Ischaemic myelopathy following aortic surgery or traumatic laceration of the aorta

Microcirculatory derangement, energy depletion and lipid peroxidation have been related to development of ischemia-reperfusion injury in the liver. This study investigates the effects of hyperbaric oxygen (HBO) on hepatic ischemia-reperfusion injury. Adult, male Sprague-Dawley rats were used. Three groups were evaluated: 1) sham-operated control (laparotomy only, no ischemia, no HBO), n=8; 2) ischemia control (1-h ischemia, 2-h reperfusion, no HBO), n=8; and 3) HBO pretreatment (100%, oxygen, 2.5 atm absolute, 90 min) plus ischemia (1-h ischemia, 2-h reperfusion), n=8. An in vivo microscope was used to investigate hepatic microcirculation. Tissue malondialdehyde (MDA) and adenosine triphosphate (ATP) were determined. In comparison with the ischemia control group, HBO significantly improved harmful insults following ischemia-reperfusion. HBO lessened adherent leukocyte count (6.00+/-1.31 cells/200 microm vs 11.38+/-2.88 cells/200 microm), and improved flow velocity (1.72+/-0.26 mm/s vs 0.83+/-0.19 mm/s) in post-sinusoidal venules. HBO also reduced MDA (1.04+/-0.24 nmol/mg protein vs 2.24+/-0.49 micromol/g protein), and increased ATP (2.03+/-0.17 micromol/g wet wt vs 0.73+/-0.11 micromol/g wet wt) levels. This study demonstrates that HBO before ischemia may ameliorate the ischemia-reperfusion injury of the liver in the rat model.     

Vasopressin reduces taurochenodeoxycholate-induced hepatotoxicity by lowering the hepatocyte taurochenodeoxycholate content

BACKGROUND/AIMS: Vasopressin has been reported to reduce bile flow, but its effects on bile acid secretion and bile acid-related hepatotoxicity are still unclear. We therefore investigated the influence of vasopressin on the hepatotoxicity and biliary excretion of taurochenodeoxycholic acid in primary cultured rat hepatocytes and isolated perfused rat liver models. METHODS/RESULTS: 1) Addition of vasopressin to hepatocyte cultures significantly decreased lactate dehydrogenase release as compared to cultures exposed to 1 mM taurochenodeoxycholic acid alone, and also reduced intracellular taurochenodeoxycholic acid content from 19.3 +/- 2.2 to 13.0 +/- 1.6 nmol/mg protein. After 30 min of preincubation with 1 mM taurochenodeoxycholic acid, rinsing and reculture of hepatocytes in bile acid-free medium resulted in gradual decrease in the intracellular level of the bile acid, and addition of vasopressin (10(-9) M) to the reculture medium accelerated this process. 2) Superimposition of vasopressin (330 pmol/l) for 10 min on taurochenodeoxycholic acid infusion (1.0 mumol/min: 25 mumol/l) caused a rapid increase in bile flow and biliary excretion of taurochenodeoxycholic acid (697 +/- 42 vs 584 +/- 27 nmol/10 min per g liver) from perfused rat livers, and significantly reduced lactate dehydrogenase release. 3) Superimposition of the PKC blocker H-7 (5 mumol/l) on taurochenodeoxycholic acid infusion (1.0 mumol/min: 25 mumol/l) caused a gradual increase in bile flow and biliary excretion of taurochenodeoxycholic acid. Furthermore, an additional infusion of vasopressin (100 pmol/l) for 10 min in the presence of H-7 produced a greater increase in bile flow and biliary excretion of taurochenodeoxycholic acid as compared with H-7 alone (754 +/- 71 vs. 657 +/- 26 nmol/g liver). 4) Continuous infusion of vasopressin (330 pmol/l) significantly increased the late peak (10-50 min) of horseradish peroxidase excretion from perfused livers (from 8.48 +/- 1.02 to 21.7 +/- 6.02 ng/g liver). CONCLUSIONS: These findings suggest that vasopressin exerts a protective effect against taurochenodeoxycholic acid-induced hepatotoxicity by stimulating the secretion of this bile acid via intracellular vesicular transport systems.     

Hydrolysis of hexose pentaacetate esters in rat pancreatic islets

The pentaacetate esters of selected hexoses were recently found to stimulate insulin release. The kinetics of their hydrolysis was now investigated in both rat pancreatic islet homogenates and intact islets. In islet homogenates, the hydrolysis of alpha-d-glucose pentaacetate, as judged from the measurement of acetate production, displayed a pH optimum of 7.4 and a Km for the ester of 0.95 mM. At pH 7.4, the reaction velocity was about 5 times higher than the rate of alpha-d-glucose pentaacetate hydrolysis by intact islets, as judged from the ester-induced increase in the acetate content of both the islet and surrounding incubation medium. Comparable results were obtained in intact islets exposed to either beta-l-glucose pentaacetate or beta-d-galactose pentaacetate. The ester content of the islets after 120 min incubation was close to 0.1 nmol/islet, yielding an apparent intracellular concentration at least one order of magnitude higher than the extracellular concentration (1.7 mM). These findings indicate that hexose esters that either stimulate insulin release or fail to do so are equally well taken up and hydrolyzed by islet cells. They are compatible, therefore, with the view that the insulinotropic action of some of these esters may be favored by the catabolism of their hexose moiety, although some other mechanisms for stimulation of insulin release must be operative in the case of beta-l-glucose pentaacetate.     

Effect of ventilation on vascular permeability and cyclic nucleotide concentrations in ischemic sheep lungs

Ventilation during ischemia attenuates ischemia-reperfusion lung injury, but the mechanism is unknown. Increasing tissue cyclic nucleotide levels has been shown to attenuate lung ischemia-reperfusion injury. We hypothesized that ventilation prevented increased pulmonary vascular permeability during ischemia by increasing lung cyclic nucleotide concentrations. To test this hypothesis, we measured vascular permeability and cGMP and cAMP concentrations in ischemic (75 min) sheep lungs that were ventilated (12 ml/kg tidal volume) or statically inflated with the same positive end-expiratory pressure (5 Torr). The reflection coefficient for albumin (sigmaalb) was 0.54 +/- 0.07 and 0.74 +/- 0. 02 (SE) in nonventilated and ventilated lungs, respectively (n = 5, P < 0.05). Filtration coefficients and capillary blood gas tensions were not different. The effect of ventilation was not mediated by cyclic compression of alveolar capillaries, because negative-pressure ventilation (n = 4) also was protective (sigmaalb = 0.78 +/- 0.09). The final cGMP concentration was less in nonventilated than in ventilated lungs (0.02 +/- 0.02 and 0.49 +/- 0. 18 nmol/g blood-free dry wt, respectively, n = 5, P < 0.05). cAMP concentrations were not different between groups or over time. Sodium nitroprusside increased cGMP (1.97 +/- 0.35 nmol/g blood-free dry wt) and sigmaalb (0.81 +/- 0.09) in nonventilated lungs (n = 5, P < 0.05). Isoproterenol increased cAMP in nonventilated lungs (n = 4, P < 0.05) but had no effect on sigmaalb. The nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester had no effect on lung cGMP (n = 9) or sigmaalb (n = 16) in ventilated lungs but did increase pulmonary vascular resistance threefold (P < 0.05) in perfused sheep lungs (n = 3). These results suggest that ventilation during ischemia prevented an increase in pulmonary vascular protein permeability, possibly through maintenance of lung cGMP by a nitric oxide-independent mechanism.     

Attenuated glycogenolysis reduces glycolytic catabolite accumulation during ischemia in preconditioned rat hearts

The aim of this work was to investigate the effect of tissue culture on the intracellular amino acid pool in both freshly isolated and surgically prepared saphenous vein segments taken from patients undergoing coronary artery bypass surgery (number of patients, n = 8). Viability of freshly isolated vein rings, indicated by ATP concentration, was maintained in culture (321 +/- 41 vs. 277 +/- 31 nmol (g wet wt)-1, 0 vs. 14 days). The initial decrease in ATP concentration in surgically prepared rings was significantly reversed following 14 days in culture from 135 +/- 26 to 201 +/- 18 nmol (g wet wt)-1 (P < 0.05). Freshly isolated vein rings maintained their intracellular free amino acid pool during the 14 days in culture (from 166 +/- 25 to 166 +/- 23 mumol (g protein)-1). Surgical preparation of vein rings induced a decrease in the intracellular free amino acid pool (from 166 +/- 25 to 87 +/- 15 mumol (g protein)-1, P < 0.05). This decrease was partially reversed after 14 days in culture (140 +/- 19 mumol (g protein)-1). Although the total amino acid pool in both types of vein rings after 14 days in culture was similar, there were variations in individual amino acid concentrations. Freshly isolated rings showed an increase in glutamine concentration and a decrease in alanine and aspartate concentrations after 14 days in culture. Surgically prepared vein rings showed a decrease in aspartate concentration and an increase in concentrations of glutamine, asparagine, glutamate and glycine. The changes in individual intracellular free amino acid concentrations, which were largely determined by the corresponding concentrations in the medium, indicates that culture media should be supplemented with taurine, aspartate and alanine.     

Verapamil protection against mercuric chloride-induced renal glomerular injury in rats

We have examined the effects of the calcium channel blocker verapamil on the renal glomerular structural damage produced by mercuric chloride in rats. Verapamil (75 micrograms/kg body wt iv) was administered 30 min prior to mercuric chloride injection (HgCl2, 5 mg/kg body wt sc). Verapamil prevented the glomerular proteinuria observed in HgCl2-treated rats. Isolated glomeruli from mercury-treated rats 1 h after injection presented a diminished cross-sectional area as compared with control glomeruli (control [micron2], 26,310 +/- 2545; HgCl2 [micron2], 18,474 +/- 1828) and increased glomerular calcium content (control, 23 +/- 6 nmol/mg protein; HgCl2, 43 +/- 7 nmol/mg protein). Verapamil pretreatment prevented glomerular cross-sectional area (GCSA) diminution and glomerular calcium content rise (GCSA [micron2] Vp + Hg, 28,281 +/- 4654, Ca2+ [nmol/mg protein] Vp + Hg, 18 +/- 5). Renal sections prepared for immunohistochemical detection and histochemical analysis showed increased deposits of fibronectin and lipids and enhanced cellularity in glomerular structures from HgCl2-treated rats. Renal sections from animals pretreated with verapamil showed fibronectin and lipid contents not different from control sections and their histological studies did not show any changes when compared with control. Verapamil pretreatment also protected glomeruli from enhanced leukocyte content (myeloperoxidase activity/mg protein): control, 59 +/- 7; HgCl2, 134 +/- 10; Vp + Hg, 79 +/- 11). HgCl2 also contracts GCSA in vitro; Vp prevented this GCSA diminution. The results described in this study indicate that mercuric chloride nephrotoxicity may be associated not only with changes in renal glomerular haemodynamics, but also with a direct effect on glomerular cells.     

Developmental differences in superoxide production in isolated guinea-pig hearts during reperfusion

The production of free radicals on reperfusion has been implicated as an important factor governing post-ischemic recovery of cardiac function. Although the response of the heart to ischemia and reperfusion is known to change during cardiac development, it is not known if different rates of free radical production play a role in these altered responses. The aim of this investigation was to determine if the production of the superoxide anion (O2-) on reperfusion differs in the immature and mature heart. Immature hearts, obtained from 3-day premature guinea pigs (delivered by cesarean section) were compared with those from adults (7 weeks old). Using the isolated Langendorff preparation. O2- production was measured during reperfusion following ischemic durations [0 (aerobic control), 15, 20, 30, and 60 min, n = 6/group] by the reduction of succinylated ferricytochrome c in the perfusate. Both immature and mature hearts exhibited bell-shaped relationship between ischemic duration and peak O2- production on reperfusion: (13.4 +/- 5.9; 22.2 +/- 5.4; 23.0 +/- 7.8; 59.3 +/- 16.2; 33.7 +/- 15.1; 32.6 +/- 8.5 nmol/min/g wet weight in the immature heart and 15.7 +/- 1.9; 55.0 +/- 30.2; 82.8 +/- 14.0; 78.8 +/- 33.8; 40.6 +/- 16.4; 45.4 +/- 13.1 nmol/min/g wet weight in the mature heart after 0; 15; 20; 30; 45 and 60 min of ischemia, respectively). A similar relationship was also demonstrated with O2- production over the 20-min reperfusion period: (134.0 +/- 57.1; 106.5 +/- 46.2; 199.3 +/- 50.6; 362.0 +/- 99.5; 375.0 +/- 60.9; 221.0 +/- 73.0 nmol/20 min/g wet weight in the immature heart and 97.8 +/- 54; 282.0 +/- 139.0; 933.3 +/- 210.3; 964.0 +/- 374.0; 443.0 +/- 106.0; 352.0 +/- 1551.0 nmol/20 min/g wet weight in the mature heart after 0, 15, 20, 30, 45 and 60 min of ischemia, respectively). Mature hearts consistently produced more O2- than immature hearts on reperfusion, while there was no significant difference in their capacity to produce O2- during aerobic perfusion. We conclude that the immature heart may be at less risk from the free radical component of reperfusion injury than the mature heart.     

Ferrous ion diminished the antiarrhythmic effect of naloxone in myocardial ischemia of isolated rat hearts

This investigation was to examine the effect of ferrous ion (a prooxidant) on the antiarrhythmic effect of naloxone (an endogenous opioid receptor antagonist) in isolated rat hearts. Isolated Sprague-Dawley rat hearts were perfused in the Langendorff mode and myocardial ischemia was performed by ligating the left descending coronary artery. Cardiac rhythm was recorded. Heart alpha-tocopherol concentrations were analyzed. Naloxone (1.2 micromol/heart) was effective in reducing the severity of arrhythmia (arrhythmia score; mean+/-S.E.M: 2.82+/-0.69 for naloxone vs. 5.18+/-0.38 for control, p<0.01). Fe2+ (100 nmol/heart) alone did not significantly affect the arrhythmia score (5.63+/-0.32) when compared with the control, however, Fe2+ administration did cause significant early onset of ventricular premature contraction and ventricular tachycardia. Additionally, Fe2+ administration diminished the naloxone's antiarrhythmic effect (arrhythmia score 4.12+/-0.40). Alpha-tocopherol, a major free radical scavenger that exerts protective functions on heart tissues during myocardial ischemia/reperfusion, was significantly higher in the naloxone-treated group (59.05+/-3.00 nmol/g wet wt) than in the control group (43.84+/-4.17 nmol/g wet wt, p<0.05). These results suggest that endogenous opioid peptides and reactive oxygen species might be related to ischemia-induced arrhythmia.     

Gender differences in the activities of aspirin-esterases in rat tissues

The activities of aspirin (acetylsalicylic acid)-esterases were measured in several tissues (liver, kidney, adrenal glands, brain and serum) from adult male and female Wistar rats. In males, both aspirin-esterase I (assayed at pH 5.5) and II (assayed at pH 7.4) activities were higher in liver homogenates when compared to females (aspirin-esterase I: males 48.9 +/- 4.8 (N = 8) and females 29.3 +/- 4.2 (N = 8) nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 41.4 +/- 4.1 (N = 8) and females 26.1 +/- 4.5 (N = 8) nmol of salicylic acid formed min-1 mg protein-1, P < 0.001). In serum, enzyme activity was higher in females than in males (aspirin-esterase I: males 0.85 +/- 0.06 (N = 6) and females 1.18 +/- 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1, aspirin-esterase II: males 1.03 +/- 0.13 (N = 6) and females 1.34 +/- 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1, P < 0.001). In the other tissues assayed, no statistically significant difference between males and females was found. There were no statistically significant differences when the enzymes were assayed in different phases of the estrous cycle in liver and serum. These results show that the differences in aspirin-esterase activity observed between males and females are not due to the estrous cycle. The gender difference obtained in our study may indicate an involvement of gonadal hormones in the control of the hydrolysis of aspirin. This possibility is currently under investigation.     

Post-ischemic stimulation of 2-deoxyglucose uptake in rat myocardium: role of translocation of Glut-4

Myocardial ischemia elicits translocation of the insulin-sensitive glucose transporter GLUT-4 from intracellular membrane stores to the sarcolemma. Because glucose metabolism is of crucial importance for post-ischemic recovery of the heart, myocardial uptake of [3H]-labeled 2-deoxyglucose and subcellular localization of GLUT-4 were determined during reperfusion in isolated rat hearts perfused with medium containing 0.4 mm palmitate and 8 mm glucose. Hearts were subjected to 20 min of no-flow ischemia, followed by reperfusion for up to 60 min. Subcellular localization of GLUT-4 was determined by cell fractionation followed by immunoblotting. After 15 and 60 min of reperfusion uptake of 2-deoxyglucose was significantly higher (91+/-9 and 96+/-8 nmol/min/g wet weight, respectively) as compared to control values (65+/-1 nmol/min/g wet weight). Ischemia elicited translocation of GLUT-4 to the sarcolemma, which persisted after 15 min of reperfusion. However, after 60 min of reperfusion the subcellular distribution of GLUT-4 was similar to control hearts. In conclusion, reversal of ischemia-induced translocation of GLUT-4 to the sarcolemma is rather slow, possibly facilitating glucose uptake early during reperfusion. However, myocardial uptake and phosphorylation of 2-deoxyglucose remains enhanced late during reperfusion, when pre-ischemic distribution of GLUT-4 is almost completely restored, indicating that additional mechanisms are likely to be involved in post-ischemic stimulation of glucose uptake.     

TrfA dimers play a role in copy-number control of RK2 replication

Heat stress pretreatment of the heart is known to protect this organ against an ischemic/reperfusion insult 24 h later. Degradation of membrane phospholipids resulting in tissue accumulation of polyunsaturated fatty acids, such as arachidonic acid, is thought to play an important role in the multifactorial process of ischemia/reperfusion-induced damage. The present study was conducted to test the hypothesis that heat stress mitigates the postischemic accumulation of arachidonic acid in myocardial tissue, as a sign of enhanced membrane phospholipid degradation. The experiments were performed on hearts isolated from rats either 24 h after total body heat treatment (42 degrees C for 15 min) or 24 h after sham treatment (control). Hearts were made ischemic for 45 min and reperfused for another 45 min. Heat pretreatment resulted in a significant improvement of postischemic hemodynamic performance of the isolated rat hearts. The release of creatine kinase was reduced from 30 +/- 14 (control group) to 17 +/- 5 units/g wet wt per 45 min (heat-pretreated group) (p < or = 0.05). Moreover, the tissue content of the inducible heat stress protein HSP70 was found to be increased 3-fold 24 h after heat treatment. Preischemic tissue levels of arachidonic acid did not differ between heat-pretreated and control hearts. The postischemic ventricular content of arachidonic acid was found to be significantly reduced in heat-pretreated hearts compared to sham-treated controls (6.6 +/- 3.3. vs. 17.8 +/- 12.0 nmol/g wet wt). The findings suggest that mitigation of membrane phospholipid degradation is a potential mechanism of heat stress-mediated protection against the deleterious effects of ischemia and reperfusion on cardiac cells.     

Hepatocellular defence against acidosis is preserved after cold storage

BACKGROUND: Primary non-function of liver allografts is related to preservation time, during which hypoxia leads to intracellular accumulation of acid. Preservation-induced failure of hepatocellular pH regulation may play a role in the pathogenesis of primary graft non-function. METHODS: Using cultured/suspended rat hepatocytes and fluorimetric determination of intracellular pH, we determined whether preservation in University of Wisconsin solution (4 degrees C) impairs hepatocellular defence mechanisms against acidosis. RESULTS: In non-preserved, 24-h-preserved and 48-h-preserved hepatocytes acidified to pH 6.7-6.8, initial Na+/H+ antiport-mediated H+ fluxes averaged 12 +/- 5, 9 +/- 5 and 12 +/- 5 nmol microL-1 min-1 and initial Na+/HCO3- symport-mediated HCO3- fluxes 7 +/- 2, 7 +/- 3 and 6 +/- 2 nmol microL-1 min respectively (P = NS). Preservation did not affect the inverse relationship between Na+/H+ antiport activity and intracellular pH. Thus, hepatocellular defence against intracellular acidosis is maintained during up to 48 h in University of Wisconsin solution. CONCLUSION: Altered pHi homeostasis is unlikely to play a role in the pathogenesis of primary non-function of liver allografts.     

Glutathione metabolism in patients with non-small cell lung cancers

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death in the United States. Because NSCLC is highly chemoresistant, it is, usually not treatable. Altered glutathione (GSH) metabolism is thought to be one major mechanism of chemoresistance, and GSH levels are reported to be elevated in NSCLC. The main objective of this study is to delineate the potential mechanisms involved in elevation of tissue GSH, including extraction from the circulation by NSCLC. Twenty consecutive patients with NSCLC were enrolled. At the time of lobectomy, pulmonary artery and vein were identified, and blood flow was measured by an electromagnetic probe. Subsequently, blood samples were drawn from pulmonary artery, the vein draining the tumor-bearing lobe, and a normal lobe. Immediately after lobectomy, tumor and lung specimens were snap frozen. NSCLC tumor specimens had higher levels of GSH compared with lung tissue (20.8 +/- 9.4 versus 11.6 +/- 3.0 nmol/mg protein, respectively; P < 0.05). The tumor demonstrated higher activity of the enzyme gamma-glutamyl transpeptidase, a membrane-bound enzyme involved in transmembrane uptake of GSH, than lung tissue (41.9 +/- 26.4 versus 22.4 +/- 12.3 units/mg protein, respectively; P < 0.05). Also, the tumor-bearing lobe showed elevated extraction of GSH and two of its component amino acids compared with lung tissue (GSH uptake: 0.60 +/- 0.67 versus 0.20 +/- 0.40 microM/min, respectively; P < 0.05). NSCLC tumors are able to extract circulating GSH and its constituent amino acids to synthesize intracellular GSH. Increased activity of gamma-glutamyl transpeptidase may be one mechanism underlying increased GSH uptake by NSCLC.     

Understanding the physiopathology of heart arrhythmias. An essential condition for good therapeutic management

OBJECTIVES: Tolerance of intravenously applied clarithromycin has been tested on marginal ear veins of rabbits. Use of human umbilical venous endothelial cells (HUVEC) for testing antibiotic solutions for intravenous compatibility provides a valuable alternate model. DESIGN AND METHODS: In order to evaluate the effect of clarithromycin on intracellular purines, reflecting cell viability, energy production, signal transduction and DNA/RNA synthesis, intracellular adenosine 5' triphosphate (ATP), adenosine 5' diphosphate (ADP), guanosine 5' triphosphate (GTP), and guanosine 5' diphosphate (GDP) levels were measured by means of high performance liquid chromatography (HPLC). RESULTS: Incubation of cells with 2 mg/mL clarithromycin resulted in a rapid decrease of the intracellular ATP from 12.6 +/- 1.1 to 8.87 +/- 0.82 nmol/million cells or 1.5 +/- 0.6 nmol/million cells, after 20 or 60 min, respectively. In addition, ADP was extensively depleted. Purine nucleotide profiles were markedly different following exposure to 1 mg/mL clarithromycin. There was no significant decline of intracellular high energy phosphate levels after 20 min. CONCLUSION: These results show that clarithromycin has a better endothelial compatibility if diluted to a final concentration of 1 mg/mL. These data are in line with our clinical observations that the occurrence of phlebitis could be minimized by diluting the manufacturers' preparation of clarithromycin to 1 mg/mL.     

Inhibitory actions of emodin on arylamine N-acetyltransferase activity in strains of Helicobacter pylori from peptic ulcer patients

Arylamine N-acetyltransferase (NAT) activities with p-aminobenzoic acid and 2-aminofluorene were determined in Helicobacter pylori, a gram-negative rod bacteria collected from peptic ulcer patients. The NAT activity was determined using a acetyl CoA recycling assay and HPLC. Cytosols or suspensions of H. pylori with and without selected concentrations of emodin co-treatment showed different percentages of 2-aminofluorene and p-aminobenzoic acid acetylation. The data indicate that there were decreased NAT activity associated with increased emodin in H. pylori cytosols. As 400 microns of emodin can obviously inhibit NAT activity both in vitro and in vivo (inhibition rate 90% and 93% for 2-aminofluorene and p-aminobenzoic acid in vitro, and 90% and 92%, respectively, for both substrate in vivo). For in vitro examination, the apparent values of Km and Vmax were 3.12 +/- 0.38 mM and 15.20 +/- 3.16 nmol/min/mg protein for 2-aminofluorene, and 0.56 +/- 0.12 mM and 0.74 +/- 0.09 nmol/min mg protein for p-aminobenzoic acid. However, when emodin was added to the reaction mixtures, the values of apparent Km and Vmax were 2.40 +/- 0.32 mM and 10.62 +/- 0.04 nmol/min/mg protein for 2-aminofluorene, and 0.23 +/- 0.02 mM and 0.62 +/- 0.08 nmol/min/mg protein for p-aminobenzoic acid. For in vivo examination, the apparent Km and Vmax were 0.82 +/- 0.18 mM and 0.92 +/- 0.21 nmol/min/10 x 10(10) colony forming units (CFU) for 2-aminofluorene, and 0.78 +/- 0.14 mM and 0.52 +/- 0.06 nmol/min/ 10 x 10(10) (CFU) for p-aminobenzoic acid. However, when emodin was added to the reaction mixtures, the values of apparent Km and Vmax were 0.50 +/- 0.08 mM and 0.62 +/- 0.22 nmol/min/ 10 x 10(10) (CFU) for 2-aminofluorene, and 0.52 +/- 0.21 mM and 0.26 +/- 0.04 nmol/min/ 10 x 10(10) (CFU) for p-aminobenzoic acid. This report is the first finding of emodin inhibition of arylamine N-acetyltransferase activity in a strain of H. pylori.     

Neonatal de-afferentation of capsaicin-sensitive sensory nerves increases in vivo insulin sensitivity in conscious adult rats

Sensory neuropeptides, released from the peripheral nervous system, might modulate glucose homeostasis by antagonizing insulin action. The effects of de-afferentation of functional small diameter unmyelinated C-fibres (sensory nerves) on in vivo insulin-mediated intracellular glucose metabolism were investigated by using euglycaemic insulin (6 and 18 mU/kg x min) clamps with [3-(3)H]-glucose infusion in 24 adult rats, treated neonatally with either capsaicin (CAP) (50 mg/kg) or vehicle (CON). Following the clamp, skeletal muscle groups, liver and adipose tissue were freeze-clamped. At plasma insulin levels of approximately 90 mU/l, CAP-rats showed a 21% increase in whole body glucose uptake compared with CON (24.4 +/- 1.6 vs 20.1 +/- 0.8 mg/kg min, p < 0.02), which was paralleled by a 20% increase in whole body glycolysis (12.6 +/- 0.8 vs 10.5 +/- 0.5 mg/ kg.min p < 0.05) (concentration of 3H2O in plasma). Whole body skeletal muscle glycogenesis was increased by 80% in CAP-rats (5.7 +/- 0.7 vs 3.1 +/- 0.7 mg/kg x min, p < 0.05) with increased muscle glycogen synthase activity. Whole body (muscle, liver and adipose tissue combined) de novo lipogenesis also was increased in CAP-rats compared with CON (0.69 +/- 0.10 vs 0.44 +/- 0.06 mg/kg x min, p < 0.05) (incorporation of [3-(3)H]-glucose counts into glycogen or fat). Hepatic glucose production was lower in CAP-rats compared with CON (0.6 +/- 0.6 vs 2.1 +/- 0.7 mg/kg x min, p < 0.05). Plasma glucagon, corticosterone, epinephrine and norepinephrine levels were reduced in CAP-rats: 43 +/- 2 compared with 70 +/- 6 pg/ml, 855 +/- 55 compared with 1131 +/- 138 nmol/l, 513 +/- 136 compared with 1048 +/- 164 pmol/l and 928 +/- 142 compared with 1472 +/- 331 pmol/l, respectively, p < 0.05. At plasma insulin levels of approximately 400 mU/l, CAP-rats showed no differences in peripheral and hepatic insulin action compared with CON. We conclude that the removal of endogenous sensory neuropeptides, by de-afferentation of capsaicin-sensitive sensory nerves, increases in vivo insulin sensitivity, but not responsiveness: 1) primarily through an increased sensitivity of skeletal muscle glycogen synthesis to insulin; 2) through a reduction in the levels of counter-regulatory hormones, thereby creating a milieu which favours overall in vivo insulin sensitivity with respect to glucose uptake, glucose production, glycolysis, glycogenesis and lipogenesis.     

Isoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes: selective catalysis by FMO3

In the present study, we expressed human flavin-containing monooxygenase 1 (FMO1), FMO3, FMO4t (truncated), and FMO5 in the baculovirus expression vector system at levels of 0.6 to 2.4 nmol FMO/mg of membrane protein. These four isoforms, as well as purified rabbit FMO2, and eleven heterologously expressed human P450 isoforms were examined for their capacity to metabolize trimethylamine (TMA) to its N-oxide (TMAO), using a new, specific HPLC method with radiochemical detection. Human FMO3 was by far the most active isoform, exhibiting a turnover number of 30 nmol TMAO/nmol FMO3/min at pH 7.4 and 0.5 mM TMA. None of the other monooxygenases formed TMAO at rates greater than 1 nmol/nmol FMO/min under these conditions. Human fetal liver, adult liver, kidney and intestine microsomes were screened for TMA oxidation, and only human adult liver microsomes provided substantial TMAO-formation (range 2.9 to 9.1 nmol TMAO/mg protein/min, N = 5). Kinetic studies of TMAO formation by recombinant human FMO3, employing three different analytical methods, resulted in a Km of 28 +/- 1 microM and a Vmax of 36.3 +/- 5.7 nmol TMAO/nmol FMO3/min. The Km determined in human liver microsomes ranged from 13.0 to 54.8 microM. Therefore, at physiological pH, human FMO3 is a very specific and efficient TMA N-oxygenase, and is likely responsible for the metabolic clearance of TMA in vivo in humans. In addition, this specificity provides a good in vitro probe for the determination of FMO3-mediated activity in human tissues, by analyzing TMAO formation at pH 7.4 with TMA concentrations not higher than 0.5 mM.     

Homeobox genes in hematopoiesis and leukemogenesis

The effect of cold and warm intermittent antegrade blood cardioplegia, on the intracellular concentration of taurine in the ischaemic/reperfused heart of patients undergoing aortic valve surgery, was investigated. Intracellular taurine was measured in ventricular biopsies taken before institution of cardiopulmonary bypass, at the end of 30 min of ischaemic arrest and 20 min after reperfusion. There was no significant change in the intracellular concentration of taurine in ventricular biopsies taken after the period of myocardial ischaemia in the two groups of patients (from 10.1 +/- 1.0 to 9.6 +/- 0.9 mumol/g wet weight for cold and from 9.3 +/- 1.3 to 10.0 +/- 1.3 mumol/g wet weight for warm cardioplegia, respectively). Upon reperfusion however, there was a fall in taurine in both groups but was only significant (P < 0.05) in the group receiving cold blood cardioplegia (6.9 +/- 0.8 mumol/g wet weight after cold blood cardioplegia versus 8.0 +/- 0.8 mumol/g wet weight following warm blood cardioplegia). Like taurine, there were no significant changes in the intracellular concentration of ATP after ischaemia in the two groups of patients (from 3.2 +/- 0.32 to 2.95 +/- 0.43 mumol/g wet weight for cold and from 2.75 +/- 0.17 to 2.62 +/- 0.21 mumol/g wet weight for warm cardioplegia, respectively). However upon reperfusion there was a significant fall in ATP in both groups with the extent of the fall being less in the group receiving warm cardioplegia (1.79 +/- 0.19 mumol/g wet weight for cold and 1.98 +/- 0.27 mumol/g wet weight for warm cardioplegia, respectively). This work shows that reperfusion following ischaemic arrest with warm cardioplegia reduces the fall in tissue taurine seen after arrest with cold cardioplegia. Accumulation of intracellular sodium provoked by hypothermia and a fall in ATP, may be responsible for the fall in taurine by way of activating the sodium/taurine symport to efflux taurine.     

Colorimetric determination of cystine (disulfide bond) in hair using dithiothreitol

We developed a simple method for the quantification of cystine (disulfide bond) content in hair by measuring the amount of oxidized dithiothreitol (lambda max 283 nm) derived from dithiothreitol (DTT) with cystine. Because the cystine content in hair is almost fixed for each animal species, it can be used as a reliable indicator of hair weight. The absorbance (A280, y) of the supernatant of the reaction mixture correlated well with hair weight (mg, x) (y = 0.10x + 0.06, r = 1.00, n = 10). Within-run and between-day reproducibilities (C.Vs., %) for the assay were 3.1 and 2.8 (n = 5 each), respectively. Hair cystine content (nmol/mg hair, mean +/- S.D.) in normal volunteers was 903 +/- 50.6 (n = 10) by the present method and 755 +/- 24.9 (n = 10) when an amino acid analyzer was used. After assay by our method, the hair sample can be washed, then used repeatedly to assay other analytes. The present method should be useful for assays of analytes present only in small amounts (2-20 mg), without the need for precise weighing of the hair samples.