CAP defines a second signalling pathway required for insulin-stimulated glucose transport

Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl protooncogene product(1). Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP(2). Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction(3). Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62940/1/407202a0.pd

Identification of host proteins interacting with Toxoplasma gondii GRA15 (TgGRA15) by yeast two-hybrid system

Background Toxoplasma gondii, an obligate intracellular protozoan parasite, possesses the remarkable ability to co-opt host cell machinery in order to maintain its intracellular survival. This parasite can modulate signaling pathways of its host through the secretion of polymorphic effector proteins localized in the rhoptry and dense granule organelles. One of such effectors is T. gondii type II-specific dense granule protein 15, TgGRA15, which activates NF-κB pathway. The aim of the present study was to identify the host interaction partner proteins of TgGRA15. Methods We screened a yeast two-hybrid mouse cDNA library using TgGRA15 as the bait. TgGRA15 (PRU strain, Type II) was cloned into the pGBKT7 vector and expressed in the Y2HGold yeast strain. Then, the bait protein expression was validated by western blotting analysis, followed by auto-activation and toxicity tests in comparison with control (Y2HGold yeast strain transformed with empty pGBKT7 vector). Results This screening led to the identification of mouse Luzp1 and AW209491 as host binding proteins that interact with TgGRA15. Luzp1 contains three nuclear localizing signals and is involved in regulating a subset of host non-coding RNA genes. Conclusions These findings reveal, for the first time, new host cell proteins interacting with TgGRA15. The identification of these cellular targets and the understanding of their contribution to the host-pathogen interaction may serve as the foundation for novel therapeutic and prevention strategies against T. gondii infection

Conversion of Vertical Banded Gastroplasty to Roux-en-Y Gastric Bypass Results in Restoration of the Positive Effect on Weight Loss and Co-morbidities: Evaluation of 101 Patients

BACKGROUND: Vertical banded gastroplasty (VBG) is a widely used restrictive procedure in bariatric surgery. However, the re-operation rate after this operation is high. In the case of VBG failure, a conversion to Roux-en-Y gastric bypass (RYGBP) is an option. A study was undertaken to evaluate the results of the conversion from VBG to RYGBP. METHODS: 101 patients had conversion from VBG to RYGBP. Patients were separated into 3 groups, based on the indication for conversion: weight regain (group 1), excessive weight loss (group 2) and severe eating difficulties (group 3). Data for the study were collected by retrospective analysis of prospectively recorded data. RESULTS: Weight regain (group 1) was the reason for conversion in 73.3% of patients. Staple-line disruption was the most important cause for the weight regain (74.3%). Excessive weight loss (group 2) affected 14% of patients and was caused by outlet stenosis in 78.6% of patients. The remaining 13% had severe eating difficulties as a result of outlet stenosis (46.1%), pouch dilatation (30.8%) and pouch diverticula (23.1%). Mean BMI before conversion to RYGBP was 40.5, 22.3 and 29.8 kg/m2 in group 1, 2 and 3, respectively. Minor or major direct postoperative complications were observed in 2.0% to 7.0%. Long-term complications were more frequent, and consisted mainly of anastomotic stenosis (22.7%) and incisional hernia (16.8%). Follow-up after conversion was achieved in all patients (100%), with a mean period of 38 +/- 29 months. BMI decreased from 40.5 to 30.1 kg/m2, increased from 22.3 to 25.3 kg/m2. and decreased slightly from 29.8 to 29.0 kg/m2 in group 1, 2 and 3, respectively. All patients in group 3 noticed an improvement in eating difficulties. CONCLUSION: Complications after conversion from failed VBG to RYGBP are substantial and need to be considered. However, the conversion itself is a successful operation in terms of effect on body weight and treating eating difficulties after VBG