In vitro association of the phosphatidylinositol 3-kinase regulatory subunit (p85) with the human insulin receptor

The insulin receptor, as a consequence of ligand binding, undergoes autophosphorylation of critical tyrosyl residues within the cytoplasmic portion of its β-subunit. The 85 kDa regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85), an SH2 domain protein, has been implicated as a regulatory molecule in the insulin signal transduction pathway. For the present study, glutathione S-transferase (GST) fusion proteins of p85 SH2 domains were used to determine if such motifs associate directly with the autophosphorylated human insulin receptor. The p85 N + C (amino plus carboxyl) SH2 domains were demonstrated to associate with the autophosphorylated β-subunit, while neither the GTPase activator protein (GAP) N SH2 domain nor the phospholipase C-γ1 (PLCγ1) N + C SH2 domains exhibited measurable affinity for the activated receptor. The p85 N SH2 domain demonstrated weak association with the insulin receptor, while the p85 C SH2 domain alone formed no detectable complexes with the insulin receptor. The association of p85 N + C SH2 domains with the autophosphorylated receptor was competed efficiently by a 15-residue tyrosine-phos-phorylated peptide corresponding to the carboxyl-terminal region of the insulin receptor, but not by phos-phopeptides of similar length derived from the juxtamembrane or regulatory regions. The insulin receptor C domain phosphopeptide inhibited the p85 N i C SH2 domain-insulin receptor complex with an IC_0.5 of 2.3 ± 0.35 μM, whereas a 10-residue phosphopeptide derived from the insulin receptor substrate 1 (IRS-1) competed with an IC_0.5 of 0.54 ± 0.10 μM. These results demonstrate that, in vitro, there is an association between the p85 regulatory protein and the carboxyl-terminal region of the activated insulin receptor that requires the presence of both the N and C SH2 domains. Furthermore, formation of the p85/insulin receptor complex may lead to signaling pathways independent of IRS-1. © Munksgaard 1995.     

RELATIVE CONTRIBUTIONS OF EXTRACELLULAR Ca2+ AND Ca2+ STORES TO SMOOTH MUSCLE CONTRACTION IN ARTERIES AND ARTERIOLES OF RAT, GUINEA-PIG DOG AND RABBIT

1. These studies describe the functional effects of modulation of the sarcoplasmic reticulum (SR) Ca²⁺ stores at three levels of the vasculature: (i) large arteries (rat and guinea-pig aorta); (ii) small resistance arteries (rat tail artery, rabbit mesenteric artery, dog mesenteric artery); and (iii) arterioles (guinea-pig submucosal arterioles of the small intestine). 2. All tissues responded to phenylephrine (PE; 10 μmol/L) with a transient contraction in Ca²⁺-free Krebs', reflecting Ca²⁺ release from PE-sensitive Ca²⁺ stores. After pretreatment with cyclopiazonic acid (CPA; 30 μmol/L) or thapsigargin (TSG; 1 μmol/L), putative SR Ca²⁺ pump inhibitors, the PE-induced contraction in a Ca²⁺-free medium was significantly inhibited in arterial tissues at all levels of the vasculature. Similarly, ryanodine (RYA; 30 μmol/L), an agonist that enhances Ca²⁺ release from the SR, also reduced the PE contraction in a Ca²⁺-free solution. 3. CPA or TSG alone in the presence of extracellular Ca²⁺, caused marked and sustained contraction in the rat and guinea-pig aorta and marked but transient or no contraction in the resistance arteries. In the rat and guinea-pig aorta, RYA caused a slowly developing tension. Little increase in basal tension was produced by RYA in resistance arteries and arterioles. 4. The findings show that an agonist-releasable Ca²⁺ pool is present at all levels of the vasculature that is independent of the size of the vessels and suggest that under normal physiological conditions there is an intimate balance between the roles of the plasma membrane and of the SR in the maintenance of vascular contractility. It appears that the role of the SR diminishes as the arteries become smaller, while Ca²⁺ fluxes across the plasma membrane predominates.     

Mortality in Systemic Sclerosis (Scleroderma)

Two hundred and thirty-seven patients with systemic sclerosiswere followed prospectively in a scleroderma clinic. The overall3, 6, and 9-year survival rates were 86, 76 and 61 per centrespectively. Renal, cardiac and pulmonary disease, and olderage at enrolment were adverse prognostic factors associatedwith reduced survival. There were no significant differencesin survival between males and females or in patients with restrictedcompared to those with diffuse skin thickening. Death from systemicsclerosis was most frequently due to pulmonary hypertension,with fewer than expected deaths from renal or cardiac causes.Twenty-eight per cent of deaths were due to causes unrelatedto systemic sclerosis, most commonly cancer and ischaemic heartdisease, and in older patients     

The value of immunohistochemical markers in the diagnosis of cervical neoplasia

Summary. This study was designed to determine whether immunohistochemical stains for tumour-associated markers may be useful in the detection and differential diagnosis of premalignant and malignant lesions of the cervix. The expression of four markers detected by monoclonal antibodies, human milk fat globule 1 and 2 (HMFG-1 and 2), Cal and anti-carcinoembryonic antigen (anti-CEA) on conventional histological sections of various cervical lesions has been investigated. None of these markers was specific for neoplastic lesions of the cervix and all four markers were expressed by metaplastic as well as neoplastic cells, and it was concluded that their application in the histopathological examination of the cervix is limited.     

Lumbar myelography with iohexol and metrizamide: a comparative multicenter prospective study

Diagnostic quality of radiographs and adverse reactions associated with the use of metrizamide and iohexol as contrast agents in lumbar myelography were compared in a prospective randomized double blind study in 350 patients at seven centers. The contrast media were administered in comparable volumes at a concentration of 180 mg I per ml. Overall quality of radiographic visualization was graded good or excellent in 95% of 175 metrizamide studies and in 98% of 175 iohexol studies. Ninety-three patients examined using metrizamide (53%) and 130 patients examined using iohexol (74%) experienced no discomfort during or after myelography. Postmyelographic headache was associated with 38% of metrizamide examinations and 21% of iohexol examinations. Nausea and vomiting were also more common with metrizamide. Five patients examined using metrizamide (3%) experienced transient confusion and disorientation following lumbar myelography. No such reactions were observed following iohexol myelography.     

A prospective trial of unilateral grommets for bilateral secretory otitis media in children

Fifty-four children with established chronic secretory otitis media, who had failed to respond to medical measures were treated with adenoidectomy and insertion of 1 grommet on a side chosen at random. Both sides improved and remained significantly improved at 12 months (P <0.001). At 3 months, the side with the grommet improved significantly more than the other side (P <0.05) but at 12 months there was no significant difference between the 2 sides (P< 0.1).