p85alpha gene generates three isoforms of regulatory subunit for phosphatidylinositol 3-kinase (PI 3-Kinase), p50alpha, p55alpha, and p85alpha, with different PI 3-kinase activity elevating responses to insulin

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by association with a variety of tyrosine kinase receptors and intracellular tyrosine-phosphorylated substrates. We isolated a cDNA that encodes a 50-kDa regulatory subunit of PI 3-kinase with an expression cloning method using 32P-labeled insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two SH2 domains and an inter-SH2 domain of p85alpha, but the SH3 and bcr homology domains of p85alpha were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by alternative splicing of the p85alpha gene product. We suggest that this protein be called p50alpha. Northern blotting using a specific DNA probe corresponding to p50alpha revealed 6.0- and 2.8-kb bands in hepatic, brain, and renal tissues. The expression of p50alpha protein and its associated PI 3-kinase were detected in lysates prepared from the liver, brain, and muscle using a specific antibody against p50alpha. Taken together, these observations indicate that the p85alpha gene actually generates three protein products of 85, 55, and 50 kDa. The distributions of the three proteins (p85alpha, p55alpha, and p50alpha), in various rat tissues and also in various brain compartments, were found to be different. Interestingly, p50alpha forms a heterodimer with p110 that can as well as cannot be labeled with wortmannin, whereas p85alpha and p55alpha associate only with p110 that can be wortmannin-labeled. Furthermore, p50alpha exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosine-phosphorylated IRS-1 than the other isoforms. Considering the high level of p50alpha expression in the liver and its marked responsiveness to insulin, p50alpha appears to play an important role in the activation of hepatic PI 3-kinase. Each of the three alpha isoforms has a different function and may have specific roles in various tissues.     

Kinetic analysis of successive reactions catalyzed by bovine cytochrome p450(17alpha,lyase)

Bovine P450(17alpha,lyase) containing an additional four histidine residues at the COOH terminus was expressed in Escherichia coli and purified by one-step column chromatography using Ni-chelate resin. The membrane enzyme was incorporated into liposome membranes having similar lipid composition to that of the endoplasmic reticulum. In the presence of excess substrate, the P450-proteoliposomes metabolize pregnenolone (Delta5-steroid) to 17alpha-hydroxypregnenolone and further to dehydroepiandrosterone. The enzyme catalyzed 17alpha-hydroxylation of progesterone (Delta4-steroid) but did not form androstenedione from progesterone, although the proteoliposomes could catalyze the conversion of 17alpha-hydroxyprogesterone to androstenedione. The kinetic analysis of rapid quenching experiments showed that about 20% of the pregnenolone consumed was converted successively to dehydroepiandrosterone via a fraction of 17alpha-hydroxypregnenolone that did not dissociate from the enzyme. The rapid quenching experiments for progesterone metabolism by the proteoliposomes revealed that the dissociation rate of 17alpha-hydroxyprogesterone was 10 times faster than that of 17alpha-hydroxypregnenolone. The release of the intermediate metabolite of Delta4-steroid is sufficiently faster than the lyase reaction to prevent further reaction by the P450. It is concluded that the dissociation rates of the first hydroxylation metabolites regulate the successive reactions of P450(17alpha,lyase).     

Activation of p21-activated protein kinase alpha (alpha PAK) by hyperosmotic shock in neonatal ventricular myocytes

Two different groups of workers involved, during the last 10 years, in the production or formulation of chlorotriazine herbicides have been retrospectively studied in order to verify the occurrence of diseases possibly related to Atrazine, Simazine, Propazine and Terbuthylazine exposure. A control group has been selected between clerical workers of the same plants. Exposure data have been collected during the last five years and some extrapolations have been performed to hypothesize the level of exposure in the preceeding years. Clinical findings and laboratory data obtained from ten year medical surveillance records have been evaluated to highlight possible differences between the investigated groups. A first close examination of the data concerning the anamnesis and clinical, hematochemical and functional reports of the three groups of investigated subjects does not show any pathological situation or symptoms specifically connected to chlorotriazine compounds exposure and/or significant differences between exposed and non exposed. A next series of statistical surveys involving some of the hematochemical indicators (RBC, WBC, Hb%, SGOT, SGPT, gamma GT) has been performed either on internal values of each group or on the average-values of the three groups differently exposed (production, formulation, control). The first survey was a longitudinal study, the second one was a cross sectional one. Both the investigations have excluded significant differences attributable to the occupational exposure.     

A critical role of the p75 tumor necrosis factor receptor (p75TNF-R) in organ inflammation independent of TNF, lymphotoxin alpha, or the p55TNF-R

Despite overwhelming evidence that enhanced production of the p75 tumor necrosis factor receptor (p75TNF-R) accompanies development of specific human inflammatory pathologies such as multi-organ failure during sepsis, inflammatory liver disease, pancreatitis, respiratory distress syndrome, or AIDS, the function of this receptor remains poorly defined in vivo. We show here that at levels relevant to human disease, production of the human p75TNF-R in transgenic mice results in a severe inflammatory syndrome involving mainly the pancreas, liver, kidney, and lung, and characterized by constitutively increased NF-kappaB activity in the peripheral blood mononuclear cell compartment. This process is shown to evolve independently of the presence of TNF, lymphotoxin alpha, or the p55TNF-R, although coexpression of a human TNF transgene accelerated pathology. These results establish an independent role for enhanced p75TNF-R production in the pathogenesis of inflammatory disease and implicate the direct involvement of this receptor in a wide range of human inflammatory pathologies.