Insulin stimulation of phosphatidylinositol 3-kinase activity maps to insulin receptor regions required for endogenous substrate phosphorylation.

We have studied the phosphatidylinositol 3-kinase (PtdIns 3-kinase) in insulin-stimulated Chinese hamster ovary (CHO) cells expressing normal (CHO/IR) and mutant human insulin receptors. Insulin stimulation of CHO/IR cells results in an increase in PtdIns 3-kinase activity associated with anti-phosphotyrosine (alpha PY) immunoprecipitates, which has been previously shown to correlate with the in vivo production of PtdIns(3,4)P2, and PtdIns(3,4,5)P3 (Ruderman, N., Kapeller, R., White, M.F., and Cantley, L.C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415). Stimulation was maximal within 1 min and showed a dose response identical to that of insulin receptor autophosphorylation. The PtdIns 3-kinase also associated with the insulin receptor in an insulin-stimulated manner, as approximately 50% of the total alpha PY-precipitable activity could be specifically immunoprecipitated with anti-insulin receptor antibody. Mutant insulin receptors displayed variable ability to stimulate the PtdIns 3-kinase, but in all cases the presence of PtdIns 3-kinase in alpha PY immunoprecipitates correlated closely with the tyrosyl phosphorylation of the endogenous substrate pp185. In CHO cells expressing a kinase-deficient mutant (IRA1018), there was no observable insulin stimulation of PtdIns 3-kinase activity in alpha PY immunoprecipitates and no tyrosyl phosphorylation of pp185. Substitution of Tyr1146 in the insulin receptor regulatory region with phenylalanine partially impaired receptor autophosphorylation, pp185 phosphorylation, and insulin-stimulated increases in alpha PY-precipitable PtdIns 3-kinase activity. In contrast, a deletion mutant lacking 12 amino acids from the juxtamembrane region (IR delta 960) displayed normal in vivo autophosphorylation but failed to stimulate the PtdIns 3-kinase or phosphorylate pp185. Finally, a mutant receptor from which the C-terminal 43 amino acids had been deleted (IR delta CT) exhibited normal insulin-stimulated autophosphorylation, pp185 phosphorylation, and stimulation of the PtdIns 3-kinase activity in alpha PY immunoprecipitates. These data suggest that the PtdIns 3-kinase is itself a substrate of the insulin receptor kinase or associates preferentially with a substrate. A comparison of the biological activities of the mutant receptors with their activation of the PtdIns 3-kinase furthermore suggests that the PtdIns 3-kinase may be linked to insulin's ability to regulate DNA synthesis and cell growth.     

Characterization of a tolerogenic T cell epitope of type II collagen and its relevance to collagen-induced arthritis.

A synthetic peptide representing sequences of type II collagen, (CII 245-270), has previously been used to induce tolerance and suppress arthritis in DBA/1 mice. To determine important residues, a series of peptides, each containing one or two site-directed substitutions, was generated. Mononuclear cells from DBA/1 mice immunized with CII were cultured in the presence of each peptide and the T cell response determined by measuring IFN-gamma in culture supernatant fluids. Substitutions within the region CII 260-270 led to significant decreases in IFN-gamma responses, identifying this sequence as a T cell epitope. To determine the effects of substitutions within this epitope on arthritis, substituted peptides were administered to neonatal mice as tolerogens. Five site-directed substitutions, four of which included the insertion of a residue found in type I collagen to replace its type II counterpart, abrogated the ability of the peptides to induce tolerance and suppress arthritis. These substitutions were located at residues 260, 261, 263, 264, and 266. Two patterns of T cell reactivity were observed. Peptides containing individual substitutions at positions 261, 264, or 266 were capable of generating a significant T lymphokine response, although those containing substitutions at residues 260 or 263 were ineffective Ag. Systematic analysis of the fine structures of T cell determinants important for autoimmune arthritis can lead to strategies for therapeutic intervention.     

Intrauterine infusion of prostaglandin E2 and subsequent luteal function in cattle.

The objective was to evaluate the effect of intrauterine infusion of prostaglandin E2 (PGE2) on luteal function in cattle. Heifers and cows were randomly assigned after two normal estrous cycles to either PGE2 or control treatment groups. Females in Treatment A were infused with 1 mg of PGE2 once daily into the uterine horn ipsilateral to the corpus luteum between days 7-10 of the estrous cycle with a 0.25 ml plastic semen straw and an artificial insemination pipette. Females in Treatment B were similarly infused with 1 mg of PGE2 once daily in 20 ml of a carrier vehicle via a catheter on days 10 and 11 of the estrous cycle. Control animals were infused with the carrier vehicle using either a semen straw (Treatment C) or via a catheter (Treatment D) on the same days of the estrous cycle. Blood samples were collected daily to monitor plasma progesterone concentrations during the treatment period. Females infused with PGE2 on days 7-10 of the estrous cycle returned to estrus in a mean of 23.5 days (range 22-25 days) and were similar (P > 0.05) to those infused on days 10 and 11 which returned to estrus in 23.5 days (range 22-25 days). Animals similarly infused with carrier vehicle on the same days of the estrous cycle returned to standing estrus in 20.2 days (range 17-23 days). Plasma progesterone concentrations indicated an extended period of elevated progesterone concentrations in PGE2-treated animals compared with control animals. These results indicate that short term administration of PGE2 early in the estrous cycle may result in extended luteal maintenance.     

Inactivation of bovine herpesvirus-1 and bovine viral diarrhea virus in association with preimplantation bovine embryos using photosensitive agents

Hematoporphyrin (HP), hematoporphyrin derivative (HPD), and thiopyronine (TP) are photosensitive agents (PSA) that have a germicidal effect when they are activated by light: helium neon laser (He Ne ) light (HP, HPD), white light (HP, HPD), and yellow-green light (TP). Experiments were conducted with appropriate controls to determine the effect of photosensitive agents a) for inactivating bovine herpesvirus-1 (BHV-1; titre 10(6) TCID(50) /ml) and bovine viral diarrhea virus (BVDV; titre 10(6) TCID(50) /ml); b) for disinfecting Day-7, zona pellucida-intact (ZP-I) bovine embryos that had been exposed to BHV-1 (titre 10(6) TCID(50) /ml) or BVDV (titre 10(6) TCID(50) /ml); and c) on the in vitro development of embryos. Exposure to HP, HPD and TP followed by light irradiation inactivated BHV-1 and BVDV. Embryos exposed to BHV-I were disinfected by HP or HPD (5 mug/ml) in combination with He Ne light, or by HP or HPD (10 mug/ml) in combination with white light. Embryos exposed to BVDV were disinfected by HPD (5 and 10 mug/ml) followed by He Ne or white light irradiation. Exposure of embryos to light alone or to light and HP or HPD had no detrimental effect on their in vitro development; however, exposure of embryos to TP (5 mug/ml) followed by irradiation caused embryonic degeneration. Exposure of embryos to 5 mug of HPD followed by He Ne light, or 10 mug/ml of HP or HPD, followed by white light, is simple methods of disinfecting them of BHV-I and BVDV.     

alpha-Conotoxins, small peptide probes of nicotinic acetylcholine receptors

alpha-Conotoxins, a family of small peptides from the venoms of the Conus marine moluscs, are selective, snake alpha-neurotoxin-competitive antagonists of the nicotinic acetylcholine receptor. A new alpha-conotoxin, SIA, has been purified, sequenced, and synthesized. Cross-linking with bivalent reagents and photoaffinity labeling of the acetylcholine receptor with alpha-conotoxin yield covalent adducts. Surprisingly, cross-linking to other subunits is considerably more efficient than to the alpha subunit. The relative efficiency of photoactivatable cross-linking to different subunits of the receptor is a function of placement of the photoactivatable group on the toxin. Since the structures of alpha-conotoxins can be solved by 2D NMR [see Pardi et al. (1989) Biochemistry 28, 5494-5508; Kobayashi et al. (1989) Biochemistry 28, 4853-4860], this family of toxins should provide a set of new ligands for probing the acetylcholine receptor with considerable precision.     

Evidence for an interaction between canine synovial cell proteoglycans and link proteins

Link proteins are glycoproteins which stabilize aggregates of proteoglycans and hyaluronic acid in cartilage. We recently identified link proteins in canine synovial cell cultures. We now find that link proteins and proteoglycans extracted from these cells under dissociative conditions sediment in the high-buoyant-density fractions of an associative cesium chloride density gradient, suggesting that link proteins interact with high-bouyant-density proteoglycans. In gradients containing [³⁵S]sulfate-labeled synovial cell extracts, 76% of the labeled sulfate and 54% of the uronic acid is found in the high-buoyant-density fractions. Under associative conditions, Sepharose 2B elution profiles of the crude synovial cell extract, synovial cell high-buoyant-density fractions, and culture medium indicate that synovial cell proteoglycans are present in monomeric form, rather than in aggregates. Synovial cell link proteins co-elute with the [³⁵S]sulfate-labeled material under the same conditions. These proteoglycans do not interact in vitro with exogenous hyaluronic acid. Dermatan sulfate, chondroitin sulfate and heparan sulfate are the major cell-associated sulfated glycosaminoglycans synthesized by cultured canine synovial cells, while hyaluronic acid is found in the culture medium. Although the proteoglycans synthesized by cultured synovial cells interact with link proteins, these data indicate that they do not interact with hyaluronic acid to form aggregates.     

Changing patterns in drug therapy for ischemic heart disease

To determine the possible influence of studies on β-blocker therapy following myocardial infarction and the introduction of calcium-channel blockers on the prescribing habits of physicians in a large urban centre, the drug therapy received by 100 patients with ischemic heart disease (IHD) (50 with myocardial infarction and 50 with unstable angina) admitted to a university teaching hospital in 1980 was compared with that received by another such group of 100 patients admitted in 1983-84. The proportion of patients with myocardial infarction receiving drug therapy was significantly higher in 1983-84, at the time of both admission (p < 0.01) and discharge (p < 0.001). Much of the increase was due to greater use of β-blockers. Of the 50 patients with unstable angina in 1983-84, 20 were taking calcium-channel blockers when admitted, and 29 were taking them when discharged. In both 1980 and 1983-84 unstable angina was treated more vigorously than myocardial infarction. The results suggest that physicians have developed a more aggressive approach to drug therapy for IHD since the publication of the β-blocker studies and the introduction of calcium-channel blockers.