No description available.

No description available.

No description available.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. The aggressiveness and therapeutic recalcitrance of this malignancy has been attributed to multiple factors including the influence of an active desmoplastic stroma. How the stromal microenvironment of PDAC contributes to the fatal nature of this disease is not well defined. In the analysis of clinical specimens, we observed diverse expression of the hypoxic marker carbonic anhydrase IX and the lactate transporter MCT4 in the stromal compartment. These stromal features were associated with the epithelial to mesenchymal phenotype in PDAC tumor cells, and with shorter patient survival. Cultured cancer- associated fibroblasts (CAFs) derived from primary PDAC exhibited a high basal level of hypoxia inducible factor 1a (HIF1 alpha) that was both required and sufficient to modulate the expression of MCT4. This event was associated with increased transcription and protein synthesis of HIF1a in CAFs relative to PDAC cell lines, while surprisingly the protein turnover rate was equivalent. CAFs utilized glucose predominantly for glycolytic intermediates, whereas glutamine was the preferred metabolite for the TCA cycle. Unlike PDAC cell lines, CAFs were resistant to glucose withdrawal but sensitive to glutamine depletion. Consistent with the lack of reliance on glucose, CAFs could survive the acute depletion of MCT4. In co-culture and xenograft studies CAFs stimulated the invasive potential and metastatic spread of PDAC cell lines through a mechanism dependent on HIF1a and MCT4. Together, these data indicate that stromal metabolic features influence PDAC tumor cells to promote invasiveness and metastatic potential and associate with poor outcome in patients with PDAC.

Pressure ischemia is a medical condition characterised by the necrosis of the skin and underlying tissues in body areas exposed to prolonged pressure. This condition leads to the development of bedsores and affects 9% of hospitalised patients, costing the NHS between £1.4 and £2.1 billion per year. The severity of pressure ischemia has been linked to the concentration of sweat lactate, a product of sweat gland metabolism under anaerobic conditions, such as hypoxia. Normal levels of lactate in human sweat are 20±7 mM, but under ischemic conditions these can rise up to approximately 70 mM. This project presents the development of a novel flexible electrochemical enzyme-based biosensor for the continuous and non-invasive monitoring of sweat lactate with the potential for becoming a body-worn device for the early detection of pressure ischemia onset. The core of the recognition system is a flexible laminate, comprising two highly porous polycarbonate membranes, which provide support for the lactate oxidase enzyme, immobilised via covalent cross-linking. Oxidation of lactate produces H2O2, which is subsequently determined electrochemically. The transducer comprises a two-electrode system on a single flexible polycarbonate membrane, sputter-coated with gold (CE/RE) and platinum (WE) to render it conductive. The developed design has been improved through investigation into different factors regarding the immobilisation method of the enzyme in the laminate and the lowering of interferences from oxidising compounds present in sweat. The sensing system exhibits lactate selectivity at physiologically relevant concentrations in sweat for pressure ischemia (0–70 mM), with good reproducibility (7.2–12.2% RSD) for a hand-manufactured device. The reliability of the sensor’s performance and the capability to detect lactate fluctuations on human sweat samples has been demonstrated. The sensing system showed excellent operational and mechanical stability. The application of Nafion® on the WE lowered interferences from ascorbic acid and uric acid by 96.7 and 81.7% respectively. These results show promise towards the further development of a body-­‐worn monitoring device for determining lactate levels in undiluted human sweat samples in a reproducible, fast and accurate manner.

Wir nahmen an, dass nach intramuskulärer Injektion von Koffein und Halothan in Schweinen mit Veranlagung (MHS) und ohne Verlangung (MHN) für eine maligne Hyperthermie, es zu einem dosisabhängigen intramuskulären Laktatanstieg kommt. Des weiteren ist die Ausdehnung des dadurch ausgelösten Hypermetabolismus nur auf ein kleines Areral um die Injektionsstelle begrenzt. Eine systemische MH-Krise wird nicht ausgelöst. Mikrodialysesonden wurden in die Hinterläufe von 7 MHN und 7 MHS Schweinen platziert und mit Ringerlösung perfundiert. Nach Einstellen eines Gleichgewichtes wurden Boli von Halothan und Koffein in aufsteigenden Konzentrationen in den Muskel appliziert. Für den zweiten Versuchsansatz, die regionale Ausbreitung betreffend, wurden wiederum Mikrodialysesonden im Abstand von 10 mm und 25 mm zum Injektionsort platziert. Das Laktat wurde photospektrometrisch im Dialysat gemessen. Durch intramuskuläre Halothan- und Koffeinapplikation kommt es zu einer dosisabhänigen Erhöhung der intramuskulären Laktatkonzentration mehr beim MHS als MHN-Tieren mit signifikantem Unterschied. Laktaterhöhungen fanden sich darüber hinaus nur an der Injektionsstelle der Trigger- substanzen, nicht jedoch in 10 und 25 mm Entfernung davon. Somit ist der Anstieg der lokalen Laktatkonzentration im Muskel nur auf ein kleines Areal um die Injektionsstelle begrenzt. / We hypothesized that IM halothan and caffein injection increases lokal lactat concentration dose-dependently in malignant hyperthermia susceptible (MHS) und non-susceptible (MHN) pigs and that the hypermetabolic reaction measured by regional distribution of lactat is limited to a small area around the injection side. Microdialysis probes were placed in the hindlimbs of 7 MHS and 7 MHN pigs and perfused with Ringers solution. After equilibration, boluses of increasing halothan and caffein concentrations were injected. For the second hypothesis regarding regional distribution, microdialysis probes were positioned in 7 MHS and 6 MHN pigs at the injection site for halothan and caffein and at a distance of 10 mm and 25 mm. Lactat were measured in the dialysate by spectrophotometry.Halothane and caffeine increased IM lactat dose-dependently in MHS significantly more than in MHN pigs. Lactat was increased only at the infection site but not at 10 mm and 25 mm distance. The increase of lactat after lokal MH trigger injection is limited to a small area around the probe.

Hui, Lai Shan. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1988. / Bibliography: leaves 83-87.

by Kwok-cheong Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1983. / Bibliography: leaves 194-221.

Thesis (Ph.D.)--Boston University / Ehrlich ascites carcinoma lactic acid dehydrogenase was isolated from an eleven-day old tumor by acid precipitation, ammonium sulfate fractionation, and chromatography on DEAE cellulose. Electrophoretic analysis indicated that the final enzyme preparation and the ammonium sulfate fraction contained a single isoenzyme, that is, one of the five possible forms of lactic acid dehydrogenase two of which are tetramers of a single but different protein while the other three are tetramer mixtures of both proteins (i.e. hybrid enzymes). Ultracentrifugal analysis indicated that the final enzyme preparation was composed of two major components with sedimentation rates of 7.3 S and 1.9 S. The enzymatic activity was associated only with the 7.3 S component. The apparent loss of enzymatic activity in 0.1 M phosphate buffer pH 7.0 and the magnitude of the value of the 1.9 S component indicated that this represented the subunit of the enzyme. [TRUNCATED]

Cancer cells utilise glycolysis to produce lactate, even in the presence of sufficient levels of oxygen. Excess lactate is removed from cancer cells by MCT1 and MCT4, to prevent intracellular acidosis, apoptosis and to aid the continuous glycolytic flux. MCT1 and MCT4 are over-expressed in many types of human cancers, which correlates with reduced overall survival and increased treatment resistance. The potential role of MCT1 and MCT4 in two EOC cell lines (Skov3 and OV90) was investigated in this study. MCT1 was expressed at similar levels in Skov3 and OV90 cells. Therefore, stable cell lines over-expressing MCT1 were produced using both cell lines. MCT4 was expressed at high levels in Skov3 cells, but very low levels in OV90 cells. Therefore, a stable cell line with MCT4 silencing under the inducible control of doxycycline was produced using Skov3 cells, and a stable cell line to over-express MCT4 was produced using OV90 cells. The consequences of these genetic modifications on the metabolic phenotype, metastatic abilities and the sensitivity of cell lines to treatment with Carboplatin and Paclitaxel, were assessed in normoxia and 1 % hypoxia and 0.1 % hypoxia. Over-expressing MCT1 in Skov3 cells, had no effect on their metabolic phenotype or the sensitivity to treatment with Carboplatin and Paclitaxel. However, over-expressing MCT1 in Skov3 cells significantly enhanced their metastatic abilities, which correlated with reduced focal adhesion size. Silencing MCT4 in Skov3 cells, had no effect on the use of glycolysis, sensitivity to treatment with Carboplatin and Paclitaxel, or their metastatic abilities. However, following MCT4 silencing there was a significant increase in the levels of intracellular ROS. Over-expressing MCT1 in OV90 cells, had no effect on lactate levels or intracellular ROS. However, there was a significant reduction in both their glycolytic activity and mitochondrial mass. Furthermore, over-expressing MCT1 in OV90 cells, increased their resistance to treatment with Paclitaxel, which correlated with increased Pgp and LDHA expression. Over-expressing MCT4 in OV90 cells, caused an increase in the use of glycolysis and increased cell survival in hypoxia. There was also a significant enhancement in the metastatic abilities of these cells following the over- expression of MCT4, which correlated with reduced focal adhesion size. Furthermore, over-expressing MCT4 in OV90 cells, increased their resistance to treatment with Paclitaxel, which correlated with an increase in the expression of Pgp and LDHA.In summary, the findings of this study revealed that MCT1 and MCT4 play a significant role in the biological function of Skov3 and OV90 cells. High expression levels of MCT4 correlated with an increase in glycolysis and cell survival in hypoxia. Whereas high expression levels of MCT1 and MCT4 in correlated with an increase in the metastatic abilities, as well as with Paclitaxel resistance and increased Pgp expression.