Inhibition of methylnitrosourea (MNU) induced oxidative stress and carcinogenesis by orally administered bee honey and Nigella grains in Sprague Dawely rats.

We studied the protective effect of bee honey and Nigella grains as nutraceuticals on the oxidative stress and carcinogenesis induced by methylnitrosourea (MNU) in Sprague Dawely rats. Four groups of animals were used and fed ad-libitum. The first group was a control (n=8), the second (n=8), the third (n=15) and the fourth groups (n=12) were injected MNU (single i.v. dose 50 mg/kg body weight). After one week the third and fourth groups were given orally 0.2 g ground Nigella grains and 0.2 g Nigella with 5 g honey/rat/day, respectively. After six months all animals were sacrificed except two from the second group (MNU-injected rats) that died one-week before the end of the experiment. We observed that MNU injected in the second group produced a variety of oxidative stresses ranging from severe inflammatory reaction in lung and skin to colon adenocarcinoma in four out of six animals. There was an associated elevation of malondialdehyde (MDA) and nitric oxide (NO) in sera obtained from animals of this group compared to the control one. Nigella sativa grains given orally protected against MNU-induced oxidative stress and carcinogenesis by 80% (12/15) and combated this effect by lowering MDA and NO. Whereas honey from bees and Nigella sativa together protected 100% (12/12) against MNU-induced oxidative stress, carcinogenesis and abolished the NO and MDA elevations shown in sera of animals who did not receive these nutrients. These results showed that supplementation of diet with honey and Nigella sativa has a protective effect against MNU-induced oxidative stress, inflammatory response and carcinogenesis.

Cell signalling and the hormonal stimulation of the hepatic glycine cleavage enzyme system by glucagon.

The glycine cleavage enzyme system (GCS) is found in mitochondria. In liver it is activated by glucagon and other hormones but it is not known how the hormonal signal is transmitted to the mitochondria. We found that the cell-permeant protein phosphatase inhibitor okadaic acid stimulated flux through GCS and could induce a significant increase in the sensitivity of GCS and of glycogenolysis to glucagon. Half-maximal stimulation of GCS by glucagon occurred at 3.2+/-0.6 nM, whereas it was fully activated at 0.3 nM in the presence of 1 microM okadaic acid. The protein kinase A agonist adenosine-3',5'-cyclic monophosphorothioate, Sp isomer (10 microM) stimulated the GCS flux by approx. 100%. This stimulation was inhibited by the protein kinase A antagonist 8-bromoadenosine-3', 5'-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-cAMPS). Although Rp-8-Br-cAMPS significantly inhibited glucagon-stimulated glycogenolysis it had no effect on the glucagon-stimulated GCS flux. These results indicate that a cytoplasmic phosphorylated protein is involved in transmitting glucagon's effect to the mitochondria. However, protein kinase A does not have a necessary role in transmitting glucagon's signal. We also examined the role of protein kinase C because angiotensin II also stimulated flux through GCS. However, the phorbol ester PMA had no effect on either GCS or on glycogenolysis.

Prevalence of hepatitis C infection and schistosomiasis in Egyptian patients with hepatocellular carcinoma.

Hepatitis C virus (HCV) infection prevalence was determined in 34 hepatocellular carcinoma (HCC) patients with underlying liver cirrhosis. Serum alfa-fetoprotein level was found to be more than 1000 ng/ml (3153 +/- 1451) in those patients. Anti-HCV antibodies were detected in 84% of patients sera negative for hepatitis B surface antigen (HBsAg), while HCV RNA was only detected in 28% of the sera of those patients. Schistosomiasis antibodies were found in 92% of the anti-HCV-positive HBsAg negative sera of HCC patients, while they were only detected in 61% of sera of control non HCC patients with anti-HCV antibodies and without HBsAg. HCV and hepatitis B virus (HBV) coinfection was found in 16% of the studied group, based on antibodies in sera, and in 9% based on the presence of HCV RNA. HBV (HBsAg) was found, on its own, in sera of 5.9% of the patients studied. These results show that HCV and schistosomiasis play a major role in the development of hepatocellular carcinoma in Egypt.

Activation of the hepatic glycine cleavage enzyme system by glucagon and glucagon-related peptides.

The hepatic glycine cleavage system (GCS) is the principal route for the catabolism of glycine in mammals. Flux through the glycine cleavage system in isolated rat hepatocytes is stimulated about 100% at 100 nM glucagon, with half-maximal stimulation at 4.3 +/- 1.3 nM. Preincubation of the hepatocytes with the glucagon antagonist des-His1-[Glu9]glucagon amide at 10 microM resulted in inhibition of the glucagon-stimulated GCS by approximately 40%, while the antagonist des-His1-[Nle9,Ala11,Ala16]glucagon amide at 10 microM inhibited the glucagon-stimulated GCS by 80%. Oxyntomodulin and glicentin, glucagon-related peptides, were found to stimulate GCS. Oxyntomodulin, 1 microM, and glicentin, 100 nM, stimulated GCS by 100 and 60%, respectively. Glucagon-like peptide 1 (7-36) amide and glucagon (19-29) (10(-10)-10(-7) M) were without effect. Des-His1-[Glu9]glucagon amide at 10 microM inhibited the oxyntomodulin and glicentin-stimulated flux through GCS by about 40%. Thus oxyntomodulin and glicentin can activate the GCS in hepatocytes via interaction with the glucagon receptor but with low affinity.

The cysteine-rich region of raf-1 kinase contains zinc, translocates to liposomes, and is adjacent to a segment that binds GTP-ras.

Different domains of the serine/threonine kinase, raf-1, were expressed as fusion proteins with glutathione S-transferase (GST) in Escherichia coli and purified to near homogeneity by affinity chromatography. A cysteine-rich domain of raf-1 was found to contain 2 mol of zinc (molar basis), similar to analogous cysteine-rich domains of protein kinase C. GST-fusion proteins, containing the cysteine-rich domain of raf-1, bound to liposomes in a phosphatidylserine-dependent manner. In contrast to protein kinase C, the translocation of raf-1 was not dependent upon diacylglycerol, phorbol ester, or calcium, nor did raf-1 bind phorbol esters. A GST-fusion protein encoding residues 1-147 of raf-1 bound to normal GTP-ras with high affinity, but not to mutant GTP-Ala35 ras; no binding was detected to GDP-ras. The binding of a smaller fusion protein (residues 1-130 of raf-1) was about 10-fold weaker, inferring that a 17-amino acid sequence represents a critical binding determinant in intact raf-1. These residues are adjacent to the amino-terminal end of, and partially extend into, the cysteine-rich domain (amino acids 139-184). A synthetic peptide corresponding to this 17-amino acid sequence blocked the interaction of raf-1 with ras. The function of the cysteine-rich region of raf-1 homologous to protein kinase C is to promote translocation of raf-1 kinase to membranes and to form part of the high affinity binding site for GTP-ras.

Acute hormonal control of acetyl-CoA carboxylase. The roles of insulin, glucagon, and epinephrine.

Acetyl-CoA carboxylase, purified from rapidly freeze-clamped livers of rats maintained on a normal laboratory diet and given 0-5 units of insulin shortly before death, gives a major protein band (Mr 265,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase from untreated rats has relatively low activity (0.8 unit/mg protein when assayed in the absence of citrate) and high phosphate content (8.5 mol of Pi/mol of subunit), while the enzyme from livers of rats that received 5 units of insulin has higher activity (2.0 units/mg protein) and lower phosphate content (7.0 mol of Pi/mol of subunit). Addition of citrate activates both preparations with half-maximal activation (K0.5) at 1.0 and 0.6 mM citrate, respectively. The enzyme from rats that did not receive insulin is mainly in the octameric state (Mr approximately 2 x 10(6)), while that from rats that received insulin is mainly in the polymeric state (Mr approximately 10 x 10(6)). Thus, short-term administration of insulin results in activation of acetyl-CoA carboxylase, lowering of its citrate requirement, and dephosphorylation and polymerization of the protein. The insulin-induced changes in the carboxylase are probably due to dephosphorylation of the protein since similar changes are observed when the enzyme from rats that did not receive insulin is dephosphorylated by the Mn2(+)-dependent [acetyl-CoA carboxylase]-phosphatase 2. The effect of glucagon or epinephrine administration on acetyl-CoA carboxylase was also investigated. The carboxylase from fasted/refed rats has a relatively high specific activity (3.4 units/mg protein in the absence of citrate), lower phosphate content (4.9 mol of Pi/mol of subunit), and is present mainly in the polymeric state (Mr approximately 10 x 10(6)). Addition of citrate activates the enzyme with K0.5 = 0.2 mM citrate. Glucagon or epinephrine injection of fasted/refed rats yielded carboxylase with lower specific activity (1.4 or 1.9 units/mg, respectively, in the absence of citrate), higher phosphate content (6.4 or 6.7 mol of Pi/mol of subunit, respectively), and mainly in the octameric state (Mr approximately 2 x 10(6)). Treatment of these preparations with [acetyl-CoA carboxylase]-phosphatase 2 reactivated the enzyme (specific activity approximately 8 units/mg protein in the absence of citrate) and polymerized the protein (Mr approximately 10 x 10(6]. These observations indicate that insulin and glucagon, by altering the phosphorylation state of the acetyl-CoA carboxylase, play antagonistic roles in the acetyl-control of its activity and therefore in the regulation of fatty acid synthesis.