No description available.

Monitoring the early responses of tumours to treatment is a crucial element in guiding therapy and increasing patient survival. To achieve this, we are using magnetic resonance imaging (MRI), which can provide detailed physiological information with relatively high temporal and spatial resolution. In combination with the dynamic nuclear polarisation (DNP) technique, high signal-to-noise is obtained, resulting in a powerful tool for in vivo 13C metabolic imaging. However, detection of hyperpolarised substrates is limited to a few seconds due to the exponential decay of the polarisation with the longitudinal relaxation time constant T1. This work aimed to improve the combination of hyperpolarisation and metabolic NMR/ MRI by extending the observation timescale of the technique. Working with quantum mechanical properties of the detected substrates, long lifetimes might be accessible by using the nuclear singlet configuration of two coupled nuclei. The singlet state is immune to intramolecular dipole-dipole relaxation processes, which is one of the main sources of signal decay in MRI. In favourable situations, the singlet relaxation time constant can be much longer than T1, so transfer of the polarisation into the singlet state may allow one to extend the usable time period of the nuclear hyperpolarisation. Here we studied the relaxation of hyperpolarised metabolites, including those found in the TCA cycle, and examined the possibility of extending their observation timescale by storing the polarisation in the long-lived singlet state. The polarisation remains in this state until it is eventually required for imaging. We also investigate how one may track polarised metabolites after injection into a subject due to the transfer of polarisation to the solvent by Overhauser cross-relaxation, so that the 13C polarisation remains untouched until imaging is required. In this way we should be able to interrogate slower metabolic processes than have been examined hitherto using hyperpolarised 13C MRS, and better understand metabolic changes induced in tumours by treatment.

Magister Scientiae - MSc / Basella alba (BA) and Hibiscus macranthus (HM) are used by traditional healers in Cameroon to treat male sexual fertility problems. Previous studies showed that in vivo administration of the leaf extracts of both plants caused a significant increase in rat seminal vesicle weight and spermatozoa numbers was accompanied by a significant increase in serum testosterone. The aim of this study was to establish the effects of BA and HM extracts on Sertoli cell functions. TM4 cell line was used in this study as it exhibited properties similar to the Sertoli cells (Mather, 1982). Sertoli cell play a key role in spermatogenesis by regulating and supporting germ cell development. Therefore, any alterations in Sertoli cell physiology or structure may lead to impaired spermatogenesis, germ cell loss and male infertility. Developing germ cells in the seminiferous tubule require a constant supply of lactate and pyruvate (Jutte et al, 1981; 1982) and toxicant induced alterations in these nutrients have been shown to induce germ cell necrosis (Monsees et al., 2000). TM4 Sertoli cells were cultured in DMEM/Ham F-12 (M) for one day and exposed to 0.01, 0.1, 1, 10, 100 μg/ml of BA and HM extracts, respectively, for four further days. The extracts were dissolved in 0.5 % DMSO in M, while 0.5 % and 2% DMSO in M were used as negative or positive controls, respectively, and 100mM ethanol as positive control where indicated. Results obtained from the Sertoli cells exposed to BA extracts, showed that the plant extract had no significant effect on the cell viability but induced a significant concentration-dependent increase in lactate (19-67%) and pyruvate levels (39-102%) and a concentration-dependent decrease in the protein content (9-42%). The H&E histological study confirmed that the BA extract had no cytotoxic effect, as there were no changes in the morphology of the cell. Likewise, apoptotic study using DAPI showed no alteration in the nucleus when compared to the negative control. The HM plant extract significantly enhanced mitochondrial dehydrogenase activity (7fold) in the Sertoli cells but caused only slight alterations in the lactate and pyruvate levels. There was no effect seen in the protein content of the Sertoli cells. H&E and DAPI staining revealed that there were neither changes in the morphology of the cells nor any alteration regarding the mitotic and apoptotic indices. Thus, the HM extract did not have a cytotoxic effect on the cells. This study demonstrated that the Basella alba methanol extract may enhance spermatogenesis as it stimulated the source of energy required for the development of germ cells without exerting a cytotoxic effect. The Hibiscus macranthus extract stimulated mitochondrial dehydrogenase activities and may thus trigger changes in Sertoli cell physiology. In summary, both plant extracts enhanced certain Sertoli cell functions and thus might explain the positive in vivo effects of the combined plant extracts on rat spermatogenesis observed by Moundipa et al. (1999). / South Africa

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22116号 / 医博第4529号 / 新制||医||1039(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 戸口田 淳也, 教授 妻木 範行, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The microbiota has a strong influence on health and disease in humans. A causative shift favoring pathobionts is strongly linked to diseases. Therefore, anti-microbial agents selectively targeting potential pathogens as well as their biofilms are urgently demanded. Here we demonstrate the impact of ethyl pyruvate, so far known as ROS scavenger and antiinflammatory agent, on planktonic microbes and biofilms. Ethyl pyruvate combats preferably the growth of pathobionts belonging to bacteria and fungi independent of the genera and prevailing drug resistance. Surprisingly, this anti-microbial agent preserves symbionts like Lactobacillus species. Moreover, ethyl pyruvate prevents the formation of biofilms and promotes matured biofilms dissolution. This potentially new anti-microbial and anti-biofilm agent could have a tremendous positive impact on human, veterinary medicine and technical industry as well.

Most cancers rely disproportionately on glycolysis for energy even in the presence of adequate oxygen supply, a condition known as "aerobic glycolysis", or the Warburg effect. Pharmacological reversal of the Warburg effect has been shown to cause selective apoptosis of tumor cells, presumably by stimulating mitochondrial respiratory chain activity and production of reactive oxygen species that, in turn, induce a caspase-mediated series of reactions leading to cell death. We reasoned that a similar effect on tumor cells might result from up-regulation of the E1α subunit gene (pda1) of the pyruvate dehydrogenase complex (PDC) that catalyzes the rate-limiting step in aerobic glucose oxidation and thus plays a major role in the control of oxidative phosphorylation. To test this postulate, we employed a self-complementary adeno-associated virus (scAAV)-based delivery and expression system for targeting pda1 to the mitochondria of primary cultures of human hepatoblastoma (HB) and hepatocellular carcinoma (HCC) cells. Serotypes 1-10 scAAV vectors that included enhanced green fluorescent (egfp) reporter gene driven by either cytomegalovirus (CMV) or chicken beta-actin (CBA) promoters were analyzed for transduction ability of HB (Huh-6) and HCC (Huh-7 and HepG2) cell lines and primary cultures of normal human hepatocytes. Serotype 3 scAAV-egfp (scAAV3-egfp) vector was the most efficient and transduced up to 90% of cells. We limited the transgene expression primarily to liver cancer cells by generating scAAV3 vectors that contained the human alpha-fetoprotein promoter (AFP)-driven reporter gene (scAAV3.AFP-egfp) and the potentially therapeutic gene scAAV3.AFP-pda1. Infection of Huh-6 cells by the scAAV3.AFP-pda1 vector increased protein expression of E1α, PDC catalytic activity, and late-stage apoptotic cell death. Apoptosis was also associated with increased protein expression of Bcl-X/S, an early marker of apoptosis, and release of cytochrome c into the cytosol of infected HB cells. These data indicate that molecular targeting of mitochondrial oxidative metabolism in liver cancer cells by AAV3-mediated delivery of pda1 holds promise as a novel and effective therapeutic approach for human hepatic tumors.

RATIONALE - Metabolic enzymes, like pyruvate kinase M2 (PKM2), play an essential role in altering endothelial cell (EC) phenotypes and behavior. Extensive research has elucidated the function of PKM2, a rate-limiting glycolytic enzyme, in the context of cancer cells and in activated immune cells, but its role in EC biology is only newly emerging. Recent findings show PKM2 acts as a key regulator of angiogenesis. Where exogenous circulating PKM2 induces EC cell proliferation leading to increased tumor angiogenesis and growth. Also, PKM2 deficient ECs exhibit decreased proliferation and migration. The relevance of PKM2 in modulating vascular barrier function is yet to be defined. OBJECTIVE -This study attempts to elucidate the role of PKM2 in regulating vascular barrier function. METHODS AND RESULTS - In vivo, EC specific deletion of PKM2 promotes increased vascular permeability in pulmonary capillary vessels and increased VEGF-induced acute vessel permeability in mouse dermal vessels. Similarly, in vitro, PKM2 deficient ECs exhibit decreased electrical resistance, disrupted VE-cadherin junctions and gap formations (illustrated via florescent VE-cadherin staining and phosphorylation of VE- cadherin protein at tyrosine residue Y658). Mechanistically, the deletion of PKM2 in ECs leads to increased angiopoietin-2 (Ang-2) expression, a well-known modulator of vascular permeability. Also, deletion of Ang-2 was sufficient to attenuate vascular leakage in PKM2 deficient endothelium, indicating that vascular leaky phenotype observed in PKM2 deficient endothelium is mediated by increased Ang-2 expression. CONCLUSIONS - PKM2, by modulating Ang-2 expression, plays a vital role in maintaining vascular barrier function. / 2019-07-11T00:00:00Z

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Regulation of the pyruvate dehydrogenase complex (PDC) is important for glucose homeostasis and control of fuel selection by tissues. Knocking out pyruvate dehydrogenase kinase 4 (PDK4), one of four kinases responsible for regulation of PDC activity, lowers blood glucose levels by limiting the supply of three carbon compounds for gluconeogenesis. Down regulation of PDK4 expression is also important for control of blood glucose by insulin. The primary goal was to determine whether PDK4 should be considered a target for the treatment of diabetes. A major concern is that inhibition of fatty acid oxidation by PDK4 deficiency may promote fat accumulation in tissues and worsen insulin sensitivity. This was examined by feeding wild type and PDK4 knockout mice a diet rich in saturated fat. Fasting blood glucose levels were lower, glucose tolerance was better, insulin sensitivity was greater, and liver fat was reduced in PDK4 knockout mice. The reduction in liver fat is contradictory to the finding that fibrate drugs increase PDK4 expression but ameliorate hepatic steatosis in rodents. To investigate this phenomenon, wild type and PDK4 knockout mice were fed the high saturated fat diet with and without clofibric acid. The beneficial effect of clofibric acid on hepatic steatosis was greater in the PDK4 knockout mice, indicating up regulation of PDK4 is not necessary and likely opposes the effect of clofibric acid on hepatic steatosis. Clofibric acid dramatically lowered the amount of hepatic CD36, a plasma membrane translocase required for fatty acid import, suggesting a novel mechanism for prevention of hepatic steatosis by fibrates. PDK4 deficiency had no effect on CD36 expression but reduced the enzymatic capacity for fatty acid synthesis, suggesting clofibric acid and PDK4 deficiency ameliorate hepatic steatosis by independent mechanisms. Investigation of the mechanism by which insulin regulates PDK4 expression revealed a novel binding site for hepatic nuclear factor 4α (HNF4α) in the PDK4 promoter. The stimulatory effect of HNF4α was sensitive to inhibition by Akt which is activated by insulin. The findings suggest PDK4 is a viable target for the treatment of hepatic steatosis and type 2 diabetes.

>Magister Scientiae - MSc / Knowledge of haematological 'normdata', of experimental animals, and the biological variables that affect it is essential in order to recognise variations from the normal. In addition, the haemopoietic system may be regarded in principle as good material for studies of the cellular events associated with ageing. These considerations, together with the well documented effects of age on various physiological processes, prompted an investigation into the effects of donor age on several blood parameters. Review of the literature revealed that age-related changes in blood parameters have been reported for several species, but the documentation thereof is incomplete, inconsistent and inconclusive in many respects. Blood samples from male Wistar rats of nine different biological ages, ranging from birth to 96 weeks of age, were analysed for haematological and biochemical parameters. These included the blood cell counts, erythrocytic indices, haemoglobin concentration, haematocrit, erythrocytic 2,3-diphosphoglycerate and adenosine triphosphate levels, and erythrocytic glucose 6-phosphate dehydrogenase and pyruvate kinase activities. Data was obtained which demonstrates that all blood parameters measured underwent significant, although not al~ays regular, age-related changes. These changes were found to be more marked during the first month of life than at any other period. Evidence is also presented to show that the depressed haemoglobin concentration during the early postnatal life may not imply a condition of 'physiologic anaemia' as was previously thought. Since the blood profile exhibits only slight changes from about 24 weeks of age, it does not seem that the haemopoietic system of the old rat deteriorates significantly as to constitute a limiting factor for the animal's life. However, the importance of taking an animal's age into account when blood parameters constitute experimental results is emphasised. The second phase of this study involved a detailed investigation of the effect of the animal's age on erythrocytes in particular. These cells have limited life-spans, and are often used as models in studies of cellular ageing. Special emphasis was therefore placed on comparing the relative effects of host and cellular ageing on the properties of these cells. Erythrocytes from rats between one and 48 weeks of age were separated into two populations by a modification of the conventional density gradient centrifugation technique. The two populations were assumed to differ in mean cell age and were analysed for erythrocytic indices, phosphate ester concentrations and the activities of glucose 6-phosphate dehydrogenase and pyruvate kinase. Evidence is presented to show that ageing rat erythrocytes exhibit a decrease in volume, phosphate ester content and enzyme activities while the cellular haemoglobin concentration increases. Differences in the mean cell age however, does not seem to account for the donor-age-related effects observed in the whole blood parameters. Rather, the significant differences found in the characteristics of similarly aged red cells, between variously aged donors, demonstrate that the biological age of the organism influences the red cells and probably the ageing thereof in vivo. The contribution of the changing status of the erythrocyte's environment of progressively older animals, to alterations which take place in the ageing red cell is discussed.