Search

Angiotensinogen fine-tunes the tightly controlled activity of the renin-angiotensin system by modulating the release of angiotensin peptides that control blood pressure. One mechanism by which this modulation is achieved is via angiotensinogen's Cys18–Cys138 disulfide bond that acts as a redox switch. Molecular dynamics simulations of each redox state of angiotensinogen reveal subtle dynamic differences between the reduced and oxidised forms, particularly at the N-terminus. Surface plasmon resonance data demonstrate that the two redox forms of angiotensinogen display different binding kinetics to an immobilised anti-angiotensinogen monoclonal antibody. Mass spectrometry mapped the epitope for the antibody to the N-terminal region of angiotensinogen. We therefore provide evidence that the different redox forms of angiotensinogen can be detected by an antibody-based detection method.

Background Lipid hydroperoxides decompose to reactive aldehydes, such as acrolein. Measurement of oxidative stress markers in the clinic could improve risk stratification for patients. Methods To aid the development of diagnostic oxidative stress markers, we defined the acrolein modifications of haemoglobin using mass spectrometry. Results Acrolein modifications have little effect on the secondary structure of haemoglobin. They do not disrupt the quaternary structure, but instead promote crosslinked octamers. For acrolein modified haemoglobin the response to O2 binding is altered such that cooperativity is lost. Mass spectrometry experiments at a 1:1 acrolein:haemoglobin molar ratio demonstrate that the α-chain quickly forms an aza-Michael adduct (+56 Da), which then forms a more stable adduct, Ne-(3-methylpyridinium)lysine (MP-lysine, +76 Da) over 7 days. The β-chain remains relatively unchanged over the duration of the 7 days and the aza-Michael adduct is dominant. At 2:1 and 5:1 molar ratios the α-chain was consistently modified at K7, H20, H50, and the β-chain at C93 and H97 with the aza-Michael adduct. Beyond 5 h, an MP-adduct (+76 Da) was located predominantly at K7 of the α-chain, while an FDP-adduct (+94 Da) was observed at K95 of the β-chain. Conclusions We have generated qualitative evidence identifying the acrolein target sites on haemoglobin, a potential oxidative stress marker that is easily measured in circulation. General significance We provide data for the community to develop targeted mass spectrometric or immunometric assays for acrolein modified haemoglobin to further validate the potential of haemoglobin as an oxidative stress marker in patients .

Female non-insulin-dependent diabetics have a high prevalence of gallstones. Treatment of hyperlipidaemia in these patients may modify the risk. Seventeen female non-insulin-dependent diabetics (age 35-65) were treated with simvastatin (n = 10) or bezafibrate (n = 7) and had the cholesterol saturation index (CSI) of bile and gall-bladder emptying measured before and after 3 months therapy. In both groups, there was a significant reduction in serum cholesterol following treatment. The mean pretreatment cholesterol saturation indices of bile did not differ between the two groups but, after 3 months therapy, there was a highly significant difference in CSI between the bezafibrate group (2.0 +/- 0.33) and the simvastatin group (1.1 +/- 0.14) P < 0.002. Whereas the increase in the CSI (42%) observed with bezafibrate therapy was significant, the decrease in the simvastatin group (14%) was only significant in those whose pretreatment cholesterol saturation indices were elevated. Despite the differences in CSI observed between the two treatment groups, no changes in gall-bladder emptying were detected.

Human bile contains a phospholipase C activity. To examine its pathophysiological importance, the effect of phospholipase C on the dynamics of lipid solubilization and nucleation (cholesterol crystal formation) were investigated in model bile. Phospholipase C from gallbladder bile from patients with gallstones was partially purified by competitively eluting from a concanavalin A (con A)-Sepharose (Sigma, St. Louis, MO) column and incubating with Pronase (Calbiochem, Behring Diagnostics, La Jolla, CA). Phospholipase C activity was resistant to Pronase digestion. When this fraction (concentrated to half the original volume) was mixed with model bile (1:1, vol/vol), a transfer of cholesterol and phospholipid from the micellar to the vesicular phase and an accelerate nucleation time were found concomitant with phospholipid hydrolysis. These effects were prevented by inhibiting the phospholipase C activity with ethylenediaminetetraacetic acid. To confirm that the results were caused by phospholipase C activity and not some other nucleation-promoting factor within the biliary con A preparation, model bile was incubated with bacterial phospholipase C. An identical cascade of events to that found with the partially purified biliary enzyme was observed. Further purification of the con A-bound proteins on DEAE-Sephadex (Pharmacia, Uppsala, Sweden) did not resolve any separate nucleation-promoting activity to that associated with phospholipase C activity. In conclusion, this study has identified phospholipase C as a/the con A nucleation-promoting activity in human gallbladder bile and has characterized a possible molecular mechanism by which cholesterol nucleation is stimulated by this fraction.

A phospholipase C in bile, free of bacterial infection, has recently been identified from cholesterol gallstone patients. Because of the importance of phosphatidylcholine in solubilizing cholesterol in bile, this study further investigates the metabolism of phosphatidylcholine in delipidated gallbladder and common bile duct biles. Phospholipase C activity, as measured by the release of phosphoryl[3H]choline from the substrate 1,2-dipalmitoyl-sn-glycero-3-phospho [N-methyl-3H]choline, was identified in both hepatic and gallbladder biles. Similar levels of activity (nmol.h-1.mg-1 of delipidated protein) were found in common bile duct (11.25 +/- 14.23) and gallbladder bile (19.07 +/- 22.24), although per milliliter of bile, the mean gallbaldder levels were 6.4 times greater than those found in common duct bile. With the tow substrates, 1-palmitoyl-2[9,10-3H] palmitoyl-sn-glycero-3-phosphocholine and 1,2(1-14C) dipalmitoyl-sn-glycero-3-phosphocholine, the majority of organically extracted label, after thin-layer chromatography, was recovered as radiolabeled diglyceride, confirming the presence of phospholipase C. Diglyceride levels were found to be closely correlated with [3H]choline (slope, 0.9820; r = 0.9844). In addition to diglyceride, both radiolabeled free fatty acid and monoglyceride were identified in common bile duct and gallbladder biles, although their levels were an order of magnitude less than measurable phospholipase C activity. To determine whether the free fatty acid release was due to either a diacylglycerol-lipase or a phospholipase A2, the effect of adding unlabeled diglyceride on free fatty acid formation from the substrate [14C]DPPC was examined. As the concentration of unlabeled diglyceride was increased, the amount of free fatty acid and monoglyceride released were both reduced in parallel. Direct measurement of diacylglycerol-lipase activity by incubating the diglyceride, sn-2[3H]dipalmitoyl, resulted in release of both products in a ratio similar to that found with sn-2[3H]DPPC. Finally, no radiolabeled lysolecithin was identified with [3H]choline-DPPC or [14C]DPPC as substrate indicating the free fatty acid was the product of a diacylglycerol-lipase rather than a phospholipase A2. Phospholipase C and diacyl-glycerol-lipase activities were significantly correlated (P less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

Apolipoproteins AI, AII and B were identified in the normal and pathological human bile duct and the gallbladder epithelium using an avidin-biotin immunoperoxidase technique. Small intestine and stomach sections served as positive and negative controls respectively. Staining was focal for apolipoproteins AI and AII, and continuous for apolipoprotein B. In addition to homogenous and granular cytoplasmic staining, foamy cytoplasmic staining, particularly for apolipoproteins AI and AII, was observed around lipid droplets in cells containing much lipid. No correlation between a particular pathological condition of the gallbladder (acute cholecystitis, mucocele, chronic cholecystitis, cholesterolosis) and staining pattern or intensity of staining was found for any of the apolipoproteins, although both apolipoproteins AI and AII stained more intensely than apolipoprotein B in each group. Positive staining was also found for all apolipoproteins in epithelial cells which had invaded the underlying connective tissue (gallbladder carcinoma), suggesting that the epithelial cells are capable of synthesizing apolipoproteins de novo. In this latter case, apolipoprotein B stained more intensely than for either AI or AII, and significantly (p less than 0.05) more strongly than that found in the other pathological groups. The identification of apolipoproteins in the gallbladder epithelium raises the interesting question of their origin and functional role.

Suspensions of isolated rat hepatocytes and adipocytes converted exogenous ATP to cyclic AMP at a rate which was about 30–50% of that observed with homogenates of isolated cells. Formation of cyclic AMP was stimulated by hormones (isoprenaline in the case of adipose tissue and glucagon in the case of liver) and sodium fluoride. Experiments with [α- 32 P]ATP indicated that the conversion of exogenous ATP to cyclic AMP did not occur within the cells. It is proposed that in isolated hepatocytes and adipocytes some catalytic units of adenylate cyclase are exposed on the outer surface of the cell membrane.

Fifty patients were given cefoperazone (1 g) intravenously over 2–3 min immediately prior to endoscopic retrograde cholangiopancreatography (ERCP). In 16 of these subjects an aliquot of common duct bile was obtained via the ERCP cannula at the commencement of the procedure. Therapeutic biliary levels of cefoperazone were found in 10 subjects. Cholestasis and sampling distal to calculi may account for those in whom undetectable levels were found. Multiple samples from one patient showed that the biliary concentration increased with time. Therapeutic serum levels were detected in all patients and no side effects were observed.

This study investigates the effect of ethanol on enzyme synthesis and secretion in rat pancreatic lobules. Ethanol caused a dose-dependent inhibition of 3H-leucine incorporation into total protein. Examination of the time dependence showed that ethanol inhibited protein synthesis at each time point. Removal of ethanol partially reversed this inhibition. An autoradiograph of the newly synthesized proteins separated on SDS-PAGE showed that ethanol inhibited synthesis of all proteins. 14C-cycloleucine uptake was not altered by ethanol, excluding inhibition of amino acid uptake as the mechanism for the decreased protein synthesis induced by ethanol. Electron microscopy revealed no ultrastructural damage. Ethanol had no effect on the stimulated release of (i) amylase from zymogen granules nor (ii) newly synthesized pulse labelled enzymes. Acetaldehyde had no inhibitory effect on enzyme synthesis or secretion indicating that ethanol per se and not its metabolite is inhibitory. The decreased synthesis after acute exposure to ethanol with preservation of exocytosis would limit the autodigestive potential of pancreatic tissue. This may explain why isolated toxic doses of ethanol are rarely if ever associated with pancreatitis.

To further investigate the nonmicelle mode of cholesterol transport in human bile, we examined its levels in relation to fasting and feeding. T-tube bile samples were collected (for 30 min) every 4 h over a 24-h period. All patients (3 with cholesterol gallstones; 1 with pigment stone) had their T tube clamped for a minimum of 4 days before the study to allow the bile acid pool to replete. Biliary lipid concentrations in all patients increased with feeding and decreased with fasting. However, because of a greater decrease in bile acid concentration relative to cholesterol concentration during the fasting period, fasting bile was consistently more saturated than bile obtained while feeding. Associated with the increase in lithogenic index with fasting was a decrease in the micelle solubilized cholesterol and an increase in nonmicelle solubilized cholesterol. At low bile acid concentrations (fasting) most biliary cholesterol is therefore transported as a nonmicelle complex, whereas at high bile acid concentrations (feeding) most of the cholesterol is transported in the mixed micelle. No nucleation of the biliary samples was observed in any of the patients over a 10-day period of observation. Thus the biliary nonmicelle complex (presumably vesicles) has a major cholesterol transport function especially at low bile acid concentrations.

Purification of glutathione S -transferases A and C from rat liver cytosol using an affinity matrix coupled with cholic acid is described. The method provides a convenient means for the rapid and homogeneous preparation of both transferases.

The phospholipid effect involves agonist-induced breakdown of phosphatidyl inositol (or polyinositides) generating second messengers followed by increased incorporation of P 32 during the resynthetic phase of the cycle. Ethanol, an aetiological factor in pancreatitis, has been shown to have various effects on pancreatic secretion. In this study ethanol decreased the incorporation of P 32 into phosphatidyl inositol but had no effect on the stimulated breakdown of prelabelled phosphatidyl inositol. However, in addition to recycling of phosphatidyl inositol stimulation of pancreatic tissue results in increased incorporation of precursors into other phospholipids. Cholecystokinin increased the incorporation of both [U- 14 C] glucose and P 32 into phosphatidyl ethanolamine 3-fold but had no effect on P 32 incorporation into phosphatidyl choline. As well as increased incorporation of P 32 into phosphatidyl inositol (8-fold) Cholecystokinin also increased the incorporation of [U- 14 C] glucose into phosphatidyl inositol (4–5-fold) implying significant de novo synthesis of 1,2 diacyl glycerol in addition to the currently accepted recycling of the 1,2 diacyl glycerol back to phosphatidyl inositol. Ethanol caused an inhibition of P 32 incorporation into total phospholipid of rat pancreas during basal and stimulated conditions. When individual phospholipids were separated ethanol was found to decrease the incorporation of P 32 into phosphatidyl choline under basal conditions and into all phospholipids during Cholecystokinin stimulation. With [U- 14 C] glucose as the precursor, ethanol inhibited its incorporation into phosphatidyl choline only. Ethanol did not alter the total P 32 radioactivity in the aqueous phase of the pancreatic extract nor the percent incorporated into nucleotides. This excluded decreased uptake of 32 P and incorporation into nucleotides as a mechanism for the differential inhibition of P 32 versus [U- 14 C] glucose incorporation into phospholipids other than phosphatidyl choline under stimulated conditions.