Evidence for clonal selection of gamma/delta T cells in response to a human pathogen.

T cells bearing gamma/delta antigen receptors comprise a resident population of intraepithelial lymphocytes in organs such as skin, gut, and lungs, where they are strategically located to contribute to the initial defense against infection. An important unsolved question about antigen-driven gamma/delta T cell responses regards the breadth of their T cell receptor (TCR) repertoire, since many specific epithelial compartments in mice display limited diversity. We have examined the diversity of TCR delta gene expression among human gamma/delta T cells from skin lesions induced by intradermal challenge with Mycobacterium leprae. We show that the vast majority of gamma/delta cells from M. leprae lesions use either V delta 1-J delta 1 or V delta 2-J delta 1 gene rearrangements and, within a given region of the lesion, display limited junctional diversity. This contrasts markedly with the extensive diversity of gamma/delta T cells from peripheral blood of these same individuals, as well as skin from normal donors. These results indicate that the gamma/delta response to M. leprae involves the selection of a limited number of clones from among a diverse repertoire, probably in response to specific mycobacterial and/or host antigens

Effect of L-Carnitine on Skeletal Muscle Lipids and Oxidative Stress in Rats Fed High-Fructose Diet

There is evidence that high-fructose diet induces insulin resistance, alterations in lipid metabolism, and oxidative stress in rat tissues. The purpose of this study was to evaluate the effect of L-carnitine (CAR) on lipid accumulation and peroxidative damage in skeletal muscle of rats fed high-fructose diet. Fructose-fed animals (60 g/100 g diet) displayed decreased glucose/insulin (G/I) ratio and insulin sensitivity index (ISI(0,120)) indicating the development of insulin resistance. Rats showed alterations in the levels of triglycerides, free fatty acids, cholesterol, and phospholipids in skeletal muscle. The condition was associated with oxidative stress as evidenced by the accumulation of lipid peroxidation products, protein carbonyls, and aldehydes along with depletion of both enzymic and nonenzymic antioxidants. Simultaneous intraperitoneal administration of CAR (300 mg/kg/day) to fructose-fed rats alleviated the effects of fructose. These rats showed near-normal levels of the parameters studied. The effects of CAR in this model suggest that CAR supplementation may have some benefits in patients suffering from insulin resistance

TARGETING THE MUC1-C ONCOPROTEIN DOWNREGULATES HER2 ACTIVATION AND ABROGATES TRASTUZUMAB RESISTANCE IN BREAST CANCER CELLS

Patients with HER2 positive breast cancer often exhibit intrinsic or acquired resistance to trastuzumab treatment. The transmembrane MUC1-C oncoprotein is aberrantly overexpressed in breast cancer cells and associates with HER2. The present studies demonstrate that silencing MUC1-C in HER2-overexpressing SKBR3 and BT474 breast cancer cells results in downregulation of constitutive HER2 activation. Moreover, treatment with the MUC1-C inhibitor, GO-203, was associated with disruption of MUC1-C/HER2 complexes and decreases in tyrosine phosphorylated HER2 (p-HER2) levels. In studies of trastuzumab-resistant SKBR3R and BT474R cells, we found that the association between MUC1-C and HER2 is markedly increased (~20-fold) as compared to that in sensitive cells. Additionally, silencing MUC1-C in the trastuzumab-resistant cells or treatment with GO-203 decreased p-HER2 and AKT activation. Moreover, targeting MUC1-C was associated with downregulation of phospho-p27 and cyclin E, which confer trastuzumab resistance. Consistent with these results, targeting MUC1-C inhibited the growth and clonogenic survival of both trastuzumab-resistant cells. Our results further demonstrate that silencing MUC1-C reverses resistance to trastuzumab and that the combination of GO-203 and trastuzumab is highly synergistic. These findings indicate that MUC1-C contributes to constitutive activation of the HER2 pathway and that targeting MUC1-C represents a potential approach to abrogate trastuzumab resistance

Measurement of dissociation rate of biomolecular complexes using CE

Fluorescence anisotropy (FA), non-equilibrium CE of equilibrium mixtures (NECEEM) and high-speed CE were evaluated for measuring dissociation kinetics of peptide–protein binding systems. Fyn-SH3-SH2, a protein construct consisting of the src homology 2 (SH2) and 3 (SH3) domain of the protein Fyn, and a fluorescein-labeled phosphopeptide were used as a model system. All three methods gave comparable half-life of∼53 s for Fyn-SH3-SH2:peptide complex. Achieving satisfactory results by NECEEM required columns over 30 cm long. When using Fyn-SH2-SH3 tagged with glutathione S -transferase (GST) as the binding protein, both FA and NECEEM assays gave evidence of two complexes forming with the peptide, yet neither method allowed accurate measurement of dissociation rates for both complexes because of a lack of resolution. High-speed CE, with a 7 s separation time, enabled separation of both complexes and allowed determination of dissociation rate of both complexes independently. The two complexes had half-lives of 22.0±2.7 and 58.8±6.1 s, respectively. Concentration studies revealed that the GST-Fyn-SH3-SH2 protein formed a dimer so that complexes had binding ratios of 2:1 (protein-to-peptide ratio) and 2:2. Our results demonstrate that although all methods are suitable for 1:1 binding systems, high-speed CE is unique in allowing multiple complexes to be resolved simultaneously. This property allows determination of binding kinetics of complicated systems and makes the technique useful for discovering novel affinity interactions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61861/1/457_ftp.pd

Cbl Enforces Vav1 Dependence and a Restricted Pathway of T Cell Development

Extensive studies of pre-TCR- and TCR-dependent signaling have led to characterization of a pathway deemed essential for efficient T cell development, and comprised of a cascade of sequential events involving phosphorylation of Lck and ZAP-70, followed by phosphorylation of LAT and SLP-76, and subsequent additional downstream events. Of interest, however, reports from our lab as well as others have indicated that the requirements for ZAP-70, LAT, and SLP-76 are partially reversed by inactivation of c-Cbl (Cbl), an E3 ubiquitin ligase that targets multiple molecules for ubiquitination and degradation. Analysis of signaling events in these Cbl knockout models, including the recently reported analysis of SLP-76 transgenes defective in interaction with Vav1, suggested that activation of Vav1 might be a critical event in alternative pathways of T cell development. To extend the analysis of signaling requirements for thymic development, we have therefore assessed the effect of Cbl inactivation on the T cell developmental defects that occur in Vav1-deficient mice. The defects in Vav1-deficient thymic development, including a marked defect in DN3-DN4 transition, were completely reversed by Cbl inactivation, accompanied by enhanced phosphorylation of PLC-γ1 and ERKs in response to pre-TCR/TCR cross-linking of Vav1-/-Cbl-/- DP thymocytes. Taken together, these results suggest a substantially modified paradigm for pre-TCR/TCR signaling and T cell development. The observed consensus pathways of T cell development, including requirements for ZAP-70, LAT, SLP-76, and Vav1, appear to reflect the restriction by Cbl of an otherwise much broader set of molecular pathways capable of mediating T cell development

Effect of L-Carnitine on Skeletal Muscle Lipids and Oxidative Stress in Rats Fed High-Fructose Diet-0

<p><b>Copyright information:</b></p><p>Taken from "Effect of L-Carnitine on Skeletal Muscle Lipids and Oxidative Stress in Rats Fed High-Fructose Diet"</p><p></p><p>Experimental Diabetes Research 2007;2007():-.</p><p>Published online 12 Apr 2007</p><p>PMCID:PMC1880866.</p><p></p