Effects of phorbol esters on insulin receptor function and insulin action in hepatocytes: evidence for heterogeneity

We investigated the effect of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator on insulin receptors and insulin action in freshly isolated and primary cultures of rat hepatocytes. PMA (1 x 10^–7 M) did not alter insulin receptor numbers or affinity either acutely or chronically but within 60 minute inactivated insulin stimulated tyrosine kinase of the insulin receptor. PKC activation inhibitied insulin (1 x 10^–7M) stimulation of glycogen and lipid synthesis with a decrease or no change in basal glycogenesis and lipogenesis respectively. However, PKC activation did not alter insulin stimulated or basal amino acid transport even though PCK activation inhibited insulin stimulation of the insulin. receptor tyrosine kinase. Thus, within one tissue, PKC activation has differential effect on insulin action depending on which pathway is examined. Furthermore, insulin stimulation of the insulin receptor tyrosine kinase may not be a necessary step for all insulin signaling pathways.     

Antioxidant depletion in aortic crossclamping ischemia: increase of the plasma alpha-tocopheryl quinone/alpha-tocopherol ratio.

To assess whether oxidative stress contributes to the ischemia/reperfusion injury of aortic surgery, the contents of alpha-tocopherol, alpha-tocopheryl quinone, ascorbate, lipid-derived malondialdehyde, protein thiols, cholesterol, and lactate were analyzed in plasma samples from 24 patients subjected to aortic crossclamping. alpha-Tocopherol, ascorbate, and protein thiols decreased during ischemia, whereas alpha-tocopheryl quinone increased in all but two cases, doubling on average in proportion to alpha-tocopherol. Upon reperfusion alpha-tocopherol, ascorbate, and protein thiols remained low, whereas alpha-tocopheryl quinone returned to the preischemic level. Lipid-derived malondialdehyde (a measure of lipid hydroperoxides) increased significantly only during reperfusion. The results suggest that oxidative stress occurs simultaneously with ischemia/reperfusion during aortic surgery, and that measurement of the tocopheryl quinone/tocopherol ratio may shed new light on the underlying pathological events.     

Comparative studies of Babesia spp. from white-tailed and sika deer

Babesia odocoilei from white-tailed deer (Odocoileus virginianus) in Texas (USA) and B. capreoli isolated from sika deer (Cervus nippon) in Ireland were compared morphologically and antigenically. Babesia odocoilei and B. capreoli paired pyriforms resembled each other closely when in sika deer, but B. odocoilei pyriforms in white-tailed deer were slightly different. Babesia odocoilei in white-tailed deer also differed from B. odocoilei and B. capreoli in sika deer in the frequency of its developmental forms. Indirect immunofluorescence antibody test titres showed that there was some antigen cross-reactivity, but not as much as between B. capreoli and the bovine parasite, B. divergens. The Babesia spp. from deer that we studied appear to be distinct but related species. The low infectivity of B. odocoilei for a splenectomised sika deer suggests that sika deer in North America are probably not very susceptible to this parasite in the wild.     

A gibberellin-regulated gene from wheat with sequence homology to cathepsin B of mammalian cells

A previous report described several cDNAs corresponding to mRNAs which accumulated in wheat aleurone layers treated with gibberellic acid (GA) (Baulcombe and Buffard, 1983). The protein sequence deduced from one of these clones (2529) has extensive similarity to the thiol protease, cathepsin B from mammalian cells. Southern analysis of wheat DNA has shown that the 2529 mRNA is encoded by a small family of genes carried on the group 4 chromosome. The nucleotide sequence of a member of the gene family expressed at a low level in aleurone layers and the use of a primer extension assay to identify a clone of a member of the gene family producing an abundant mRNA are reported. The 2529 mRNA accumulates in the scutellum and the aleurone layer of germinating grains where its expression is regulated by GA. In the scutellum the expression was restricted to the parenchyma, suggesting that the 2529 product may have a role other than for mobilization of the endosperm.     

p lambda Zd39: a new type of cDNA expression vector for low background, high efficiency directional cloning.

We have developed a new type of bacteriophage lambda vector which provides a strong biological selection against non-recombinants that is independent of the sequences immediately surrounding the cloning site. This system, which we call 'selective substitution', is ideally suited for cDNA expression vectors where it is necessary to flank the cDNA insert with sequence elements (promoters etc.) required to produce a biologically active mRNA in vivo. Selective substitution is a general method, which may be applied to many types of vectors. In this report, we have specifically applied selective substitution to the construction of a new mammalian retrovirus expression vector. The level of background obtained with this vector (that is, the number of plaques obtained when the vector is ligated in the absence of insert DNA) is 0.02% when compared to ligation with restriction fragments and 0.1% to 0.4% when compared to ligation with newly synthesized cDNA. These features have allowed us to easily and efficiently generate several large cDNA libraries using total and size selected cDNA.