The protein product of the proto-oncogene c-cbl forms a complex with phosphatidylinositol 3-kinase p85 and CD19 in anti-IgM-stimulated human B-lymphoma cells

Multiple signal transduction cascades, consisting of multiple interacting proteins, are activated following stimulation through most cell surface receptors, including the immunoglobulin receptor of B lymphocytes. In this report, we investigated the multimolecular complexes formed following anti-Ig stimulation of a human B-lymphoma cell line, resulting in activation of phosphatidylinositol 3-kinase (PI3K). PI3K is a lipid kinase that consists of an 85-kD regulatory subunit, bound to a 110-kD catalytic subunit. CD19 is a 95-kD B-cell surface marker that contains a consensus binding motif for PI3Kp85 in the cytoplasmic domain and recruits PI3K activity in activated B cells. The protein product of the c-cbl protooncogene is a 120-kD protein that is expressed in early B-lineage cells and in myeloid cells and is phosphorylated on tyrosine following receptor-mediated signaling in T and B lymphocytes. We demonstrate here that phosphorylated c-cbl complexes with CD19 and with PI3Kp85 via its C-terminal SH2 domain, and that both c-cbl and CD19 are associated with active PI3K in anti-Ig- stimulated cells. Although we cannot differentiate between a three- component, c-cbl/CD19/p85 complex and individual two-component complexes, these studies suggest that c-cbl may function as a docking protein, possibly linking distinct signal transduction pathways.     

Ileorectostomy in the older patient

Surgeons have always been wary of performing abdominal colectomy and ileorectostomy in the older patient for fear of excessive stool frequency and fecal incontinence. Thirty-two patients, aged 60 years or over, underwent abdominal colectomy and ileorectostomy and were closely questioned regarding their preoperative, early postoperative, and late postoperative bowel habits. These patients were compared with a group of age- and sex-matched controls who had undergone right hemicolectomy. In both groups, the ileocecal valve had been resected, but only the ileorectostomy group had the entire colon resected. Immediately after ileorectostomy, patients underwent an average increase in bowel movements of 3.6 movements per day. This gradually decreased over time, so that, after five years, older patients with ileorectostomy had an average of 1.5 more bowel movements per day than they had had preoperatively. There were similar increases in the right hemicolectomy patient group: 0.9 bowel movements per day immediately after right hemicolectomy and 0.2 bowel movements per day after five years. Incontinence was an uncommon problem in both groups. This study suggests that elderly patients undergoing abdominal colectomy and ileorectostomy have an increase in daily bowel movements, which is not solely attributable to the loss of the right colon. However, it is a procedure that is well tolerated, with a low risk of incontinence and only a mild increase in stool frequency.     

Alteration of the amino terminus of the mature sequence of a periplasmic protein can severely affect protein export in Escherichia coli.

Escherichia coli alkaline phosphatase, coded for by the phoA gene, is normally translocated across the cytoplasmic membrane into the periplasm with high efficiency. We have constructed a series of derivatives of the phoA gene that code for a wild-type signal sequence but result in altered amino acid sequences at the amino terminus of the mature alkaline phosphatase. Our results suggest that the presence of two positively charged amino acids very early in the mature sequence interferes significantly with protein export. In one case, phoA2AB, the presence of the sequence Arg-Ile-Arg at the amino terminus of alkaline phosphatase results in a 50-times reduction in the export of the protein. By using oligonucleotide-directed mutagenesis, we have constructed mutant derivatives of phoA2AB that are greatly enhanced for export. In all cases, these derivatives reduce the net positive charge in the region. Our results may explain the failure of E. coli to export a number of proteins coded for by artificial constructs and suggest a way to improve export in these cases.     

[99mTc]TRODAT-1: A novel technetium-99m complex as a dopamine transporter imaging agent

Technetium-99m is the most commonly used radionuclide in routine nuclear medicine imaging procedures. Development of^99mTc-labeled receptor-specific imaging agents for studying the central nervous system is potentially useful for evaluation of brain function in normal and disease states. A novel^99mTc-labeled tropane derivative, [^99mTc]TRODAT 1, which is useful as a potential CNS dopamine transporter imaging agent, was evaluated and characterized. After i.v. injection into rats, [^99mTc]TRODAT-1 displayed specific brain uptake in the rat striatal region (striatum-cerebellum/cerebellum ratio 1.8 at 60 min), where dopamine neurons are concentrated. The specific striatal uptake could be blocked by pretreating rats with a dose of competing dopamine transporter ligand, ß-CIT (or RTI-55, i.v., 1 mg/kg). However, the specific striatal uptake of [^99mTc]TRODAT-] was not affected by co-injection of excess free ligand (TRODAT-1, up to 200 g per rat) or by pretreating the rats with haloperidol (i.v., 1 mg/kg). The specific uptake in striatal regions of rats that had prior 6-hydroxydopamine lesion in the substantia nigra area showed a dramatic reduction. The radioactive material recovered from the rat striatal homogenates at 60 min after i.v. injection of [^99mTc]TRODAT-1 showed primarily the original compound (>95%), a good indication of in vivo stability in brain tissue. Similar and comparable organ distribution patterns and brain regional uptakes of [^99mTc]TRODAT-1 were obtained for male and female rats. Ex vivo autoradiography results of rat brain sections further confirmed the high uptake and retention of [^99mTc]TRODAT-1 in the striatal region. In vitro binding studies measuring the affinity to dopamine transporters for the free ligand, TRODAT-1, and a nonradioactive rhenium derivative, Re-TRODAT-1, showed K _i values of 9.7 nM and 14.1 nM, respectively. Behavioral studies in rats using the free ligand, TRODAT-1 and Re-TRODAT-1 indicated that, unlike other tropane derivatives, they displayed no effect on locomotor activity, suggesting low toxicity. These results strongly support the conclusions that this novel^99mTc radioligand binds selectively to dopamine transporters in the brain and that is is potentially useful for in vivo assessment of the loss of dopamine neurons in Parkinson's and other neurodegeneralive diseases.     

Cloning, expression, and catalytic activity of Helicobacter hepaticus urease

Helicobacter hepaticus causes disease in the liver and lower intestinal tract of mice. It is strongly urease positive, although it does not live in an acidic environment. The H. hepaticus urease gene cluster was expressed in Escherichia coli with and without coexpression of the Helicobacter pylori nickel transporter NixA. As for H. pylori, it was difficult to obtain enzymatic activity from recombinant H. hepaticus urease; special conditions including NiCl2 supplementation were required. The H. hepaticus urease cluster contains a homolog of each gene in the H. pylori urease cluster, including the urea transporter gene ureI. Downstream genes were homologs of the nik nickel transport operon of E. coli. Nongastric H. hepaticus produces urease similar to that of H. pylori.     

Erythropoietin and erythropoietin receptor expression in human cancer

Erythropoietin (EPO) stimulates the growth of erythroblasts in the bone marrow (C. Lacombe and P. Mayeux, NEPHROL: DIAL: TRANSPLANT:, 14 (SUPPL: 2): 22-28, 1999). We report basal and hypoxia-stimulated expression of EPO and its receptor, EPOR, in human breast cancer cells, and we demonstrate EPO-stimulated tyrosine phosphorylation and the proliferation of these cells in vitro. In 50 clinical specimens of breast carcinoma, we report high levels of EPO and EPOR associated with malignant cells and tumor vasculature but not with normal breast, benign papilloma, or fibrocystic tissue. Hypoxic tumor regions display the highest levels of EPO and EPOR expression. Enhanced EPO signaling may contribute to the promotion of human cancer by tissue hypoxia.     

Linkage to treatment and supportive services among HIV-positive ex-offenders in Project Bridge

HIV-positive inmates often have histories of substance use, mental illness, and homelessness. Access to supportive services is important for members of this population upon their release from prison to improve continuity of medical care in the community. This paper briefly reviews Project Bridge, a federally funded demonstration project that provided intensive case management for HIV positive ex-offenders. METHODS: Ex-offenders received 18 months of intensive case management by teams of a professional social worker and an outreach worker between May 2003 and December 2005. Client contacts were weekly for 12 weeks and, at a minimum, monthly thereafter. RESULTS: Most clients (95%) received medical care throughout their enrollment. Of all clients in Project Bridge, 45.8% secured housing, 71% were linked to mental health care, and 51% were linked to addiction services. CONCLUSION: Despite high levels of addiction (97%) and mental health disorders (34% on medication), ex-offenders were retained in health care for a year after being released from incarceration.     

Glucose turnover in type I diabetic subjects during exercise. Effect of selective and nonselective beta-blockade and insulin withdrawal.

OBJECTIVE--To assess the effect of selective beta 1-blockade (atenolol and betaxolol) and nonselective beta-blockade (propranolol) on glucose turnover in subjects with insulin-dependent (type I) diabetes mellitus during moderate exercise. RESEARCH DESIGN AND METHODS--Five subjects with type I diabetes were infused with insulin and then exercised for 1 h, after pretreatment with each of the three drugs or saline and, on a separate day, after withdrawal of insulin. Glucose turnover was measured using tritiated glucose. RESULTS--Plasma glucose, initially 9.2 +/- 0.5 mmol/L (mean +/- SE) when insulin infused and 14.0 +/- 0.8 when insulin was withdrawn, fell on exercise by 3.4 +/- 1.1 mmol/L (P < 0.05) saline, 4.0 +/- 0.8 mmol/L (P < 0.01) with betaxolol, 3.8 +/- 0.7 mmol/L (P < 0.01) with atenolol, 5.0 +/- 0.6 mmol/L (P < 0.005) with propranolol, and 1.7 +/- 1.0 mmol/L (NS) when insulin was withdrawn. Propranolol, but not the other beta-blockers, caused a significantly greater fall in glucose on exercise than during the control study. Glucose appearance rate (Ra) was similar basally and rose to an almost identical level in all five groups during exercise. Glucose disappearance rate (Rd) rose similarly during exercise, except after propranolol when the rise was significantly greater than with saline (P < 0.01). Failure of glucose to change significantly during exercise when insulin had been withdrawn was associated with the smallest rise in Rd and the highest nonesterified fatty acid concentrations. Propranolol and betaxolol, but not atenolol, reduced nonesterified fatty acids. CONCLUSIONS--We conclude that the greater fall in glucose on exercise after beta-blocking drugs is probably largely a direct effect of beta 2-blockade on muscle, increasing the exercise-induced rise in Rd glucose. This offers support to the use of beta 1-specific drugs, where beta-blockade is necessary in type I diabetes.