Nitric oxide inhibits angiotensin II-induced activation of the calcium-sensitive tyrosine kinase proline-rich tyrosine kinase 2 without affecting epidermal growth factor receptor transactivation.

In a previous study, we showed that nitric oxide donors and N-acetylcysteine, either alone or in combination, inhibited the activation of several mitogen-activated protein kinases by angiotensin II in rat cardiac fibroblasts (Wang, D., Yu, X., and Brecher, P. (1998) J. Biol. Chem. 273, 33027-33034). In the present study, we have focused on the mechanism by which nitric oxide exerts this effect on the activation of extracellular signal-regulated kinase (ERK). We contrasted the effects of nitric oxide on ERK activation by angiotensin II and epidermal growth factor (EGF), since the transactivation of the EGF receptor has been implicated as a response to angiotensin II. We found that nitric oxide inhibited ERK activation by angiotensin II but did not inhibit the relatively slight but significant transactivation of the EGF receptor by angiotensin II. The tyrphostin AG1478, known to inhibit EGF receptor phosphorylation, also inhibited the angiotensin II and EGF-induced activation of ERK, the phosphorylation of the EGF receptor, and the subsequent association of Shc and Grb2. Nitric oxide did not affect either EGF receptor phosphorylation or Shc-Grb2 activation induced by either Ang II or EGF. However, the activation of the calcium-sensitive tyrosine kinase PYK2, which occurred in response to angiotensin II, but not EGF, was inhibited by nitric oxide. The data suggested that PYK2 activation may be an important inhibitory site in signaling pathways affected by nitric oxide.     

AMP-activated protein kinase is required for the lipid-lowering effect of metformin in insulin-resistant human HepG2 cells

The antidiabetic drug metformin stimulates AMP-activated protein kinase (AMPK) activity in the liver and in skeletal muscle. To better understand the role of AMPK in the regulation of hepatic lipids, we studied the effect of metformin on AMPK and its downstream effector, acetyl-CoA carboxylase (ACC), as well as on lipid content in cultured human hepatoma HepG2 cells. Metformin increased Thr-172 phosphorylation of the alpha subunit of AMPK in a dose- and time-dependent manner. In parallel, phosphorylation of ACC at Ser-79 was increased, which was consistent with decreasing ACC activity. Intracellular triacylglycerol and cholesterol contents were also decreased. These effects of metformin were mimicked or completely abrogated by adenoviral-mediated expression of a constitutively active AMPKalpha or a kinase-inactive AMPKalpha, respectively. An insulin-resistant state was induced by exposing cells to 30 mm glucose as indicated by decreased phosphorylation of Akt and its downstream effector, glycogen synthase kinase 3alpha/beta. Under these conditions, the phosphorylation of AMPK and ACC was also decreased, and the level of hepatocellular triacylglycerols increased. The inhibition of AMPK and the accumulation of lipids caused by high glucose concentrations were prevented either by metformin or by expressing the constitutively active AMPKalpha. The kinase-inactive AMPKalpha increased lipid content and blocked the ability of metformin to decrease lipid accumulation caused by high glucose concentrations. Taken together, these results indicate that AMPKalpha negatively regulates ACC activity and hepatic lipid content. Inhibition of AMPK may contribute to lipid accumulation induced by high concentrations of glucose associated with insulin resistance. Metformin lowers hepatic lipid content by activating AMPK, thereby mediating beneficial effects in hyperglycemia and insulin resistance.     

Esterification of cholesterol and 25-hydroxycholesterol by rat liver microsomes

The properties of an enzyme in rat liver microsomes was described that catalyzed the formation of 25-hydroxycholesteryl ester in the presence of labeled sterol and oleoyl-CoA. The reaction was similar in several respects to that of cholesteryl ester formation by acyl-CoA: cholesterol acyltransferase. Trypsin pretreatment of microsomes inhibited the esterification of both sterols and a similar dose-dependent inhibition was produced by addition of progesterone and several androgens. Microsomes with an enhanced cholesterol content resulting from in vivo treatment with ethinyl estradiol showed increased esterifying activity towards both cholesterol and 25-hydroxycholesterol. Esterification of endogenous microsomal cholesterol was increased by the addition of 25-hydroxycholesterol, concomitant with 25-hydroxycholesteryl ester formation. To assess the relationship between the association of sterols with membranes and sterol ester formation, microsomes were preincubated with either sterol, reisolated by ultracentrifugation in a density gradient and then analyzed chemically or enzymatically. Cholesterol and 25-hydroxycholesterol both associated with microsomes and the added sterol was subsequently esterified. Maximal esterification was only partially dependent on the amount bound. Progesterone, which inhibited sterol esterification, did not bind to microsomes and no inhibition was observed in reisolated microsomes, indicating that the inhibition produced by progesterone was reversible.     

Regulation of the production of murine IgG2a by an H-2-linked gene and other unlinked genes

Alleles of at least two loci (rig-1 and Rig-2) regulate the levels of serum immunoglobulin of the Igh-1b class and allotype in BALB/c Ig^b (BAB/14) and (BALB/c × BAB/14)F₁ mice. The combined effect of the BALB/c alleles at these two loci is to lower Igh-1b levels significantly below those observed in other strains and below their own levels of Igh-1a in allotype heterozygous mice. The rig-1 locus is closely linked to or within the H-2 complex. Two alleles have been defined: rig-1 ^d and rig-1 ^b in H-2 ^d and H-2 ^b haplotypes, respectively. Homozygous rig-1 ^d d animals heterozygous for the BALB/c Rig-2 allele(s) have very low levels of Igh-1b. The designation of Rig-2 is provisional since it has not been mapped or defined as a single locus.     

Die Puppenfärbungen des Kohlweißlings,Pieris brassicae L. Sechster Teil: Chemismus der Farbanpassung

Ohne Zusammenfassung Mit Tafel II. Ein Auszug dieser Arbeit erschien mit gleichlautendem Titel als Mitteilung Nr. 41 aus der Biologischen Versuchsanstalt der Akademie der Wiss., Zool. Abteil., Vorstand H. Przibram, im Akad. Sitzungsanzeiger Nr. 18, 1919.     

Refolding of a fully functional flavivirus methyltransferase revealed that S‐adenosyl methionine but not S‐adenosyl homocysteine is copurified with flavivirus methyltransferase

Methylation of flavivirus RNA is vital for its stability and translation in the infected host cell. This methylation is mediated by the flavivirus methyltransferase (MTase), which methylates the N7 and 2′‐O positions of the viral RNA cap by using S‐adenosyl‐l ‐methionine (SAM) as a methyl donor. In this report, we demonstrate that SAM, in contrast to the reaction by‐product S‐adenosyl‐l ‐homocysteine, which was assumed previously, is copurified with the Dengue (DNV) and West Nile virus MTases produced in Escherichia coli (E. coli). This endogenous SAM can be removed by denaturation and refolding of the MTase protein. The refolded MTase of DNV serotype 3 (DNV3) displays methylation activity comparable to native enzyme, and its crystal structure at 2.1 Å is almost identical to that of native MTase. We characterized the binding of Sinefungin (SIN), a previously described SAM‐analog inhibitor of MTase function, to the native and refolded DNV3 MTase by isothermal titration calorimetry, and found that SIN binds to refolded MTase with more than 16 times the affinity of SIN binding to the MTase purified natively. Moreover, we show that SAM is also copurified with other flavivirus MTases, indicating that purification by refolding may be a generally applicable tool for studying flavivirus MTase inhibition.