Molecular mapping with functional antibodies localizes critical sites on the human IL receptor common gamma (gamma c) chain

The IL receptor common gamma (gamma c) chain is required for the formation of high affinity cytokine receptor complexes for IL-2, IL-4, IL-7, IL-9, and IL-15, and for signals regulating cell survival, growth, and differentiation. Our current understanding of how gamma c chain associates with multiple ligands and receptor subunits is drawn largely from its structural homology to the human growth hormone (hGH) receptor and known structure of the hGH/hGH receptor complex. These receptors share distinct features in their extracellular portions and are believed to function by a mechanism of ligand-induced association of receptor subunits. Here, we report the first directed mutational analysis of the human gamma c chain by alanine scanning conducted across seven regions likely to contain residues required for intermolecular contact. Functionally distinct, neutralizing anti-gamma c mAbs were employed to define critical residues. One particular mAb, CP.B8, unique in its ability to inhibit IL-2-, IL-4-, IL-7-, and IL-15-induced proliferation and high affinity cytokine binding of normal T cells as an intact mAb and as a Fab fragment, localized critical residues to four noncontinuous stretches, namely residues in loops AB and EF of domain 1, in the interdomain segment, and in loop FG of domain 2. Notably, these residues form a contiguous patch on the gamma c chain surface in a three-dimensional structural model. These results provide functional evidence for the location of contact points on gamma c chain required for its association with multiple ligands.     

Superfluid Helium Between Rotating Cylinders

The isotopic ordering of H2–D2 mixtures adsorbed at low temperature on graphite in the monolayer range is measured by small angle neutron scattering (SANS). We show that below 8 K, solid mixtures exhibit isotopic clustering at large density (monolayer completion) and a tendency towards the formation of ordered compounds for smaller density (near the commensurate structure).     

The enzymic release of fatty acids from phosphatidylcholine in green peas (Pisum sativum)

‘Phospholipid acyl-hydrolase’ (PLAH), an enzymic activity releasing fatty acid from phosphatidylcholine (PC), has been identified and characterised in green peas. The K_m value for PC dipalmitoyl ester was 0·167 mm. The enzymic activity possessed a pH optimum of 5·6 and was stable for 20 min only at that pH value. The optimum temperature was 45°C and thermal sensitivity was indicated by a 94% decrease in activity upon exposure of the enzyme to 55°C for 3 min, and by an exponential decrease in activity upon storage at 4°C for 1 week. The enzyme was optimally activated by 2·0 mm calcium chloride at pH 5·6, and the optimal concentration of sodium dodecyl sulphate was 0·75 mg ml^?1. Pea PLAH was non-competitively inhibited by sodium cyanide, EDTA and p-chloromercuribenzoate, with no activity in the presence of mercuric chloride. The results from this study are related to those of other workers on lipid-degrading enzymes in peas, and a pathway is proposed for the enzymic degradation of endogenous lipids in fresh or unblanched frozen peas during post-harvest storage.     

The Veterans Affairs Implantable Insulin Pump Study: effect on cardiovascular risk factors

OBJECTIVE: To determine whether implantable insulin pump (IIP) and multiple-dose insulin (MDI) therapy have different effects on cardiovascular risk factors in insulin-requiring patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: A randomized clinical trial was conducted at seven Veterans Affairs medical centers in 121 male patients with type 2 diabetes between the ages of 40 and 69 years receiving at least one injection of insulin per day and with HbA1c, levels of > or =8% at baseline. Weights, blood pressures, insulin use, and glucose monitoring data were obtained at each visit. Lipid levels were obtained at 0, 4, 8, and 12 months, and free and total insulin levels were obtained at 0, 6, and 12 months. All medications being taken were recorded at each visit. RESULTS: No difference in absolute blood pressure, neither systolic nor diastolic, was seen between patients receiving MDI or IIP therapy, but significantly more MDI patients required anti-hypertensive medications. When blood pressure was modeled against weight and time, IIP therapy was significantly better than MDI therapy for systolic blood pressure in patients with BMI <33 and for diastolic blood pressure in patients with BMI >34 kg/m2. Total cholesterol levels decreased in the overall sample, but IIP patients exhibited significantly higher levels than MDI patients. Triglyceride levels increased over time for both groups, with IIP patients having significantly higher levels than patients in the MDI group. BMI was a significant predictor of, and inversely proportional to, HDL cholesterol level. No difference in lipid-lowering drug therapy was seen between the two groups. Free insulin and insulin antibodies tended to decrease in the IIP group as compared with the MDI group. C-peptide levels decreased in both groups. CONCLUSIONS: IIP therapy in insulin-requiring patients with type 2 diabetes has advantages over MDI therapy in decreasing the requirement for antihypertensive therapy and for decreasing total and free insulin and insulin antibodies. Both therapies reduce total cholesterol and C-peptide levels.     

Somatostatin receptor desensitization in NG108-15 cells. A consequence of receptor sequestration

In NG108-15 cells inhibition of both N-type calcium channel current and adenylyl cyclase by somatostatin (SRIF) was not sustained but rapidly desensitized in the continued presence of the drug. The degree and rate of desensitization were concentration-dependent, and the desensitization was homologous with respect to the delta-opioid receptor. We have been unable to obtain evidence for the involvement of G protein-coupled receptor kinases (GRKs) in this desensitization. SRIF-induced desensitization of N-type calcium channel currents was not reduced in cells stably overexpressing a dominant negative mutant of GRK2 or following intracellular dialysis with GRK2- and GRK3-blocking peptides or with heparin. Inhibitors of protein kinase A, protein kinase C, and protein kinase G were also without effect. In contrast, both the rate and degree of SRIF-induced desensitization were reduced by pretreatment with phenylarsine oxide or concanavalin A, both inhibitors of receptor endocytosis. Furthermore, SRIF-induced desensitization was enhanced by monensin, which prevents receptor recycling back to the plasma membrane. Similarly, SRIF-induced desensitization of adenylyl cyclase inhibition was not reduced in cells stably overexpressing dominant negative mutant GRK2 but was reduced in cells pretreated with the receptor endocytosis inhibitor hyperosmotic sucrose or concanavalin A. These data are consistent with the view that SRIF-induced desensitization in NG108-15 cells results from receptor internalization.