BBA - Molecular Basis of Disease (v.1740, #3)

Anti-diabetic effect of ginsenoside Re in ob/ob mice by Jing-Tian Xie; Sangeeta R. Mehendale; Xinmin Li; Richard Quigg; Xiaoyu Wang; Chong-Zhi Wang; Ji An Wu; Han H. Aung; Paul A. Rue; Graeme I. Bell; Chun-Su Yuan (319-325).
We evaluated the anti-diabetic effects of ginsenoside Re in adult male C57BL/6J ob/ob mice. Diabetic ob/ob mice with fasting blood glucose levels of approximately 230 mg/dl received daily intraperitoneal injections of 7, 20 and 60 mg/kg ginsenoside Re for 12 consecutive days. Dose-related effects of ginsenoside Re on fasting blood glucose levels were observed. After the 20 mg/kg treatment, fasting blood glucose levels were reduced to 188±9.2 and 180±10.8 mg/dl on Day 5 and Day 12, respectively (both P<0.01 compared to vehicle group, 229±9.5 and 235±13.4 mg/dl, respectively). The EC70 of ginsenoside Re was calculated to be 10.3 mg/kg and was used for subsequent studies. Consistent with the reduction in blood glucose, there were significant decreases in both fed and fasting serum insulin levels in mice treated with ginsenoside Re. With 12 days of ginsenoside treatment, glucose tolerance of ob/ob mice increased significantly, and the area under the curve for glucose decreased by 17.8% (P<0.05 compared to vehicle treatment). The hypoglycemic effect of the ginsenoside persisted even at 3 days of treatment cessation (blood glucose levels: 198±13.1 with ginsenoside treatment vs. 253±20.3 mg/dl with vehicle, P<0.01). There were no significant changes in body weight or body temperature. Preliminary microarray analysis revealed differential expression of skeletal muscle genes associated with lipid metabolism and muscle function. The results suggest that ginsenoside Re may prove to be useful in treating type 2 diabetes.
Keywords: Diabetes; Ginsenoside Re; Intraperitoneal glucose tolerance test; Insulin; Microarray; ob/ob mouse;

Up-regulation of mitogen activated protein kinases in mdx skeletal muscle following chronic treadmill exercise by Akinori Nakamura; Kunihiro Yoshida; Hideho Ueda; Shin'ichi Takeda; Shu-ichi Ikeda (326-331).
Dystrophin, a product of the Duchenne muscular dystrophy gene, is a cytoskeletal protein of skeletal and cardiac muscle fibers. Dystrophin-deficient muscle fibers are abnormally vulnerable to mechanical stress including physical exercise, which is a powerful stimulator of mitogen-activated protein kinases (MAPKs). To examine how treadmill exercise affects MAPK family members in dystrophin-deficient skeletal muscle, we subjected both mdx mice, an animal model for Duchenne muscular dystrophy, and C57BL/10 mice to treadmill exercise and examined the phosphorylated protein levels of extracellular-signal regulated kinase (ERK1/2), p38 MAPK and c-Jun N terminal kinase 1 and 2 (JNK1 and JNK2) in the gastrocnemius muscle. Phosphorylation of ERK1/2, p38 MAPK and JNK2, but not JNK1, increased more in the muscles of exercise trained mdx mice than in muscles of trained C57BL/10 or untrained mdx mice. These results show that physical exercise aberrantly up-regulates the phosphorylated form of ERK1/2, p38 MAPK and JNK2 in dystrophin-deficient skeletal muscle and that their up-regulation might play a role in the degeneration and regeneration process of dystrophic features.
Keywords: Dystrophin; Treadmill exercise; Mitogen-activated protein kinase;

Effect of acetaminophen on the membrane anchoring of Na+, K+ATPase of rat renal cortical cells by Laura Trumper; Gabriela Coux; Liliana A. Monasterolo; Sara Molinas; Verónica M.C. García; M. Mónica Elías (332-339).
In previous works we reported that the administration of a toxic dose of acetaminophen (APAP) induces acute renal failure (ARF) and promotes changes on Na+, K+ATPase distribution in renal proximal plasma membranes. In the present work, we analyzed if APAP could promote the dissociation of Na+, K+ATPase from its membrane anchorage. The participation of calpain activation was also evaluated. We analyzed the Triton X-100 extractability of Na+, K+ATPase in freshly isolated cortical cell suspensions incubated with different APAP concentrations (0.1, 1, 10 and 100 mM). Both α1 and β1 subunits were studied by Western blot. APAP promoted the increment of both subunits abundance in the Triton-soluble fraction. Calpain activation was detected in the membrane fractions of cells incubated with APAP. Incubation with APAP 0.1, 1 and 10 mM did not promote an increment in LDH release compared with controls, while APAP 100 mM promoted an increased LDH release. Our results show that incubation of proximal cells with sublethal and lethal APAP concentrations promotes the detachment of Na+, K+ATPase from its membrane anchoring. Inhibition of calpain activation by SJA 7029 protected against APAP-induced membrane damage but not against APAP-induced increase of the Triton X-100 extractability of Na+, K+ATPase.
Keywords: Acetaminophen; Na+; K+ATPase; Triton X-100 extractability; Calpain; Renal cell suspension;

The effect of short-term kaempferol exposure on reactive oxygen levels and integrity of human (HL-60) leukaemic cells by Charles S. Bestwick; Lesley Milne; Lynn Pirie; Susan J. Duthie (340-349).
Flavonoids may be a principal contributor to the cancer preventative activity of fruit- and vegetable-rich diets and there is interest in their use as dietary supplements. However, there is potential conflict between the cytoprotective and cytotoxic activities of flavonoids, and their efficacy as anti-cancer agents is unresolved. Here, the integrity and survival of HL-60 promyelocytic leukaemia cells following short-term (90 min) exposure to the dietary abundant flavonoid kaempferol (1–100 μM) is reported. Supplementation initially decreased reactive oxygen levels but, paradoxically, a dose-dependent increase in single-strand DNA breakage occurred. However, there was no increase in oxidised DNA purines or membrane damage. Following a 24-h recovery period in non-kaempferol supplemented media, DNA single-strand breakage had declined and kaempferol exposed and control cultures possessed similar reactive oxygen levels. A reduction in 3H-thymidine incorporation occurred with ≥10 μM kaempferol. One hundred micromolar kaempefrol increased the proportion of cells in G2-M phase, the proportion of cells with a sub-G1 DNA content and enhanced ‘active’ caspase-3 expression but only induced a loss of mitochondrial membrane potential within a minority of cells. The relevance of induced DNA damage within a non-overtly oxidatively stressed environment to the disease preventative and therapeutic use of kaempferol is discussed.
Keywords: Cell cycle; DNA damage; Flavonoid; HL-60; Kaempferol; Oxidative stress;

Expression of apolipoprotein AI mRNA in peripheral white blood cells of patients with alcoholic liver disease by Patricia Nuño-González; Bertha Ruíz-Madrigal; Blanca E. Bastidas-Ramírez; Erika Martínez-López; Jorge E. Segura; Arturo Panduro (350-356).
Because (i) changes in plasma and liver mRNA of apolipoprotein (apo) AI have been observed in patients with alcoholic liver disease, (ii) apo AI mRNA can be induced in non-hepatic tissues, and (iii) apolipoproteins expression is influenced by plasma colloid osmotic pressure (P CO) and viscosity (η), we analyzed the Apo AI mRNA expression in the peripheral white blood cells (PWBC), P CO, and η in control volunteers (C), patients with liver cirrhosis (LC), and cirrhotic patients with superimposed alcoholic hepatitis (LC+AH). We found that apo AI mRNA is expressed in the PWBC in 20% of C and it is induced 1.5 fold in 66.6% of LC and 1.95 fold in 85% of LC+AH. A significant decrease of P CO in LC and LC+AH (14.8±2.4 and 16.2±2.4 mm Hg, respectively) compared to C (27.9±2 mm Hg) was observed. By contrast, η was mildly increased from 1.7389±0.07 in C to 1.8022±0.154 in LC and 1.9030±0.177 in LC+AH. No significant correlation was found between P CO and η with apo AI mRNA but with lipid profile.In conclusion, apo AI mRNA expression in PWBC is associated to liver disease severity and could be an indirect indicator of alcoholic liver damage.
Keywords: Apolipoprotein AI; Gene expression; Liver cirrhosis; Alcoholic hepatitis;

Curcumin, a natural, biologically active compound extracted from rhizomes of Curcuma species, has been shown to act as a biological response modifier in various disorders. We have reported previously that the dietary supplementation of curcumin enhances the activities of antioxidant and phase II metabolizing enzymes in mice (M. Iqbal, S.D. Sharma, Y. Okazaki, M. Fujisawa, S. Okada, Dietary supplementation of curcumin enhances antioxidant and phase II metabolizing enzymes in ddY mice: possible role in protection against chemical carcinogenesis and toxicity, Pharmacol and Toxicol. 92 (2003) 33_38.) and inhibits ferric nitrilotriacetate (Fe-NTA) induced oxidative injury of lipids and DNA in vitro (M. Iqbal, Y. Okazaki, S. Okada, In vitro curcumin modulates Ferric Nitrilotriacetate (Fe-NTA) and hydrogen peroxide (H2O2)-induced peroxidation of microsomal membrane lipids and DNA damage, Teratogenesis Carcinogenesis and Mutagenesis Supplement 23 (2003) 151-160.). In our present study, Fe-NTA, a known complete renal carcinogen, which generate ROS in vivo, was given intraperitoneally to mice and curcumin was tested for its ability to inhibits oxidative stress and the activity of ornithine decarboxylase (ODC) as well as histopathological changes in the kidney. Substantial changes in glutathione, antioxidant enzymes as well as changes in phase II metabolizing enzymes were observed in the kidney at 12 h after treatment with Fe-NTA (9.0 mg Fe/kg body weight). Effect of oxidative stress induced by Fe-NTA were also demonstrated by the increase in lipid peroxidation as monitored by formation of thiobarbituric acid-reactive substances and 4-hydroxy-2-nonenal (HNE)-modified proteins in kidney. Likewise, the level of protein carbonyl contents, an indicator of protein oxidation was also increased after Fe-NTA administration. However, the changes in these parameters were restored to normal in curcumin-pretreated mice. The ODC activity in the kidney was significantly increased by Fe-NTA, while the increased ODC activity induced by Fe-NTA was normalized in curcumin-pretreated mice. In addition, curcumin pretreatment almost completely prevented kidney biomolecules from oxidative damage and protected the tissue against observed histopathological alterations.
Keywords: Ferric nitrilotriacetate; Antioxidant enzyme; Ornithine decarboxylase; Oxidative stress; Renal tubular necrosis; Lipid peroxidation; Protein carbonyl; Curcumin; 4-hydroxy-2-nonenal-modified protein;

Apoptosis vs. necrosis: glutathione-mediated cell death during rewarming of rat hepatocytes by Mariapia Vairetti; Andrea Ferrigno; Roberta Bertone; Plinio Richelmi; Francantonio Bertè; Isabel Freitas (367-374).
Hypothermia induces injury in its own right, but the mechanisms involved in the cell damage are still unclear. The aim of this study was to test the effects that glutathione (GSH) depletion induces on cell death in isolated rat hepatocytes, kept at 4 °C for 20 h, by modulating intracellular GSH concentration with diethylmaleate and buthionine sulfoximine (DEM and BSO). Untreated hepatocytes showed Annexin V stained cells (AnxV+), scarce propidium iodide stained cells (PI+) and presented a low level of lactate dehydrogenase (LDH) leakage after 20 h at 4 °C and rewarming at 37 °C. When DEM and BSO were added before cold storage, we observed a few AnXV+ cells and an increase in PI+ cells associated with LDH release in the incubation medium. Conversely, the addition of DEM and BSO only during rewarming caused a marked increase in cell death by apoptosis. Production of reactive oxygen species (ROS) and thiobarbituric acid species (TBARS), associated with a decrease in GSH concentrations, was higher when DEM and BSO were added before cold storage. Cells treated with DEM and BSO before cold storage showed lower ATP energy stores than hepatocytes treated with DEM and BSO only during rewarming. Pretreatment of hepatocytes with deferoxamine protected against apoptotic and necrotic morphology in conditions of GSH depletion. These results suggest that pretreatment of hepatocytes with DEM and BSO before cold storage induces necrosis, while the treatment of hepatocytes only during rewarming increases apoptosis. In both conditions, iron represents a crucial mediator of cell death.
Keywords: Glutathione; Necrosis; Apoptosis; Reactive oxygen species (ROS); Iron; Liver;

Insulin resistance is polygenic in origin, and can be observed at an early age. We have shown that variations in APOC3−482T>C and hepatic lipase (LIPC)-514C>T), individually (APOC3 alone) and interactively, modulate insulin and glucose levels after an OGTT in young healthy men participating in the European Atherosclerosis Research Study II (EARSII). Variation in the insulin gene (INS) variable number tandem repeat (VNTR) has been found to predispose to type 1 and type 2 diabetes. We have evaluated the HphI site 23 bp upstream of the INS gene (a surrogate marker for the VNTR) in EARSII (n=822), to determine if variation in INS contributes to insulin resistance. Carriers of the INS VNTR class III (HphI−) allele (frequency=0.29 (95%CI 0.27, 0.31)) had significantly higher 60-min insulin concentrations after the OGTT (P=0.014) and a marginally higher AUC insulin (P=0.07), compared to class I (HphI+) homozygotes. However, this effect on AUC insulin was modified by the level of physical activity, displaying significant gene:environment interaction (P=0.03). We tested for gene:gene interaction between the INS VNTR and both the LIPC−514C>T and APOC3−482T>C. While there was a significant interaction between INS VNTR and LIPC−514C>T on AUC glucose (P=0.013) and on AUC insulin (P=0.015), there was no interaction with APOC3−482T>C. Thus, despite a modest effect of the INS VNTR alone, the influence of this variant on insulin sensitivity was modified by gene:environment and gene:gene interactions, illustrating the biological complexity of insulin resistance.
Keywords: Insulin; Hepatic lipase; Glucose;

Skeletal muscles, heart, and lung are the main sources of oxygen radicals in old rats by Andrey V. Kozlov; Laszlo Szalay; Fraz Umar; Karl Kropik; Katrin Staniek; Hans Niedermüller; Soheyl Bahrami; Hans Nohl (382-389).
The aim of this study was to compare rat tissues with respect to their reactive oxygen and nitrogen species (RONS) generating activities as a function of age. We quantified the RONS generation in vivo in young (6 months) and in old (30 months) male Sprague–Dawley rats using the recently developed spin trap 1-hydroxy-3-carboxy-pyrrolidine, applied intravenously. This spin trap reacts with superoxide radical and peroxynitrite yielding a stable spin adduct which is detectable by means of electron paramagnetic resonance (EPR) spectroscopy in frozen tissues. In old rats RONS generation was significantly increased compared to their young counterparts in the following order: blood<skeletal muscle<lung<heart, but did not change in intestine, brain, liver, and kidney. Experiments with isolated heart mitochondria showed a significant rate of RONS generation in succinate-supplemented mitochondria from old rats while no RONS were detected in mitochondria from young rats. This study identifies heart, lung, and skeletal muscle as the tissues with increased RONS formation as a function of age.
Keywords: Reactive oxygen and nitrogen species; Aging; Spin trapping; Electron paramagnetic resonance; Mitochondria; Rat;

Among the more than 75 known variants of α1-proteinase inhibitor, a sub-population of rare, point mutations causing single amino acid replacements have been identified and classified as “at risk” alleles for development of pulmonary disease. In most cases, it is not clear how the amino acid replacements typical of these variants change the properties of the inhibitor to increase risk of disease in the affected individuals. To begin to address this question, we mutagenized a wild type α1-proteinase inhibitor cDNA to encode a panel of eight different point mutants reported to be associated with increased risk for development of pulmonary disease. These variants were then expressed in COS-l cells transiently transfected with plasmids containing the altered cDNAs. The effects of the mutations on the rates of secretion, cellular location, intracellular degradation, activity, stability, and tendency to aggregate were determined. Results of these studies show that, in some cases, the mutations affect the rate of secretion, the activity or both of these properties of α1-proteinase inhibitor in a manner consistent with its designation as an “at-risk” allele. In other cases, the mutations do not significantly change the properties of the inhibitor, suggesting that these may be normal variants and that their expression may not increase the risk of disease.
Keywords: α1-proteinase inhibitor; Point mutants; Elastase; Binding-activity; Secretion; Stability;

Characterization of the first described mutation of human red blood cell phosphoglycerate mutase by Pedro de Atauri; Ada Repiso; Baldomero Oliva; Joan Lluis Vives-Corrons; Fernando Climent; José Carreras (403-410).
In a patient with clinical diagnosis of Hereditary Spherocytosis and partial deficiency (50%) of red blood cell phosphoglycerate mutase (PGAM) activity, we have recently reported [A. Repiso, P. Pérez de la Ossa, X. Avilés, B. Oliva, J. Juncá, R. Oliva, E. Garcia, J.L.L. Vives-Corrons, J. Carreras, F. Climent, Red blood cell phosphoglycerate mutase. Description of the first human BB isoenzyme mutation, Haematologica 88 (2003) (03) ECR07] the first described mutation of type B PGAM subunit that as a dimer constitutes the PGAM (EC isoenzyme present in red blood cells. The mutation is the substitution c.690G>A (p.Met230Ile). In this report, we show that the mutated PGAM possesses an abnormal behaviour on ion-exchange chromatography and is more thermo-labile that the native enzyme. We also confirm that, similar to the PGAM isoenzymes from other sources, the BB-PGAM from human erythrocytes has a ping pong or phosphoenzyme mechanism, and that the mutation does not significantly change the K m and K i values, and the optimum pH of the enzyme. The increased instability of the mutated enzyme can account for the decreased PGAM activity in patient's red blood cells. However, the implication of a change of the k cat produced by the mutation cannot be discarded, since we could not determine the k cat value of the mutated PGAM.
Keywords: Phosphoglycerate mutase; Erythrocyte; Mutation; Kinetic property; Heat stability; Modelation;

The protective effect of α-crystallin against acute inflammation in mice by J. Gunasingh Masilamoni; E. Philip Jesudason; S. Nirmala Bharathi; R. Jayakumar (411-420).
Acute inflammation can activate macrophages or monocytes and subsequently release several inflammatory cytokines and reactive oxygen species (ROS). Oxidative stress triggered by the production of ROS plays deleterious role leading to multiple organ failure. This study was designed to investigate the prophylactic effect of α-crystallin, a major chaperone lens protein comprising of α-A and α-B subunits in inflammation-induced mice. Mice were divided into three groups (n=6 in each): control, inflammation and α-crystallin-treated. Results show that ROS was significantly higher in the lymphocytes, hepatocytes and astrocytes (P<0.05) of inflammation-induced mice when compared to control, but no significant changes were observed in the α-crystallin-treated group. Increased level of lipid peroxidation (LPO) and decreased activities of antioxidant such as superoxide dismutase (SOD), catalase, glutathione peroxidase and glutathione were observed in the inflammation-induced mice when compared to control, whereas the activities of these were found to be normal followed by α-crystallin treatment. We also observed a reduction in reduced glutathione levels in hepatocytes of inflammation-induced mice, which were normalized on α-crystallin treatment. The in vitro study has shown that α-crystallin treatment not only suppresses the increase in LPO levels but also inhibits the lipid breakdown resulting from autooxidation in mouse cerebral cortex homogenate, and strongly suggests that α-crystallin therapy may serve as a potent pharmacological agent in systemic inflammation.
Keywords: Acute inflammation; α-crystallin; ROS; LPO; Antioxidant; ATPase; Lymphocyte; Hepatocyte; Astrocyte; Mouse;

Enzymes that hydrolyze adenine nucleotides in platelets from breast cancer patients by Maria do Carmo Araújo; João Batista Teixeira Rocha; André Morsch; Rafael Zanin; Rita Bauchspiess; Vera Maria Morsch; Maria Rosa Chitolina Schetinger (421-426).
The activities of NTPDase (EC, apyrase, CD39) and 5′-nucleotidase (EC, CD73) enzymes were analyzed in platelets from breast cancer patients. Initially, patients were compared in terms of length (years) of tamoxifen use. The following groups were studied: breast cancer patients who did not use tamoxifen, patients using tamoxifen for 1–48 months, patients using tamoxifen for 49–84 months, and controls (healthy subjects). Results demonstrated that adenosine triphosphate (ATP) hydrolysis was enhanced (F(3,114)=8.53; P<0.001) and adenosine diphosphate (ADP) hydrolysis was reduced (F(3,106)=5.09, P=0.002) as a function of tamoxifen use, while adenosine monophosphate (AMP) hydrolysis was unchanged. Next, patients were compared statistically according to disease stage, determined by the tumor–node–metastasis (TNM) staging system for classifying breast tumor. ATP hydrolysis was significantly elevated in patients with stage I and II breast cancer (F(4,113)=4.35; P=0.003), but was normal in patients with stage III and IV cancer. ADP hydrolysis was reduced in stages II to IV (F(4,105)=3.88, P=0.006) and AMP hydrolysis was elevated in stage II (F(4,105)=3.45 P=0.01), but was normal in stages III and IV. Platelet aggregation time was similar in all patients regardless of tamoxifen use or disease stage. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were also within the normal range and similar among all groups. Similarly, fibrinogen and fibrin degradation product (FDP) were unchanged in all groups. In conclusion, our study demonstrated for the first time that hydrolysis of adenine nucleotides is modified in platelets from breast cancer patients taking tamoxifen.
Keywords: Breast cancer; Tamoxifen; NTPDase; 5′-nucleotidase; Platelet;

Transgenic mice overexpressing SREBP-1a under the control of the PEPCK promoter exhibit insulin resistance, but not diabetes by Akimitsu Takahashi; Hitoshi Shimano; Yoshimi Nakagawa; Takashi Yamamoto; Kaori Motomura; Takashi Matsuzaka; Hirohito Sone; Hiroaki Suzuki; Hideo Toyoshima; Nobuhiro Yamada (427-433).
Sterol regulatory element-binding protein-1 (SREBP-1) is a transcription factor which regulates genes involved in the synthesis of fatty acids and triglycerides. The overexpression of nuclear SREBP-1a in transgenic mice under the control of the PEPCK promoter (TgSREBP-1a) caused a massively enlarged fatty liver and disappearance of peripheral white adipose tissue. In the current study, we estimated the impact of this lipid transcription factor on plasma glucose/insulin metabolism in vivo. TgSREBP-1a exhibited mild peripheral insulin resistance as evidenced by hyperinsulinemia both at fasting and after intravenous glucose loading, and retarded glucose reduction after insulin injection due to decreased plasma leptin levels. Intriguingly, hyperinsulinemia in TgSREBP-1a mice was markedly exacerbated in a fed state and sustained after intravenous glucose loading, and paradoxically decreased after the portal injection of glucose. TgSREBP-1a mice consistently showed very small plasma glucose increases after portal glucose loading because of a large capacity for hepatic glucose uptake. These data suggested that hepatic insulin resistance emerges postprandially. In addition, pancreatic islets from TgSREBP-1a were enlarged. These data demonstrate that SREBP-1a activation in the liver has a strong impact on plasma insulin levels, implicating the potential role of SREBPs in hepatic insulin metabolism relating to insulin resistance.
Keywords: Transcription factor; SREBP-1a transgenic mouse; Liver steatosis; Lipodystrophy; Insulin resistance;

Magnetic characterisation of rat muscle tissues after subcutaneous iron dextran injection by F.J. Lázaro; A.R. Abadía; M.S. Romero; L. Gutiérrez; J. Lázaro; M.P. Morales (434-445).
Ex vivo freeze-dried rat muscle tissues, collected at different times t after a single dose of subcutaneously injected iron dextran, have been magnetically characterised. The AC susceptibility of the tissues shows an overall superparamagnetic behaviour and the dependence on t of, especially, the out-of-phase component is remarkably systematic despite the fact that each tissue originates in a different rat individual. The experiments show that the akaganéite (β-FeOOH) nanoparticles contained in the injected drug are progressively degraded in the living tissue and, at times of the order of 1 month and for all the analysed rat individuals, converge to a magnetically well-defined species with much narrower magnetic activation energy distribution than iron dextran. Thorough transmission electron microscopy experiments of the same tissues indicate the presence of oxyhydroxide particles, whose size decreases for increasing t in agreement with the interpretation of the magnetic susceptibility. The conclusions drawn from the magnetic study do well correspond to the properties of the whole tissue since no biochemical extraction work has been done. The AC susceptibility appears to be a valuable and complementary tool in pharmacological studies of iron-containing drugs.
Keywords: Iron dextran; AC susceptibility; Superparamagnetism; Iron speciation; Iron metabolism; Pharmacokinetics;

Gut-derived norepinephrine plays an important role in up-regulating IL-1β and IL-10 by Mian Zhou; Padmalaya Das; H. Hank Simms; Ping Wang (446-452).
Previous studies have shown that the gut is a major source of norepinephrine (NE) released in early sepsis and that gut-derived NE plays an important role in up-regulating TNF-α expression in Kupffer cells (KC) via an α2-adrenoceptor (α2-AR) pathway. However, it remains unknown whether NE affects the release of other inflammatory cytokines such as IL-1β and IL-10 and, if so, whether α2-AR is also involved in such a process. To study this, a branch of the portal vein in normal adult male rats was cannulated under anesthesia. NE (20 μM in ascorbate saline), NE plus yohimbine (YHB, a specific α2-AR antagonist, 1 mM) or vehicle (0.1% ascorbate saline) was infused at a rate of 13 μl/min for 2 h. The above rate of NE infusion was used to increase the portal level of NE to approximately 20 nM, similar to that observed in sepsis. Blood samples were then collected and serum levels of IL-1β and IL-10 were measured. In addition, the KC was isolated from normal rats and stimulated with either NE (20 nM) or NE plus YHB (1 μM). The gene expression of IL-1β and IL-10 in KC and their supernatant levels were assessed. The results indicate that serum levels of IL-1β and IL-10 increased significantly after the intraportal infusion of NE. Co-administration of NE and YHB, however, significantly attenuated IL-1β and IL-10 production. Similarly, IL-1β and IL-10 gene expression and release from KC were up-regulated by NE stimulation, whereas YHB attenuated both cytokines. Thus, gut-derived NE up-regulates IL-1β and IL-10 expression and release in the liver through an α2-AR pathway. Since adenylate cyclase activator forskolin prevents the increase in NE-induced IL-1β and IL-10, the up-regulatory effect of NE on those cytokines appears to be mediated, at least in part, by inhibition of adenylate cyclase and reduction in intracellular cyclic AMP levels.
Keywords: Catecholamine; Small intestine; IL-1β; IL-10; IL-6; α2-Adrenoceptor; Yohimbine; Kupffer cell;

The presence of leukocyte CC-chemokine receptor 2 in CCR2 knockout mice promotes atherogenesis by Jian Guo; Miranda Van Eck; Vivian de Waard; Nobuyo Maeda; G. Martin Benson; Pieter H.E. Groot; Theo J.C. Van Berkel (453-459).
To selectively determine the role of leukocyte CC-chemokine receptor 2 (CCR2) in atherogenesis.Bone marrow progenitor cells harvested from CCR2(+/+) mice were transplanted into irradiated CCR2(−/−) mice, representing the whole-body absence of CCR2 except in leukocytes. Transplantation of CCR2(−/−) bone marrow into CCR2(−/−) mice served as control. Eight weeks after bone marrow transplantation, the diet of regular chow was switched to a high-cholesterol diet for another 10 weeks in order to induce atherosclerosis. No significant differences in serum cholesterol and triglyceride levels were observed between the two groups. However, the mean cross-sectional aortic root lesion area of CCR2(+/+)→CCR2(−/−) mice amounted up to 12.28±3.28×104 μm2, compared with only 3.08±0.74×104 μm2 observed in the CCR2(−/−)→CCR2(−/−) group. Thus, the presence of CCR2 exclusively on leukocytes induces a fourfold increase in aortic lesion area. This extent of lesion development was comparable to C57Bl/6 mice receiving CCR2(+/+) bone marrow (10.08±3.30×104 μm2).These results point at a dominant role of leukocyte CCR2 in atherogenesis, implying that CCR2 from nonleukocyte sources, like endothelial cells or smooth muscle cells, is less critical in the initiation of atherosclerosis. Pharmacological inhibition of leukocyte CCR2 function might be a promising strategy to prevent atherosclerosis.
Keywords: CCR2; Chemokine receptor; Bone marrow transplantation; Atherosclerosis;

Propionic and methylmalonic acids increase cAMP levels in slices of cerebral cortex of young rats via adrenergic and glutamatergic mechanisms by Samanta Oliveira Loureiro; Priscila de Lima Pelaez; Luana Heimfarth; Diogo Onofre Souza; Moacir Wajner; Regina Pessoa-Pureur (460-466).
We have previously described that propionic (PA) and methylmalonic (MMA) acids increased the in vitro phosphorylation of cytoskeletal proteins through cAMP-dependent protein kinase and glutamate. In the present study we investigated the in vitro effects of 1 mM glutamate, 2.5 mM MMA and 2.5 mM PA on cAMP levels in the slices of cerebral cortex of young rats. Results showed that PA, MMA and glutamate increased cAMP levels after 30 min of incubation, while the β-adrenergic agonist epinephrine elicited a similar effect only at a shorter incubation time. Then effects were prevented by the β-adrenergic antagonist propranolol, rather than by glutamate antagonists (AP5, CNQX and MCPG), suggesting that they were mediated by β-adrenergic receptors. In addition, glutamate antagonists per se induced increased cAMP levels; however propranolol prevented only the effect elicited by the metabotropic glutamate antagonist MCPG. Taken together, it is feasible that PA and MMA increase cAMP synthesis via a β-adrenergic/G protein coupled pathway, in a glutamate-dependent manner. Although additional studies will be necessary to evaluate the importance of these observations for the neuropathology of propionic and methylmalonic acidemias, it is possible that high brain cAMP levels may contribute to a certain extent to the neurological dysfunction of the affected individuals.
Keywords: Propionic acid; Methylmalonic acid; cAMP; β-Adrenergic receptor; Glutamate;

Glucose phosphate isomerase deficiency: enzymatic and familial characterization of Arg346His mutation by Ada Repiso; Baldomero Oliva; Joan-Lluis Vives Corrons; Josep Carreras; Fernando Climent (467-471).
Homozygous glucose phosphate isomerase (GPI) deficiency is one of the most important genetic disorders responsible for chronic non-spherocytic hemolytic anemia (CNSHA), a red blood cell autosomal recessive genetic disorder which causes severe metabolic alterations. In this work, we studied a patient with CNSHA due to an 82% loss of GPI activity resulting from the homozygous missense replacement in cDNA position 1040G>A, which leads to substitution of the protein residue A346H mutation. The enzyme is present in a dimeric form necessary for normal activity; the A346H mutation causes a loss of GPI capability to dimerize, which renders the enzyme more susceptible to thermolability and produces significant changes in erythrocyte metabolism.
Keywords: Glucose phosphate isomerase; GPI; Deficiency; Mutation; Hemolytic anemia;

Effect of quinones on microtubule polymerization: a link between oxidative stress and cytoskeletal alterations in Alzheimer's disease by Ismael Santa-María; Mark A. Smith; George Perry; Félix Hernández; Jesús Avila; Francisco J. Moreno (472-480).
Increases in the concentration of quinones, such as benzoquinone, in pathological processes mediated by oxidative imbalance play a role in the disorganization and disassembly of the microtubule network in both non-neural and neural cells. In this study, we show that the effects on microtubules appear to be a direct result of the action of the quinones on tubulin, the main component of microtubules, since tubulin modification by quinones, including benzoquinone and juglone, leads to aggregation into dimers and other oligomers. Therefore, quinones and quinone-mediated effects provide a mechanistic link between oxidative stress, microtubule disruption, neuronal dysfunction and death, i.e., key salient feature of Alzheimer's disease.
Keywords: Quinone; Tubulin; Microtubule; Alzheimer's disease; Oxidative damage;

We show that rat neonatal cardiac myocytes exposed to 1 μmol/l of the calcium ionophore A23187 respond with an enhanced production of reactive oxygen species (ROS). This dose is not cytotoxic to the myocytes. A higher concentration (10 μmol/l) evokes less ROS production and is significantly cytotoxic 24 h after exposure, but not immediately after removal of the A23187, when ROS are measured. Both cell death and the decrease in mitochondrial potential are only partially sensitive to MPT inhibitor cyclosporin A. Experiments performed to elucidate the sources of ROS included use of the nitric oxide synthase (NOS) inhibitor L-NAME; NOS involvement was excluded. Experiments with the oxidative phosphorylation uncoupler CCCP revealed that mitochondria are at least partially responsible for the observed effect. Further studies with cyclooxygenase (COX) and lipoxygenase (LOX) inhibitors (indomethacin and MK886, respectively) showed that these enzymes could also be sources of ROS when the calcium level is elevated. Their effect appeared to be independent of phospholipase A2 inhibition, suggesting that COX and LOX stimulation is not due to elevated substrate (arachidonic acid) concentration but rather to a direct effect of calcium.
Keywords: Calcium; Reactive oxygen species; Cardiac myocyte; Ischemia and reperfusion;

Towards a model to explain the intragenic complementation in the heteromultimeric protein propionyl-CoA carboxylase by Pilar Rodríguez-Pombo; Celia Pérez-Cerdá; Belén Pérez; Lourdes R. Desviat; Luis Sánchez-Pulido; Magdalena Ugarte (489-498).
Mutations in the PCCA or PCCB genes coding for α and β subunits of propionyl CoA carboxylase can cause propionic acidemia. To understand the molecular basis of the intragenic complementation previously reported at the PCCB locus, we now examine the complementation behaviour of four carboxy-terminal and 11 amino-terminal naturally occurring mutant alleles both using cell fusion and reconstructing the complementation event by transfecting the mutant cDNAs to generate multimeric hybrid proteins. Alleles carrying mutations p.R410W and p.W531X are able to complement with 10 out of 11 amino-terminal mutations assayed. Only the unstable p.R512C, p.L519P and p.G112D mutants fail to complement. The results analyzed in the framework of the crystal structure of the homologous 12S transcarboxylase from Propionibacterium shermanii show that all mutant alleles studied are located at β subunits interfaces, complementing alleles at the inter-trimer interface, where the catalysis probably happens, and non-complementing alleles at the intra-trimer interface, probably disrupting the trimer formation. Our results also show a remarkable stabilization effect when p.R410W is cotransfected with p.G246V. We propose a model for intragenic complementation requiring the production of two different β subunits carrying carboxy and amino-terminal mutations that allow regenerating functional active sites and in which a stabilization effect between subunits could be relevant to ameliorate the biochemical phenotype of each mutation separately.
Keywords: Intragenic complementation; Propionyl-CoA carboxylase; Somatic cell complementation; Thermal stability; PCCB; 12S transcarboxylase crystal structure; Propionic acidemia;

To determine the temporal changes in oxidative stress, mitogen-activated protein (MAP) kinases and mitochondrial apoptotic proteins, and their relationship to myocyte apoptosis in the remote noninfarcted myocardium after myocardial infarction (MI), rabbits were randomly assigned to either coronary artery ligation to produce MI or sham operation. The animals were sacrificed at 1, 4, 8, or 12 weeks after coronary artery occlusion. Sham rabbits were sacrificed at 12 weeks after surgery. MI rabbits exhibited progressive increases of left ventricular (LV) end-diastolic pressure and end-diastolic dimension, and progressive decreases of LV fractional shortening and dP/dt over 12 weeks. The LV remodeling with LV chamber dilation and LV systolic dysfunction was temporally associated with progressive increases of cardiac oxidative stress as evidenced by decreased myocardial reduced-to-oxidized-glutathione ratio and increased myocardial 8-hydroxydeoxyguanosine and myocyte apoptosis. The ERK and JNK activities were decreased while p38 MAP kinase activity was increased with age of MI. The extent of p38 MAP kinase activation correlated with Bcl-2 phosphorylation. Bcl-2 protein was decreased in both mitochondrial and cytosolic fractions with age of MI. Bax protein was increased in both mitochondrial and cytosolic fractions. Cytochrome c was reduced in mitochondrial fraction and increased in cytosolic fraction in a time-dependent manner after MI. Cleaved caspase 9 and caspase 3 proteins were time-dependently increased after MI. These data suggest that p38 MAP kinase activation is not only time-dependent after MI, but also correlates with oxidative stress, Bcl-2 phosphorylation, and myocyte apoptosis. These changes in the remote noninfarcted myocardium may contribute to LV remodeling and dysfunction after MI.
Keywords: Apoptosis; Infarction; MAP kinase; Mitochondria; Oxygen radical;

Cumulative Contents (514-517).