Myocardial Mitochondrial and Contractile Function Are Preserved in Mice Lacking Adiponectin

Adiponectin deficiency leads to increased myocardial infarct size following ischemia reperfusion and to exaggerated cardiac hypertrophy following pressure overload, entities that are causally linked to mitochondrial dysfunction. In skeletal muscle, lack of adiponectin results in impaired mitochondrial function. Thus, it was our objective to investigate whether adiponectin deficiency impairs mitochondrial energetics in the heart. At 8 weeks of age, heart weight-to-body weight ratios were not different between adiponectin knockout (ADQ^-/-) mice and wildtypes (WT). In isolated working hearts, cardiac output, aortic developed pressure and cardiac power were preserved in ADQ^-/- mice. Rates of fatty acid oxidation, glucose oxidation and glycolysis were unchanged between groups. While myocardial oxygen consumption was slightly reduced (-24%) in ADQ^-/- mice in isolated working hearts, rates of maximal ADP-stimulated mitochondrial oxygen consumption and ATP synthesis in saponin-permeabilized cardiac fibers were preserved in ADQ^-/- mice with glutamate, pyruvate or palmitoyl-carnitine as a substrate. In addition, enzymatic activity of respiratory complexes I and II was unchanged between groups. Phosphorylation of AMP-activated protein kinase and SIRT1 activity were not decreased, expression and acetylation of PGC-1α were unchanged, and mitochondrial content of OXPHOS subunits was not decreased in ADQ^-/- mice. Finally, increasing energy demands due to prolonged subcutaneous infusion of isoproterenol did not differentially affect cardiac contractility or mitochondrial function in ADQ^-/- mice compared to WT. Thus, mitochondrial and contractile function are preserved in hearts of mice lacking adiponectin, suggesting that adiponectin may be expendable in the regulation of mitochondrial energetics and contractile function in the heart under non-pathological conditions.     

Alcohol Dehydrogenase Accentuates Ethanol-Induced Myocardial Dysfunction and Mitochondrial Damage in Mice: Role of Mitochondrial Death Pathway

Objectives

Binge drinking and alcohol toxicity are often associated with myocardial dysfunction possibly due to accumulation of the ethanol metabolite acetaldehyde although the underlying mechanism is unknown. This study was designed to examine the impact of accelerated ethanol metabolism on myocardial contractility, mitochondrial function and apoptosis using a murine model of cardiac-specific overexpression of alcohol dehydrogenase (ADH).

Methods

ADH and wild-type FVB mice were acutely challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Myocardial contractility, mitochondrial damage and apoptosis (death receptor and mitochondrial pathways) were examined.

Results

Ethanol led to reduced cardiac contractility, enlarged cardiomyocyte, mitochondrial damage and apoptosis, the effects of which were exaggerated by ADH transgene. In particular, ADH exacerbated mitochondrial dysfunction manifested as decreased mitochondrial membrane potential and accumulation of mitochondrial O₂^•−. Myocardium from ethanol-treated mice displayed enhanced Bax, Caspase-3 and decreased Bcl-2 expression, the effect of which with the exception of Caspase-3 was augmented by ADH. ADH accentuated ethanol-induced increase in the mitochondrial death domain components pro-caspase-9 and cytochrome C in the cytoplasm. Neither ethanol nor ADH affected the expression of ANP, total pro-caspase-9, cytosolic and total pro-caspase-8, TNF-α, Fas receptor, Fas L and cytosolic AIF.

Conclusions

Taken together, these data suggest that enhanced acetaldehyde production through ADH overexpression following acute ethanol exposure exacerbated ethanol-induced myocardial contractile dysfunction, cardiomyocyte enlargement, mitochondrial damage and apoptosis, indicating a pivotal role of ADH in ethanol-induced cardiac dysfunction possibly through mitochondrial death pathway of apoptosis.     

Selenium and Vitamin E Modulates Cigarette Smoke Exposure-Induced Oxidative Stress in Blood of Rats

Cigarette smoke contains about 5,000 chemicals that include organic and metallic compounds. The current study was undertaken to investigate the effects of selenium and vitamin E on oxidative stress-induced damage in rats exposed to cigarette smoke. Forty male rats were equally divided into four groups. The first and second groups were used as control and cigarette smoke groups, respectively. Selenium was administered to rats constituting the third group for 27 days. The Se and vitamin E combination was given to animals in fourth group for 27 days. All groups except the control, were exposed to cigarette smoke starting at the third day of the experiment and continuing for 27 days. The blood samples from all groups were taken at the end of 27 days. Plasma lipid peroxidation, triacylglycerol, and total cholesterol levels were higher in the cigarette smoke group than in the control, although erythrocytic superoxide dismutase and glutathione peroxidase activities were lower in the cigarette smoke group than in the control. The plasma lipid peroxidation, triacylglycerol, and total cholesterol levels were lower in cigarette smoke+Se+VE group than in the cigarette smoke group, although erythrocytic superoxide dismutase activity and glutathione peroxidase activity in selenium and vitamin E-administered groups were higher than in the exposed to cigarette smoke group. High-density lipoprotein-cholesterol level was not affect by selenium and vitamin E administrations. In conclusion, selenium and vitamin E seem to have protective effects on the cigarette smoke-induced blood toxicity by supporting the enzymatic antioxidant redox systems.     

Overexpression of Mitochondrial Leishmania major Ascorbate Peroxidase Enhances Tolerance to Oxidative Stress-Induced Programmed Cell Death and Protein Damage

Ascorbate peroxidase from Leishmania major (LmAPX) is one of the key enzymes for scavenging of reactive oxygen species generated from the mitochondrial respiratory chain. We have investigated whether mitochondrial LmAPX has any role in oxidative stress-induced apoptosis. The measurement of reduced glutathione (GSH) and protein carbonyl contents in cellular homogenates indicates that overexpression of LmAPX protects Leishmania cells against depletion of GSH and oxidative damage of proteins by H₂O₂ or camptothecin (CPT) treatment. Confocal microscopy and fluorescence spectroscopy data have revealed that the intracellular elevation of Ca²⁺ attained by the LmAPX-overexpressing cells was always below that attained in control cells. Flow cytometry assay data and confocal microscopy observation strongly suggest that LmAPX overexpression protects cells from H₂O₂-induced mitochondrial membrane depolarization as well as ATP decrease. Western blot data suggest that overexpression of LmAPX shields against H₂O₂- or CPT-induced cytochrome c and endonuclease G release from mitochondria and subsequently their accumulation in the cytoplasm. Caspase activity assay by flow cytometry shows a lower level of caspase-like protease activity in LmAPX-overexpressing cells under apoptotic stimuli. The data on phosphatidylserine exposed on the cell surface and DNA fragmentation results show that overexpression of LmAPX renders the Leishmania cells more resistant to apoptosis provoked by H₂O₂ or CPT treatment. Taken together, these results indicate that constitutive overexpression of LmAPX in the mitochondria of L. major prevents cells from the deleterious effects of oxidative stress, that is, mitochondrial dysfunction and cellular death.In multicellular organisms, mitochondria are the major physiological source of reactive oxygen species (ROS) within cells and also are important checkpoints for the control of programmed cell death (27). There are increasing numbers of reports that describe apoptosis- or programmed cell death-like processes in unicellular organisms also, such as trypanosomatids (4, 60), bacteria (20, 25), yeasts (34), and Plasmodium (3). Among the kinetoplastid parasites, Trypanosoma and Leishmania are the most carefully studied genera where apoptotic features are well established (49). Several reports have shown that mitochondrial dysfunction or an imbalance of antioxidant homeostasis causes an increase in mitochondrion-generated ROS, which include H₂O₂, superoxide radical anions, singlet oxygen, and hydroxyl radicals. These species have all been implicated in apoptosis (16, 26, 28, 41). Increasing evidence has been presented to support that ROS homeostasis regulates two major types of important physiological processes and exerts diverse functions within cells. One type of function includes damage or oxidation of cellular macromolecules (DNA, proteins, and lipids), which can lead to necrotic cell death or protein modification (7). The second type of function includes the activation of cellular signaling cascades that regulate proliferation, detoxification, DNA repair, or apoptosis (11). The detoxification of toxic mitochondrial ROS in cells occurs through a variety of cellular antioxidant enzymes, such as superoxide dismutase, which detoxifies cells from superoxide released into the mitochondrial matrix, and several other antioxidant proteins, such as catalase, glutathione (GSH) peroxidase, and peroxiredoxins, which are known to catalyze further degradation of H₂O₂ (44). During its life cycle, the Leishmania sp. encounters a pool of ROS that is generated either by its own physiological processes or as a result of host immune reaction and drug metabolism. However, unlike most eukaryotes, Leishmania lacks catalase- and selenium-containing GSH peroxidases, enzymes that play a front-line role in detoxifying ROS. Hence, the mechanism by which it resists the toxic effects of H₂O₂ remains poorly understood.Recently, we cloned, expressed and characterized the unusual heme-containing ascorbate peroxidase from Leishmania major (LmAPX) and observed that the expression of LmAPX is increased when Leishmania cells are treated with exogenous H₂O₂ (1, 18). This enzyme is a functional hybrid between cytochrome c peroxidase and APX, owing to its ability to use both ascorbate and cytochrome c as reducing electron donors (58). Colocalization studies by confocal microscopy, submitochondrial fractionation analysis of the isolated mitochondria, and subsequent Western blot analysis with anti-LmAPX antibody have confirmed that the mature enzyme is present in intermembrane space side of the inner membrane. It has also been shown that overexpression of LmAPX causes a decrease in the mitochondrial ROS burden, an increase in tolerance to H₂O₂, and protection against cardiolipin oxidation under oxidative stress (18). Although previous studies have shown that Leishmania species use superoxide dismutase (23), peroxiredoxins (8), intracellular thiols (14), lipophosphoglycan (13), trypanothione (5), HSP 70 (a heat shock protein) (36), tryparedoxin peroxidase (29), and APX (18) for detoxification of ROS, it is still unclear how the antioxidants protect against oxidative stress-induced apoptotic events in the unicellular organism Leishmania.Since the LmAPX protein is localized in the mitochondria, we hypothesized that it would be a key protein for the maintenance of mitochondrial functions due to its antioxidant properties via its ROS-scavenging function (18). To test this hypothesis, we overexpressed LmAPX in Leishmania major cells and investigated whether overexpression of LmAPX can confer resistance to oxidant-mediated mitochondrial damage as well as oxidative stress-induced cell death. In this study, we provide evidence that the overexpression of LmAPX in Leishmania cells can indeed protect against camptothecin (CPT) or H₂O₂-mediated mitochondrial damage as measured by various parameters, including disruption of mitochondrial membrane potential (Δψ_m), decrease of ATP production, and cytochrome c and endonuclease G release from mitochondria. Cells overexpressing LmAPX were also protected against oxidative stress-induced protein carbonylation, DNA fragmentation, and apoptosis. To the best of our knowledge, this is the first report of a mitochondrial hemeperoxidase that controls the ROS-induced mitochondrial death pathway.     

Myocardial Overexpression of Mecr,a Gene of Mitochondrial FAS II Leads to Cardiac Dysfunction in Mouse

It has been recently recognized that mammalian mitochondria contain most, if not all, of the components of fatty acid synthesis type II (FAS II). Among the components identified is 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (Etr1/Mecr), which catalyzes the NADPH-dependent reduction of trans-2-enoyl thioesters, generating saturated acyl-groups. Although the FAS type II pathway is highly conserved, its physiological role in fatty acid synthesis, which apparently occurs simultaneously with breakdown of fatty acids in the same subcellular compartment in mammals, has remained an enigma. To study the in vivo function of the mitochondrial FAS in mammals, with special reference to Mecr, we generated mice overexpressing Mecr under control of the mouse metallothionein-1 promoter. These Mecr transgenic mice developed cardiac abnormalities as demonstrated by echocardiography in vivo, heart perfusion ex vivo, and electron microscopy in situ. Moreover, the Mecr transgenic mice showed decreased performance in endurance exercise testing. Our results showed a ventricular dilatation behind impaired heart function upon Mecr overexpression, concurrent with appearance of dysmorphic mitochondria. Furthermore, the data suggested that inappropriate expression of genes of FAS II can result in the development of hereditary cardiomyopathy.     

Overexpression of Mitochondrial Sirtuins Alters Glycolysis and Mitochondrial Function in HEK293 Cells

SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.     

Practice and Attitude of Cigarette Smoking: A Community-Based Study

Background

In Saudi Arabia many studies have addressed cigarette smoking from various perspectives. Most of these studies, however, were conducted among males and confined to Riyadh, the capital city. Such limitations have enhanced the need for community-based epidemiological studies that include both genders and various age groups and socio-demographic features, as well as different regions.

Objective

This cross-sectional study aims to assess the prevalence of cigarette smoking and to discuss the association between cigarette smoking habits and socio-demographic factors among community members of the Jazan area in southwest Saudi Arabia.

Methods

A pre-coded questionnaire was designed and tested for data consistency. A well-trained health team was assigned to gather the data from the 30 primary healthcare centers distributed across eight provinces. The response rate was 92.8% (4,326 respondents ≥13 years old). The associations among the subjects'' socio-demographic characteristics were examined by the chi-square test. A multiple logistic regression and odds ratios were calculated as well.

Results

A total of 1,017 (23.5%), 1,042 (24.1%), and 3,284 (75.9%) respondents were, respectively, current smokers (TCS), ever-smokers (TES), and non-smokers (TNS). Though current smokers seem to be more prevalent in urban populations (13.8%) than in rural populations (9.7%), the association of urbanization with a current smoking habit is insignificant.

Conclusion

Having fun, relieving stress, and the influence of parents, particularly of mothers, were the main motives that encouraged participants'' cigarette-smoking habits. This situation was worsened by the fact that accessing cigarettes was either very easy or easy for over 90% of the respondents.     

Genome-Wide and Candidate Gene Association Study of Cigarette Smoking Behaviors

The contribution of common genetic variation to one or more established smoking behaviors was investigated in a joint analysis of two genome wide association studies (GWAS) performed as part of the Cancer Genetic Markers of Susceptibility (CGEMS) project in 2,329 men from the Prostate, Lung, Colon and Ovarian (PLCO) Trial, and 2,282 women from the Nurses'' Health Study (NHS). We analyzed seven measures of smoking behavior, four continuous (cigarettes per day [CPD], age at initiation of smoking, duration of smoking, and pack years), and three binary (ever versus never smoking, ≤10 versus >10 cigarettes per day [CPDBI], and current versus former smoking). Association testing for each single nucleotide polymorphism (SNP) was conducted by study and adjusted for age, cohabitation/marital status, education, site, and principal components of population substructure. None of the SNPs achieved genome-wide significance (p<10⁻⁷) in any combined analysis pooling evidence for association across the two studies; we observed between two and seven SNPs with p<10⁻⁵ for each of the seven measures. In the chr15q25.1 region spanning the nicotinic receptors CHRNA3 and CHRNA5, we identified multiple SNPs associated with CPD (p<10⁻³), including rs1051730, which has been associated with nicotine dependence, smoking intensity and lung cancer risk. In parallel, we selected 11,199 SNPs drawn from 359 a priori candidate genes and performed individual-gene and gene-group analyses. After adjusting for multiple tests conducted within each gene, we identified between two and five genes associated with each measure of smoking behavior. Besides CHRNA3 and CHRNA5, MAOA was associated with CPDBI (gene-level p<5.4×10⁻⁵), our analysis provides independent replication of the association between the chr15q25.1 region and smoking intensity and data for multiple other loci associated with smoking behavior that merit further follow-up.     

Cigarette Smoking and the Risk of Adult Myeloid Disease: A Meta-Analysis

Background

The adult myeloid diseases, myelodysplastic syndrome and acute myeloid leukemia, have been reported to be associated with cigarette smoking, but the results have been conflicting. Previous studies may have ignored the relationship between myelodysplastic syndrome and acute myeloid leukemia, where approximately one-third of myelodysplastic syndrome cases will progress to acute myeloid leukemia, which could induce a serious bias in independent analyses. For the purposes of researching pathogenesis, we suggest that myelodysplastic syndrome and acute myeloid leukemia should be regarded as a single class of adult myeloid disease, and herein assessed the relationship between cigarette smoking and the risk of adult myeloid disease.

Methods

The PubMed, Cochrane Library, EBSCO, and EMBASE databases were systematically searched for reports published from 1990 to 2015. Two authors independently assessed the methodological quality and the extracted data. The odds ratios and adjusted odds ratios (OR), a sensitivity analysis, and the publication bias were analyzed using the CMA v2 (Comprehensive Meta Analysis Version 2) software program.

Results

Twenty-five studies were included in this meta-analysis. The publication dates ranged from 1990 to 2014. The pooled OR in current smokers and ever-smokers showed an increased risk of adult myeloid disease, with ORs of 1.45 (95% CI, 1.30–1.62; p<0.001) and 1.23 (95% CI 1.15–1.32; p<0.001) versus non-smokers, respectively. In the subset analyses, the OR of adult myeloid disease was increased regardless of the form of disease, geographical region, NOS (Newcastle Ottawa Scale) score, and source of controls. The smoking status was divided into <20 and ≥20 cigarettes per day, and these groups had ORs of developing adult myeloid disease of 1.24 (95% CI, 1.09–1.40; p = 0.001) and 1.32 (95% CI, 1.14–1.53; p<0.001), respectively. In the groups divided based on the number of years the subjects had smoked (<20 and ≥20 years), the ORs were 1.05 (95% CI, 0.90–1.23; p = 0.25) and 1.30 (95% CI, 1.16–1.45; p<0.001), respectively. Similarly, <20 and ≥20 pack-years were associated with ORs of 1.15 (95% CI, 1.03–1.29; p = 0.017) and 1.34 (95% CI, 1.18–1.52; p<0.001), respectively.

Conclusions

This meta-analysis, for the first time, combined myelodysplastic syndrome with acute myeloid leukemia to assess the overall risk of adult myeloid disease, and it demonstrated that cigarette smoking is associated with a significantly increased risk of adult myeloid disease.     

A Meta-Analysis of the Relationship between Cigarette Smoking and Incidence of Myelodysplastic Syndromes

Background

In recent years, epidemiologic studies have reported controversial results relating cigarette smoking to myelodysplastic syndromes (MDS) risk. A meta-analysis was performed to assess such potential relationship between cigarette smoking and incidence of MDS.

Methods

A search of literature published before October 2012 for observational studies evaluating the association between cigarette smoking and MDS, returned 123 articles and of these, 14 were selected for this study. The outcomes from these studies were calculated and reported as odds ratios (OR). Quality assessments were performed with the Newcastle-Ottawa Scale. Heterogeneity was evaluated by the I² index and source of heterogeneity was detected by sensitivity analyses. Finally, publication bias was assessed through visual inspection of funnel plots and Egger’s test.

Results

The pooled OR of developing MDS in ever-smokers was 1.45 (95% CI, 1.25 to 1.68) versus non-smokers. Current and former smokers had increased risks of MDS, with ORs of 1.81 (95% CI, 1.24 to 2.66) and 1.67 (95% CI, 1.42 to 1.96), respectively. In subset analyses, ever-smokers had increased risks of developing MDS if they were living in the United States, or in Europe, female in gender, had refractory anemia (RA)/RA with ringed sideroblasts (RARS) or RA with excess blasts (RAEB)/RAEB in transformation (RAEBt), respectively. Our results demonstrated that the association was stronger in individuals who smoked ≥20 cigarettes/day (OR, 1.62; 95% CI, 1.03 to 2.55) versus those who smoked <20 cigarettes/day (OR, 1.36; 95% CI, 1.13 to 1.64). Moreover, individuals who smoked more than 20 pack-years had increased MDS risk (OR, 1.94; 95% CI, 1.29 to 2.92).

Conclusion

Our outcomes show that smoking increases the risk of developing MDS in ever-smokers who are current or former smokers. We also demonstrate here that positive association between cigarette smoking and risk of MDS exists, and occurs in a dose-dependent manner.     

Oxidative Damage and Fragmentation of Mitochondrial DNA in Cellular Apoptosis

The molecular genetics and bioenergetics of oxidative damage, fragmentation, and fragility of mitochondrial DNA in cellular apoptosis is reviewed in connection with the redox mechanism of ageing.     

Cigarette Smoking and Risk of Breast Cancer in a New Zealand Multi-Ethnic Case-Control Study

Background

The association between breast cancer and tobacco smoke is currently unclear. The aim of this study was to assess the effect of smoking behaviours on the risk of breast cancer among three ethnic groups of New Zealand women.

Methods

A population-based case-control study was conducted including breast cancer cases registered on the New Zealand Cancer Registry between 2005 and 2007. Controls were matched by ethnicity and 5-year age-group. Logistic regression was used to estimate the association between breast cancer and smoking at different time points across the lifecourse, for each ethnic group. Estimated odds ratios (OR) were adjusted for established risk factors.

Results

The study comprised 1,799 cases (302 Māori, 70 Pacific, 1,427 non-Māori/non-Pacific) and 2,540 controls (746 Māori, 191 Pacific, 1,603 non-Māori/non-Pacific). There was no clear association between smoking and breast cancer for non-Māori/non-Pacific women, although non-Māori/non-Pacific ex-smokers had statistically significant increased risk of breast cancer when smoking duration was 20 years or more, and this remained significant in the fully adjusted model (OR 1.31, 95% CI 1.03 to 1.66). Māori showed more consistent increased risk of breast cancer with increasing duration among current smokers (<20 years OR 1.61, 95% CI 0.55 to 4.74; 20+ years OR 2.03, 95% CI 1.29 to 3.22). There was a clear pattern of shorter duration since smoking cessation being associated with increased likelihood of breast cancer, and this was apparent for all ethnic groups.

Conclusion

There was no clear pattern for cigarette smoking and breast cancer incidence in non-Māori/non-Pacific women, but increased risks were observed for Māori and Pacific women. These findings suggest that lowering the prevalence of smoking, especially among Māori and Pacific women, could be important for reducing breast cancer incidence.     

An Anthelmintic Drug,Pyrvinium Pamoate,Thwarts Fibrosis and Ameliorates Myocardial Contractile Dysfunction in a Mouse Model of Myocardial Infarction

Metabolic adaptation to limited supplies of oxygen and nutrients plays a pivotal role in health and disease. Heart attack results from insufficient delivery of oxygen and nutrients to the heart, where cardiomyocytes die and cardiac fibroblasts proliferate – the latter causing scar formation, which impedes regeneration and impairs contractility of the heart. We postulated that cardiac fibroblasts survive metabolic stress by adapting their intracellular metabolism to low oxygen and nutrients, and impeding this metabolic adaptation would thwart their survival and facilitate the repair of scarred heart. Herein, we show that an anthelmintic drug, Pyrvinium pamoate, which has been previously shown to compromise cancer cell survival under glucose starvation condition, also disables cardiac fibroblast survival specifically under glucose deficient condition. Furthermore, Pyrvinium pamoate reduces scar formation and improves cardiac contractility in a mouse model of myocardial infarction. As Pyrvinium pamoate is an FDA-approved drug, our results suggest a therapeutic use of this or other related drugs to repair scarred heart and possibly other organs.     

Influence of Smoking Procedures on Combustion Temperature of Cigarettes and the Nicotine Content of Cigarette Smoke

In order to establish fundamental knowledge on the combustion mechanism of tobacco, the effects of smoking procedures on both combustion temperature of cigarettes and the amount of nicotine transferred into cigarette smoke were investigated. The combustion temperatures measured with fine thermocouples specially devised, and an excellent responsive autorecording potentiometer, were 794~827°C, irrespective of smoking procedures. The free burning temperature obtained was 746°C.

When the cigarette was smoked up to a definite length (45 mm) from the lighted end, the amount of nicotine in cigarette smoke increased with increment in puff velocity, showing almost a linear curve.     

MitoQ Blunts Mitochondrial and Renal Damage during Cold Preservation of Porcine Kidneys

Cold preservation has greatly facilitated the use of cadaveric kidneys for transplantation but damage occurs during the preservation episode. It is well established that oxidant production increases during cold renal preservation and mitochondria are a key target for injury. Our laboratory has demonstrated that cold storage of renal cells and rat kidneys leads to increased mitochondrial superoxide levels and mitochondrial electron transport chain damage, and that addition of Mitoquinone (MitoQ) to the preservation solutions blunted this injury. In order to better translate animal studies, the inclusion of large animal models is necessary to develop safe preclinical protocols. Therefore, we tested the hypothesis that addition of MitoQ to cold storage solution preserves mitochondrial function by decreasing oxidative stress, leading to less renal tubular damage during cold preservation of porcine kidneys employing a standard criteria donor model. Results showed that cold storage significantly induced oxidative stress (nitrotyrosine), renal tubular damage, and cell death. Using High Resolution Respirometry and fresh porcine kidney biopsies to assess mitochondrial function we showed that MitoQ significantly improved complex II/III respiration of the electron transport chain following 24 hours of cold storage. In addition, MitoQ blunted oxidative stress, renal tubular damage, and cell death after 48 hours. These results suggested that MitoQ decreased oxidative stress, tubular damage and cell death by improving mitochondrial function during cold storage. Therefore this compound should be considered as an integral part of organ preservation solution prior to transplantation.     

SUMOylation of Nuclear γ-Actin by SUMO2 supports DNA Damage Repair against Myocardial Ischemia-Reperfusion Injury

Myocardial infarction triggers oxidative DNA damage, apoptosis and adverse cardiac remodeling in the heart. Small ubiquitin-like modifier (SUMO) proteins mediate post-translational SUMOylation of the cardiac proteins in response to oxidative stress signals. Upregulation of isoform SUMO2 could attenuate myocardial injury via increasing protein SUMOylation. The present study aimed to discover the identity and cardioprotective activities of SUMOylated proteins. A plasmid vector for expressing N-Strep-SUMO2 protein was generated and introduced into H9c2 rat cardiomyocytes. The SUMOylated proteins were isolated with Strep-Tactin^® agarose beads and identified by MALDI-TOF-MS technology. As a result, γ-actin was identified from a predominant protein band of ~42 kDa and verified by Western blotting. The roles of SUMO2 and γ-actin SUMOylation were subsequently determined in a mouse model of myocardial infarction induced by ligating left anterior descending coronary artery and H9c2 cells challenged by hypoxia-reoxygenation. In vitro lentiviral-mediated SUMO2 expression in H9c2 cells were used to explore the role of SUMOylation of γ-actin. SUMOylation of γ-actin by SUMO2 was proven to be a new cardioprotective mechanism from the following aspects: 1) SUMO2 overexpression reduced the number of TUNEL positive cells, the levels of 8-OHdG and p-γ-H2ax while promoted the nuclear deposition of γ-actin in mouse model and H9c2 cell model of myocardial infarction; 2) SUMO-2 silencing decreased the levels of nuclear γ-actin and SUMOylation while exacerbated DNA damage; 3) Mutated γ-actin (K⁶⁸R/K²⁸⁴R) void of SUMOylation sites failed to protect cardiomyocytes against hypoxia-reoxygenation challenge. The present study suggested that SUMO2 upregulation promoted DNA damage repair and attenuated myocardial injury via increasing SUMOylation of γ-actin in the cell nucleus.     

Dexamethasone Rescues Neurovascular Unit Integrity from Cell Damage Caused by Systemic Administration of Shiga Toxin 2 and Lipopolysaccharide in Mice Motor Cortex

Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) that can lead to fatal encephalopathies. Neurological abnormalities may occur before or after the onset of systemic pathological symptoms and motor disorders are frequently observed in affected patients and in studies with animal models. As Stx2 succeeds in crossing the blood-brain barrier (BBB) and invading the brain parenchyma, it is highly probable that the observed neurological alterations are based on the possibility that the toxin may trigger the impairment of the neurovascular unit and/or cell damage in the parenchyma. Also, lipopolysaccharide (LPS) produced and secreted by enterohemorrhagic Escherichia coli (EHEC) may aggravate the deleterious effects of Stx2 in the brain. Therefore, this study aimed to determine (i) whether Stx2 affects the neurovascular unit and parenchymal cells, (ii) whether the contribution of LPS aggravates these effects, and (iii) whether an inflammatory event underlies the pathophysiological mechanisms that lead to the observed injury. The administration of a sub-lethal dose of Stx2 was employed to study in detail the motor cortex obtained from a translational murine model of encephalopathy. In the present paper we report that Stx2 damaged microvasculature, caused astrocyte reaction and neuronal degeneration, and that this was aggravated by LPS. Dexamethasone, an anti-inflammatory, reversed the pathologic effects and proved to be an important drug in the treatment of acute encephalopathies.