required for K/X, to achieve identical statistical sensitivity. These data demonstrate the importance of anesthetic selection for relevant and reliable ECG analysis in mice and illustrate the selective influences of anesthetics and age on cardiac conductance in this species.     

Compartmentalization of membrane trafficking,glucose transport,glycolysis, actin,tubulin and the proteasome in the cytoplasmic droplet/Hermes body of epididymal sperm

Discovered in 1909 by Retzius and described mainly by morphology, the cytoplasmic droplet of sperm (renamed here the Hermes body) is conserved among all mammalian species but largely undefined at the molecular level. Tandem mass spectrometry of the isolated Hermes body from rat epididymal sperm characterized 1511 proteins, 43 of which were localized to the structure in situ by light microscopy and two by quantitative electron microscopy localization. Glucose transporter 3 (GLUT-3) glycolytic enzymes, selected membrane traffic and cytoskeletal proteins were highly abundant and concentrated in the Hermes body. By electron microscope gold antibody labelling, the Golgi trafficking protein TMED7/p27 localized to unstacked flattened cisternae of the Hermes body, as did GLUT-3, the most abundant protein. Its biogenesis was deduced through the mapping of protein expression for all 43 proteins during male germ cell differentiation in the testis. It is at the terminal step 19 of spermiogenesis that the 43 characteristic proteins accumulated in the nascent Hermes body.     

3D-QSAR CoMFA study of benzoxazepine derivatives as mGluR5 positive allosteric modulators

Positive allosteric modulation of the metabotropic glutamate receptor subtype 5 was studied by conducting a comparative molecular field analysis on 118 benzoxazepine derivatives. The model with the best predictive ability retained significant cross-validated correlation coefficients of q² = 0.58 (r² = 0.81) yielding a standard error of 0.20 in pEC₅₀ for this class of compounds. The subsequent contour maps highlight the structural features pertinent to the bioactivity values of benzoxazepines.     

cPGES/p23 is required for glucocorticoid receptor function and embryonic growth but not prostaglandin E2 synthesis

A number of studies have identified cytosolic prostaglandin E(2) synthase (cPGES)/p23 as a cytoplasmic protein capable of metabolism of prostaglandin E(2) (PGE(2)) from the cyclooxygenase metabolite prostaglandin endoperoxide (PGH(2)). However, this protein has also been implicated in a number of other pathways, including stabilization of the glucocorticoid receptor (GR) complex. To define the importance of the functions assigned to this protein, mice lacking detectible cPGES/p23 expression were generated. cPGES/p23(-/-) pups die during the perinatal period and display retarded lung development reminiscent of the phenotype of GR-deficient neonates. Furthermore, GR-sensitive gluconeogenic enzymes are not induced in the prenatal period. However, unlike GR-deficient embryos, cPGES/p23(-/-) embryos are small and a proliferation defect is observed in cPGES/p23(-/-) fibroblasts. Analysis of arachidonic acid metabolites in embryonic tissues and primary fibroblasts failed to support a function for this protein in PGE(2) biosynthesis. Thus, while the growth retardation of the cPGES/p23(-/-) pups and decreased proliferation of primary fibroblasts identify functions for this protein in addition to GR stabilization, it is unlikely that these functions include metabolism of PGH(2) to PGE(2).     

Molecular characterization of the effects of Y-27632

Many key cellular functions, such as cell motility and cellular differentiation are mediated by Rho-associated protein kinases (ROCKs). Numerous studies have been conducted to examine the ROCK signal transduction pathways involved in these motile and contractile events with the aid of pharmacological inhibitors such as Y-27632. However the molecular mechanism of action of Y-27632 has not been fully defined. To assess the relative contribution of these Rho effectors to the effects of Y-27632, we compared the cytoskeletal phenotype, wound healing and neurite outgrowth in cells treated with Y-27632 or subjected to knockdown with ROCK-I, ROCK-II or PRK-2- specific siRNAs. Reduction of ROCK-I enhances the formation of thin actin-rich membrane extensions, a phenotype that closely resembles the effect of Y-27632. Knockdown of ROCK II or PRK-2, leads to the formation of disc-like extensions and thick actin bundles, respectively. The effect of ROCK-I knockdown also mimicked the effect of Y-27632 on wound closer rates. ROCK-I knockdown and Y-27632 enhanced wound closure rates, while ROCK-II and PRK-2 were not appreciably different from control cells. In neurite outgrowth assays, knockdown of ROCK-I, ROCK-II or PRK-2 enhances neurite lengths, however no individual knockdown stimulated neurite outgrowth as robustly as Y-27632. We conclude that several kinases contribute to the global effect of Y-27632 on cellular responses.     

AIDS-related vasculopathy: evidence for oxidative and inflammatory pathways in murine and human AIDS

Increased life expectancy of human immunodeficiency virus (HIV)-positive patients has led to evidence of complications apparently not directly related to immunodeficiency or opportunistic infection, including increased cardiovascular risk. We tested the hypothesis that vascular dysfunction occurs in the murine acquired immune deficiency syndrome (AIDS) model and evaluated potential mechanisms in murine AIDS tissues and relevant human HIV/AIDS vascular tissues. We also investigated endothelial activation and/or endothelial protein nitration and their association with time-dependent vascular dysfunction. At 1 and 5 wk of murine AIDS, statistically significant decreases in KCl contractility and time-dependent contractile deficits in response to phenylephrine were observed. The maximal response (E(max)) was reduced by approximately 40% at 10 wk, and EC(50) values were significantly changed: 102 +/- 7.3 ng for control vs. 190 +/- 37 and 130 +/- 22 ng at 5 and 10 wk, respectively (P < 0.05). Endothelium-dependent relaxation to ACh was decreased (EC(50) = 120 +/- 27 and 343 +/- 94 nM for control and at 10 wk, respectively), whereas the response to an exogenous nitric oxide donor, sodium nitroprusside, remained unchanged, suggesting a specific endothelial dysfunction. Histochemical investigations of the same vascular tissues as well as corresponding coronary endothelium showed an increase in protein 3-nitrotyrosine, intercellular adhesion molecule, and nitric oxide synthase isoforms 2 and 3. These findings were corroborated in concurrent experiments in a cohort of well-cataloged human cardiac microvascular tissues. We have demonstrated, for the first time, a specific functional vasculopathy with endothelial involvement in a murine model of AIDS that was also associated with and correlated to increased oxidative stress and specific endothelial activation. This finding was echoed in a relevant population of human HIV/AIDS patients. Research into sources and intracellular targets of oxidants in this disease could provide important mechanistic insights and may reveal new therapeutic opportunities for this increasingly important cardiovascular disease state.     

14-3-3 Protects against stress-induced apoptosis

Expression of human Bax, a cardinal regulator of mitochondrial membrane permeabilization, causes death in yeast. We screened a human cDNA library for suppressors of Bax-mediated yeast death and identified human 14-3-3β/α, a protein whose paralogs have numerous chaperone-like functions. Here, we show that, yeast cells expressing human 14-3-3β/α are able to complement deletion of the endogenous yeast 14-3-3 and confer resistance to a variety of different stresses including cadmium and cycloheximide. The expression of 14-3-3β/α also conferred resistance to death induced by the target of rapamycin inhibitor rapamycin and by starvation for the amino acid leucine, conditions that induce autophagy. Cell death in response to these autophagic stimuli was also observed in the macroautophagic-deficient atg1Δ and atg7Δ mutants. Furthermore, 14-3-3β/α retained its ability to protect against the autophagic stimuli in these autophagic-deficient mutants arguing against so called ‘autophagic death''. In line, analysis of cell death markers including the accumulation of reactive oxygen species, membrane integrity and cell surface exposure of phosphatidylserine indicated that 14-3-3β/α serves as a specific inhibitor of apoptosis. Finally, we demonstrate functional conservation of these phenotypes using the yeast homolog of 14-3-3: Bmh1. In sum, cell death in response to multiple stresses can be counteracted by 14-3-3 proteins.