Seit Anfang der Siebzigerjahre werden weltweit in Rinderbetrieben in vielfältigen Variationen und Anwendungen Stoffwechseluntersuchungen durchgeführt. Das Hauptaugenmerk lag dabei stets auf Seiten der Energie- und Mineralstoffversorgung. Die Superoxid-Dismutase (SOD) fand hier bislang keine Berücksichtigung. Ziel der vorliegenden Untersuchung war es, die SOD-Aktivität in Stoffwechsel-untersuchungen bei klinisch gesunden Kühen mit ein zu beziehen sowie den Einfluss von Laktation und Jahreszeit zu prüfen. Versuchsanordnung: Insgesamt wurden bei 125 SB/HF-Kühen (7990 kg fettkorregierte Milch/Jahr) folgende drei Gruppen analysiert: Gruppe 1: Im Verlauf eines Jahres wurden im Abstand von 6 Wochen jeweils 10 gesunde Kühe, die sich alle 1-2 Wochen post partum (pp) befanden, untersucht. Zusätzlich wurden die Stall- und die Außentemperaturen berücksichtigt. Gruppe 2: Zur Kontrolle des Laktationsverlaufes wurden 10 Kühe zum Zeitpunkt 4-5 Wochen ante partum (ap), 1 Woche ap, 1-2 Wochen pp, 4 Wochen pp und 8-12 Wochen pp untersucht. Gruppe 3: Um jahreszeitlich bedingte Schwankungen des Stoffwechsels und der SOD-Aktivität in Gruppe 2 auszuschließen, wurden an einem Entnahmetag jeweils sieben verschiedene Kühe zum Zeitpunkt 4-5 Wochen ap, 1 Woche ap, 1-2 Wochen pp, 4 Wochen pp, und 8-12 Wochen pp geprüft. Die Tiere aller drei Gruppen wurden nach der klinischen Untersuchung hämatologisch sowie klinisch-chemisch (SOD, β-Hydroxybutyrat (BHB), Glucose, Cholesterol, Bilirubin, Glutamat-Dehydrogenase (GLDH), Aspartat-aminotransferase (ASAT), Creatinkinase (CK), Protein, Albumin, Harnstoff, Calcium, anorganisches Phosphat, Magnesium, Natrium, Kalium, Chlorid, Eisen) getestet. Auch die Fütterung fand durch Anfertigung einer Rationsberechnung Berücksichtigung. Ergebnisse: Die Ergebnisse der Stoffwechseluntersuchungen und der SOD-Aktivität im Jahresverlauf (Gruppe 1) deuten auf eine verminderte Futteraufnahme in den Sommermonaten infolge Hitzestresses hin. Vor allem ein Absinken der Harnstoff-, Glucose- (positive Korrelation zur SOD-Aktivität), Phosphat- (positive Korrelation zur SOD-Aktivität) und Cholesterolkonzentrationen bei gleichzeitig ansteigenden BHB- (negative Korrelation zur SOD-Aktivität) und Bilirubinkonzentrationen weisen auf eine negative Energiebilanz in diesen Monaten hin. Die Calcium- und Magnesiumkonzentrationen liegen bei allen Tieren im unteren Referenzbereich. Die SOD-Aktivität klinisch gesunder Kühe im Jahresverlauf beträgt 501 bis 978 U/ml Erythrozytenlysat (Interzentilbereich). In Gruppen 2 und Gruppe 3 sind ebenfalls deutliche Schwankungen der Stoffwechselparameter Protein, Harnstoff, Bilirubin, Glucose, BHB und Cholesterol zu beobachten, wobei die stärksten Schwankungen im Zeitraum 1 Woche ap bis 4 Wochen pp vorhanden sind. Auch dies ist durch die reduzierte Futteraufnahme peripartal sowie steigende Futteraufnahme in der Frühlaktation zu erklären. Die SOD-Aktivität der Kühe aus Gruppe 2 zeigt die entsprechenden Schwankungen mit niedrigen Aktivitäten 4-5 Wochen ap bis 1-2 Wochen pp und einen deutlichen Anstieg bis 4 Wochen pp. Die Calcium- und Magnesiumkonzentrationen befinden sich auch in dieser Gruppe im unteren Referenzbereich. In der Gruppe 3 fällt bei den Kühen 4 Wochen pp eine deutliche Stoffwechselbelastung mit erhöhten Bilirubin- und BHB-Konzentrationen sowie ASAT- und GLDH-Aktivitäten auf. In dieser Gruppe sind die Calcium- und Magnesiumkonzentrationen physiologisch. Die SOD-Aktivitäten differieren nur gering mit einer tendenziell höheren Aktivität 4 Wochen pp. Insgesamt fallen im Vergleich zu Gruppe 1 und 2 deutlich höhere SOD-Aktivitäten zwischen 835 und 1758 U/ml Erythrozytenlysat (Interzentilbereich) auf, welche mit den höheren Calcium- und Magnesiumkonzentrationen in Beziehung stehen können. Schlussfolgerungen: Schwankungen von Stoffwechselparametern im Jahres- und Laktationsverlauf betreffen vor allem fütterungsabhängige Parameter. Die SOD-Aktivität verhält sich entsprechend dieser fütterungsabhängigen Parameter mit niedrigerer Aktivität bei längeren Belastungsphasen sowie im geburtsnahen Zeitraum. Statistisch gesicherte Korrelationen ergaben sich mit folgenden fütterungs-abhängigen Parametern: im Jahresverlauf mit BHB, Glucose und anorganischem Phosphat, im Laktationsverlauf in Gruppe 2 mit BHB, Cholesterol, Protein, Glucose, Magnesium und Milchharnstoff und in Gruppe 3 zu BHB und Magnesium. Die Analyse der SOD-Aktivität bereichert die Informationen bei Stoffwechselkontrollen sinnvoll.

Superoxide dismutase scavenges oxygen radicals, which have been implicated in ischemia/reperfusion (I/R) injury in the heart. Our experiments were designed to study the effect of a moderate increase of copper/zinc superoxide dismutase (CuZnSOD) on myocardial I/R injury in TgN(SOD1)3Cje transgenic mice. A species of 0.8 kb human CuZnSOD mRNA was expressed, and a 273% increase in CuZnSOD activity was detected in the hearts of transgenic mice with no changes in the activities of other antioxidant enzymes. Furthermore, immunoblot analysis revealed no changes in the levels of HSP-70 or HSP-25 levels. Immunocytochemical study indicated that there was increased labeling of CuZnSOD in the cytosolic fractions of both endothelial cells and smooth muscle cells, but not in the myocytes of the hearts from transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x LVDP, was 48 ± 3% in the transgenic hearts as compared to 30 ± 5% in the nontransgenic hearts (p < .05). The improved cardiac function was accompanied by a significant reduction in lactate dehydrogenase release from the transgenic hearts. Our results demonstrate that overexpression of CuZnSOD in coronary vascular cells renders the heart more resistant to I/R injury.

Sponges are the descendants of the oldest members of the metazoan phylogenetic lineage and their genome contains animal specific genes but lack true tissues, organ systems, and neurons. Thus, the sponge model system can be used to elucidate origin of developmental processes. The PSED (RDGN) network (Pax/Six/Eya/Dac) is important in development of eyes, muscles, and other structures in Bilaterians. Similarly, sponges contain a precursor Pax-Six gene network. The Ephydatia muelleri (Em) PaxB protein binds to a Pax2/5/8 consensus sequence site and two cis-regulatory elements upstream and one intron sequence of EmSix1/2 (Rivera et al., 2013). This study aimed to determine if transcription factor EmSix1/2 binds upstream of EmPaxB using gel shift mobility assays, identify other downstream targets of EmSix1/2 using DNA immunoprecipitation, and to identify the recognition sequence of Six in sponges through sequencing. In conclusion, purified EmSix binds to DNA specific fragments (1 and 3), which may contain enhancer sequences located in the PaxB promoter region.Possible consensus recognition sequence of Six in sponges were also identified. The host immune response has various mechanisms to protect the organism from infections and invasions of microorganisms and cell damaging chemicals. One such mechanism is the elimination of reactive oxygen species aided by superoxide dismutase (SOD). A study showed that anti-neutrophil chemotactic factor antibodies recognize Tritrichomonas foetus SOD (Granger et al., 1997). During first casualties, SOD is released and triggers a host immune response. The parasites could use SOD to counter oxidative attacks by the leukocytes and damaged cells to protect surrounding parasites. We used the parasite Trichomonas vaginalis that causes trichomoniasis to determine if SOD acts a neutrophil (or other leukocyte) chemotactic factor in this parasite and characterize the expression and secretions of epithelial cells when treated with SOD6. HeLa cells treated with SOD6 showed an increase in the expression of IL-8 chemokine relative to TrxR treated cells. Scratch test assays showed that SOD may act as a macrophage chemoattractant as compared to TrxR but further tests are needed to confirm this.

Dysregulation of the autonomic cardiovascular control is a complication of diseases including diabetes, hypertension, sleep apnea, and aging. A common factor in these conditions is an increase in reactive oxygen species (ROS) in neural, cardiac, and endothelial tissues. Cu/Zn superoxide dismutase (SOD1) is an intracellular anti-oxidant enzyme that catalyzes dismutation of the superoxide anion (O2.-) to hydrogen peroxide (H2O2). Expression and function of this enzyme are diminished in pathologies that impair cardiovascular autonomic control. This study employed mice overexpressing a transgene for human SOD1 (hSOD1) to determine if its overexpression would alter autonomic regulation of BP, HR, and BRS in healthy animals, and if this animal line (C57B6SJL-Tg (SOD1)2 Gur/J) could be used in future studies to determine if hSOD1 overexpression can preserve cardiac autonomic function in disease models. To accomplish this aim, using anesthetized SOD1 and C57 (control) mice, we recorded HR, and aortic depressor nerve (ADN) activity changes in response to pharmacologically-induced BP changes in order to measure baroreflex and baroreceptor sensitivity, respectively. In order to identify any alterations in central, efferent, and cardiac components of the baroreflex arc, we electrically stimulated the left ADN and left cervical vagus and compared the reductions in BP and HR between the C57 and SOD1 mice. Time- and frequency-domain analysis of heart rate variability (HRV) was performed using pulse pressure recordings prior to pharmacologic or surgical procedures. We found that hSOD1 overexpression in the SOD1 mouse line, in comparison to C57 controls did not significantly affect resting HR (C57: 558 ± 8 vs. SOD1:553 ± 13 beats-per-minute) or blood pressure (C57: 88.8 ± 2.9 vs.SOD1: 85.8 ± 2.1 mmHg). hSOD1 overexpression did not affect the decrease in average mean arterial pressure (MAP) following injection of sodium nitroprusside (SNP) (C57: 38.7 ± 1.4 vs. SOD1: 39.5 ± 1.3 mmHg) or increase in average MAP (C57: 135.8 ± 3.1 vs. SOD1: 136.6 ± 3.5 mmHg) following injection of phenylephrine (PE). BRS, as measured by the averaged regression lines for ΔHR/ΔMAP for the SNP-induced tachycardic baroreflex (C57: 0.57 ± 0.06 bpm/mmHg, SOD1: 0.61 ± 0.08 bpm/mmHg)) and the PE-induced bradycardic baroreflex (C57: -2.9 ± 0.57 bmp/mmHg, SOD1: -4.3 ± 0.84 bpm/mmHg) are not significantly different between C57 and SOD1. Baroreceptor activation showed a significant increase in gain (C57: 5.4 ± 0.3 vs. SOD1: 7.4 ± 0.5 %/mmHg, P < 0.01) in the SOD1 transgenic mice. Heart rate depression in response to electrical stimulation of the left ADN and cervical vagus was comparable between C57 and SOD1, though MAP reduction in response to ADN stimulation is slightly, but significantly increased at 50 Hz in SOD1 animals. Time- domain analysis of HRV did not reveal any significant difference in beat-to-beat variability between SOD1 and C57 (SDNN: C57: 2.78 ± 0.20, SOD1: 2.89 ± 0.27), although frequency-domain analysis uncovered a significant reduction in the low-frequency power component of the HRV power spectral distribution (C57: 1.19 ± 0.11, SOD1: 0.35 ± 0.06, P < 0.001). This study shows that although hSOD1 overexpression does not affect overall baroreflex function, it does potentiate baroreceptor sensitivity and brain stem control of arterial pressure, and reduces low-frequency beat-to-beat variations in HR, without affecting total HRV.

Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals to oxygen and hydrogen peroxide. Mn SOD is localized in mitochondria, Cu-Zn SOD is in the cytosol and chloroplast, and Fe SOD is in chloroplasts. The effects of a chloroplast-localized oxidative stress, caused by methyl viologen or 3-(3, 4-dichlorphenyl)-1-1′ dimethylurea (DCMU) on SOD populations were investigated. A cloned Arabidopsis thaliana Fe SOD gene was expressed in Escherichia coli and was purified from transformed cells. This protein was used to raise antibodies against A. thaliana Fe SOD which in turn were used to quantify the effects of oxidative stress on Fe SOD protein. Effects of oxidative stress on enzyme activity were measured in native gels. Fe SOD responded to oxidative stress with an increase in activity, but not in antibody reactive protein. Two novel forms of Fe SOD activity, with faster migration rates in activity gels, were detected. Mn SOD, a mitochondrial enzyme, responded to the stress with an increase in activity. In contrast, the activity or amount of Cu-Zn SOD protein did not respond to this oxidative stress. In light of these results, we propose that SODs respond to oxidative stress at the enzyme and gene levels. Mitochondrial Mn SOD responded to a chloroplast-localized stress with an increase in activity, suggesting either that the site of action for methyl viologen is not exclusively in the chloroplast or that there are other signals among the compartments of the cell. Fe SOD, but not Cu-Zn SOD responded to stress, suggesting that Fe SOD may be the stress responsive enzyme in this organelle. Evolutionary relationships among different isoforms were investigated based on the known primary, secondary, and tertiary structures of these isoforms. The three dimensional structure of A. thaliana Fe SOD was modeled by using structures of crystallized E. coli and Pseudomonas ovalis Fe SODs as templates. Comparison of prokaryotic Fe SOD with eukaryotic isoforms showed that Fe and Mn SODs are structurally homologous, whereas Cu Zn SOD is not. / Ph. D.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24839号 / 医博第5007号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上, 治久, 教授 寺田, 智祐, 教授 林, 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

<i>Brucella abortus</i> is capable of resisting the microbicidal mechanisms of phagocytic cells and growing within phagocytic cells, usually macrophages. <I>B. abortus</i>, like several other intracellular bacteria responds to the hostile environment in macrophages by producing heat shock proteins (HSPs) which are induced by environmental stresses. Bacterial HSPs are very immunogenic, eliciting both cellular and humoral immune responses in the infected host. The significance of host cellular and protective immune responses directed against these proteins is currently unresolved. Baculovirus recombinants were generated in <i>Sf9</i> insect cells for <i>B. abortus</i> HSPs and the protein expression was optimized. Humoral (Western blot), cell mediated (CMI, IFN-g- release by splenocytes, and CD3+CD4+, CD3+CD8+ T cell/ total splenocytes ratios) and protective immune responses of BALB/c mice (challenge with virulent <i>B. abortus</i> 2308) against these recombinants, against <i>B. abortus</i> superoxide dismutase (SOD) and ribosomal L7/L12 proteins, inoculated alone or in various combinations with complete Freund's, Ribi and recombinant IL-12 as adjuvants, were analyzed. Vaccinia virus-GroEL recombinant as priming immunogen, followed by baculovirus-GroEL-Ribi booster, was explored. Androstenediol, an immune up-regulator, was tested for its ability to induce resistance against challenge. None of the mice inoculated with individual, divalent or trivalent HSP-expressing <i>Sf9</i> cells combined with Freund's were protected against challenge and the <i>Sf9</i> cell-induced response masked the recombinant protein-specific CMI responses. Recombinant HSPs were purified and combined with Ribi. Although significant IFN-g release was induced by immunization with the HtrA-Ribi combination, no mice were protected against challenge. Priming with vaccinia virus-GroEl recombinant and boosting with purified baculovirus-GroEL protein-Ribi combination did not induce protection. Androstenediol did not enhance in vivo resistance to challenge. IL-12 alone did not activate splenocytes but induced significant IFN-g release in mice when combined with killed <i>B. abortu</i>s RB51 vaccine, purified recombinant HtrA or purified SOD proteins, or L7/L12 expressing <i>Escherichia coli</i> cells. Significant protection was induced by SOD combined with IL-12. No correlation was seen between IFN-g release by splenocytes and protection against challenge in the SOD/IL-12-immunized mice. The results suggest that <i>B. abortus</i> HSPs are not highly immunogenic in mice and though various immune responses may be induced by one or another HSPs, protective immune response, unfortunately, is not among them. The results of this study reflect the difficulties in experimenting with immune responses against single or a limited number of recombinant <i>B. abortus</i> proteins. This is particularly true when the task includes induction of a protective immune response and finding significant correlation between different types of immune response assays. / Ph. D.

The effect of Mn(III)-salophen, a superoxide scavenger, against oxidative stress was evaluated in <i>Escherichia coli sodA- sodB-</i>. Oxidative stress was imposed by exposure of the cells to paraquat or hyperoxia. Cells were grown in LB medium overnight, washed and resuspended in the indicated glucose/salts medium supplemented with casamino acids. The effect of Mn(III)-salophen in the oxidative stress model <i>in vivo</i> was measured in terms of the cell growth. Mn(III)-salophen ( 60 nM) completely protected <i>E. coli</i>JI132<i>sodA- sodB-</i>against 1.0 μM paraquat. Equivalent amounts of Mn(III) acetate, a Mn(III)-salophen component, also protected against paraquat toxicity in aerobic <i>E. coli</i> JI132<i>sodA- sodB-</i>. Fe(III)-salophen which has no superoxide scavenging activity, did not protect the cells against paraquat toxicity. The protective effect of Mn(III)-salophen against the paraquat toxicity was proposed to come from the intracellular superoxide scavenging activity of either the complex itself, its component Mn(III), or both, but not by inhibiting the uptake of paraquat. The protective effect of Mn(III)-salophen and Mn(III) in the glucose/salts medium containing casamino acids was also observed in <i>E. coli sodA- sodB-</i> in 100% and 50% oxygen. Hyperoxia increases intracellular levels of superoxide radicals that are intercepted by Mn(III)salophen and Mn(III). / Master of Science

Water stress is the most critical environmental factor limiting crop production in the US Piedmont. The presence of humic substances in composted organic amendments may increase crop tolerance to water stress through their hormone-like effects on plant metabolism. The objectives of this study were to calculate N mineralization rates of composted and non-composted organic materials used in this long-term field study, and to determine differences in soil physiochemical properties, corn and soybean leaf physical and biochemical properties yield and seed quality between organically amended and inorganically fertilized treatments. Nitrogen mineralization rates were greatest in the poultry litter (21%) and Panorama yard waste compost (4.5%) amended plots. Nitrogen uptake (120 mg/pot, 133 mg/pot, respectively) in these treatments were greater than that in the control (0N) (91.3 mg/pot) treatment. Wolf Creek biosolids compost and Huck's Hen Blend yard waste compost induced N immobilization (-5.0% and 0.18%, respectively), and had N uptake values similar to the control (92.6 mg/pot and 95.7 mg/pot). Rivanna biosolids compost immobilized N (-14.8%) but N uptake (136 mg/pot) was greater than that in the control due to the relatively high inorganic N content in the amendment. The total N concentration and C:N values were less reliable variables in predicting N mineralization when a significant portion of the total N was in the inorganic form. The annual application of poultry litter, Rivanna biosolids compost, and Panorama yard waste compost at 100% agronomic nitrogen and 30 % agronomic nitrogen rates in the field study improved soil fertility and increased total organic and humified carbon contents relative to the inorganically fertilized and control treatments. The amended treatments had slightly greater plant available water contents (average 10.0 cm/15 cm) than the control (8.38 cm/15 cm). Leaf water potential measurements revealed that neither crop experienced water stress during the sampling season. Treatment differences in leaf antioxidant activity were only observed in corn. All corn plants that were fertilized with amendments supplying the crop's nitrogen needs, regardless of the source, had greater leaf nitrogen (+29%), chlorophyll (+33%), and protein contents (+37%), lower superoxide dismutase (-29%) and ascorbate peroxidase (-17%) activities, and lower malondialdehyde (-33%) contents relative to the control and low nitrogen treatments. There were no observed differences in catalase activity, which was likely due to the evolutionary advantage of C4 metabolism. Yield was strongly related to midseason leaf nitrogen contents (R2=0.87, p<0.0001) and not soil humified carbon (R2=0.02, p=0.0543). There were no observed treatment differences in soybean leaf physiology and metabolism. Differences, however, were observed over time. As the leaves senesced, leaf chlorophyll, protein, superoxide dismutase and catalase activities decreased, and the malondialdehyde content increased. Ascorbate peroxidase activity slightly increased with time. Catalase activity in soybean was primarily driven by the oxidation of glycolate, a product of photorespiration, and not the formation of reactive oxygen species in the chloroplasts. The organically amended treatments had higher yields (9-21% increase), greater protein contents (4-9% increase), and seed weights (5-14% increase) relative to the fertilizer and control treatments. It was concluded that differences in soybean yield and seed quality were due to non-nutritive benefits of the organic amendments and not available water or plant nutrition. / Master of Science