Effects of exercise and the relative condition of exercised fish were viii evaluated for two strains of rainbow trout. Circular tanks fitted with a paddle were used to continuously exercise the fish and a stamina tunnel was used to evaluate the condition of exercised trout. Trout were exercised continuously at one fish-length per second and one-half fish length per second. Serum LDH activity in Shephard-of-the-Hills (Missouri) rainbow trout acclimated to 10 C increased two fold during the first four days of exercise. Serum LDH activity returned to control levels by the tenth day of exercise. Serum LDH activity in Shephard-of-the-Hills rainbow trout exercised at one-half length per second did not increase significantly. Serum LDH activity in Fish Lake stock acclimated to 17 C did not increase significantly. Shephard-of-the-Hills and Fish Lake rainbow trout exercised at one length per second were better conditioned to water velocities of two lengths per second than were controls.

Intertidal habitats are characterized by dynamic, tidally-driven fluctuations in abiotic and biotic factors. Many of the environmental stressors that vary across the intertidal (e.g., temperature, oxygen, food availability, predation pressure) are strong drivers of metabolic rate in ectotherms. As such, we predicted that there may be pronounced differences in the metabolic and stress physiology of conspecific sessile invertebrates occupying at different relative tidal heights. The common acorn barnacle Balanus glandula represents an ideal model organism in which to investigate the possibility of tidal height-dependent physiological differences, owing to their wide distribution in the intertidal zone and their eurytolerant nature. In the first chapter of my thesis, we investigate the hypothesis that B. glandula anchored in the low intertidal have a greater capacity for anaerobic metabolism than conspecifics in the high intertidal, and that this is due to increased predation pressure during submersion. Further, we explore the temporal and spatial fidelity of certain tidal-height driven trends in lactate dehydrogenase activity previously observed in our lab (i.e., higher LDH activity in low intertidal barnacles; Horn et al., 2021), and attempt to identify environmental variables that drive plasticity in LDH activity. We found that, in general, there were higher densities of B. glandula and gastropod whelk predators in the low intertidal compared to the high intertidal, but follow-up studies in the lab revealed that opercular closure in B. glandula was induced by predator exposure (Acanthinucella spirata) for less than 24h. This time frame for shell closure is unlikely to result in internal hypoxia or enhance capacity for anaerobic metabolism. We were therefore not surprised to find that LDH activity in B. glandula was likewise not affected by predator exposures (48h) carried out in the lab. After failing to find an effect of predators on LDH activity in B. glandula, we attempted to replicate the previous finding that LDH activity was highest in low intertidal populations of B. glandula. We did this at the original location in San Luis Obispo Bay, CA as well as at three novel field sites and across seasons and years. While we did observe variation in LDH activity over time and between sites, we did not consistently observe the same trend in LDH activity whereby low intertidal barnacles had the highest activity. In response to these variable patterns, we attempted to identify what environmental parameters, other than predation, might be responsible for plasticity in LDH activity. Unfortunately, neither temperature nor emersion stress – the two variables we examined – had any significant an effect on LDH activity in B. glandula. These data suggest that there must be multiple, interacting stressors – including tidal position - that influence the anaerobic metabolic capacity of B. glandula. In the second chapter of my thesis, we went on to investigate how the response to thermal stress might differ between populations of B. glandula from different vertical heights in the intertidal zone. To this end, we assessed how aerial temperature stress affected oxygen consumption rates (MO2), superoxide dismutase (SOD) activity, and time to mortality in B. glandula collected from both low and high intertidal positions. We found that barnacles from the low intertidal showed a significant increase in MO2 with higher temperature, while MO2 was unaffected by temperature in B. glandula from the high intertidal. We also observed that SOD activity levels were higher in the high intertidal barnacles compared to the low intertidal barnacles, although neither group was increasing SOD activity under higher temperature. Finally, we observed significantly longer survival times during thermal stress in barnacles from the high intertidal zone (e.g., LT50 = 8.75 h vs 5 h at 33˚C for the high and low barnacles, respectively), although this advantage seemed to be lost with the addition of desiccation stress at these same temperatures. It is evident that life in highest reaches of the intertidal zones is physiologically challenging, and this has resulted in a population of B, glandula barnacles that are less sensitive to and better suited to tolerate temperature extremes than conspecifics in the lowest intertidal regions. Understanding how habitat variation may differentially impact the metabolic and thermal stress physiology of B. glandula is increasingly important as climate change progresses. This is particularly significant considering that organisms in the intertidal already reside within a relatively stressful environment and may be living closer to their thermal tolerance limits than animals from less extreme habitats.

no / Background Plasmodium falciparum drug resistance is a major public health challenge in sub-Sahara Africa. Many people are now resorting to the use of herbs in managing malaria due to the increasing treatment failures with the conventional drugs. In this study the ethanolic extract of Polyalthia longifolia (Sonn) Thw. var. pendula, a variety fondly used in folklore medicine in Ghana was investigated for potential antimalarial drug development. Method The ethanolic extract of P. longifolia (Sonn) Thw. var. pendula stem bark was screened against the multidrug resistant, K1 strain of P. falciparum by the parasite lactate dehydrogenase (pLDH) assay and a good antiplasmodial activity (IC50 22.04 ± 4.23 µg/ml) was observed which led to further chromatographic analysis in search for actives. Results Bioassay guided fractionation of the extract yielded; three clerodane diterpenes [16-hydroxycleroda-3,13-dien-16,15-olide (1), 16-oxocleroda-3,13E-dien-15-oic acid (2) and 3,16-dihydroxycleroda-4(18),13(14)Z-dien-15,16-olide (3)], a steroid [beta-stigmasterol (4)] and two alkaloids [darienine (5) and stepholidine (6)]. While compounds 4, 5 and 6 exhibited weak antiplasmodial activity (IC50 22–105 µg/ml), the clerodane diterpenes exhibited significantly potent (p<0.005) blood schizonticidal activity (IC50: 3–6 µg/ml). This is the first report of the antiplasmodial activity of compounds 2 and 3. In combination assay with chloroquine, compounds 1, 2, 3 and 5 antagonized the antiplasmodial activity of chloroquine while 4 and 6 demonstrated a synergistic action. Conclusion The potent antiplasmodial activity of the extract of P. longifolia (Sonn) Thw. var. pendula and compounds therein strongly suggests its usefulness as an antimalarial agent and supports its inclusion or exploitation in formulations of herbal remedies for malaria in Ghana.

Hypoxia refers to any condition in which the water is less than fully saturated with oxygen. Although it is generally accepted that adults are more tolerant of hypoxic conditions than larval stages, there is little information to support this assumption. To determine whether reduced concentrations of dissolved oxygen (DO) affect fishes differently during various early life stages, I examined the responses of two species of fish (fathead minnows (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss)) exposed to low dissolved oxygen concentrations at different ages during the first 100 days post-hatch. The changes in oxygen requirements and respiratory patterns that occur during ontogeny and exposure to hypoxia were observed. The results of this study suggest that the early larval stages appear to be at least as tolerant of short-term exposure to low dissolved oxygen concentrations as the older, more developed stages. Fathead minnows underwent a gradual transition from being metabolic conformers to regulators during development. Hemoglobin appeared to be playing a larger role in oxygen supply in the early post-hatch trout than in the minnows. Fathead minnow larvae produced relatively low concentrations of lactate upon exposure to hypoxia. Conversely, rainbow trout larvae exhibited significant increases in lactate concentration under similar conditions. This implies that there is a threshold oxygen concentration below which trout larvae utilize anaerobic metabolism to provide additional energy. Lactate dehydrogenase activity increased as the rainbow trout larvae aged, suggesting that they develop an anaerobic capacity which could be used to provide additional energy during hypoxia. The minnows did not exhibit this increase in activity. The ability of larval fishes to detect and avoid hypoxic conditions was also examined. The overall trends suggest that throughout this period of development, both fish species gradually leave an area as the dissolved oxygen concentration decline. Both species appeared to leave the hypoxic areas with deliberate motions, indicating that a directed sensor system allowed them to detect oxygen gradients. The results suggest that a combination of physiological, biochemical, and behavioral mechanisms may allow fishes to cope with hypoxia. / Ph. D.

No description available.

Die von Tumorzellen exzessiv exprimierte Laktat-Dehydrogenase 5 (LDH-A) ist das Schlüssel-Enzym für den katalytischen Stoffwechsel um Pyruvat in Laktat unter Gewinnung von Energie umzuwandeln. Bei LDH handelt es sich um einen wichtigen prognostischen Marker für die Einstufung der Aggressivität und die Behandlung von Tumoren. Ziel dieser Arbeit war es, das Wissen um die Funktion des Genproduktes von LDH-A (LDH-V) in Bezug auf die Proliferation von Tumorzellen, die Expression Angiogenese-Gene, maligne Konversion von Tumoren und die Metastasierung zu vertiefen. Zusätzlich sollte die Rolle von LDH auf die Genexpression HIF-regulierter Proteine, die das Überleben von Tumorzellen und vor allem der Glykolyse fördern, untersucht werden. Hierfür wurden LDH-A knockdown-Klone von dem murinen B16F10 Melanom und Lewis Lung Karzinom sowie dem humanem HT29 Kolonkarzinom generiert und in vitro und in vivo in den drei verschiedenen Tumormodellen untersucht. Der knockdown von LDH-A führte in vitro zu einer Hemmung der Proliferation in Lewis Lung und B16F10, während bei HT29 Tumorzellen kein solcher Effekt beobachtet werden konnte. Interessanterweise ließen sich nicht bei allen drei Zelllinien die durch limitierte LDH Aktivität ausgelösten Effekte auf die in vitro Proliferation auf das Tumorwachstum in vivo übertragen: Das Wachstum der Lewis Lung als auch der HT29 Tumoren in vivo war durch die Reduktion von LDH-A drastisch vermindert, während die Größe der B16F10 Tumoren davon nicht beeinflusst war. Es konnte gezeigt werden, dass B16F10 Tumoren unter LDH Suppression verstärkt VEGF exprimieren. Dadurch waren diese Zellen in vivo in der Lage, durch verstärkte Vaskularisierung des Tumors der durch LDH-A knockdown ausgelösten Hemmung des Tumorwachstums zu entkommen. Bei Lewis Lung Tumorzellen hingegen, führte die Unterdrückung von LDH-V zu einer beeinträchtigten Glukoseaufnahme unter normoxischen sowie hypoxischen Bedingungen. Die reduzierte Aufnahme von Glukose hatte in vivo, in einer vielschichtigen komplexen Struktur, deutlich größere Auswirkungen als auf einer eindimensionalen Zellkulturschale. Die Ergebnisse der vorliegenden Arbeit liefern neue Erkenntnisse zur Rolle von LDH-V während der Tumorprogression und können als Grundlage für zukünftige, neue Therapiestrategien bei Krebspatienten dienen. / LDH is an important prognostic marker in cancer staging and therapy. High serum LDH activity is associated with poor patient prognosis in different tumor types. LDH-A has been shown to play a major role in tumor malignancy, but the molecular mechanism behind this role is only starting to be understood. The objective of this work was to broaden our knowledge about the role of LDH-A gene product (LDH-V) in tumor cell proliferation, angiogenesis-related gene expression, tumor malignancy and metastasis. Herein, LDH-A shRNA knockdown clones of murine B16F10 melanoma, Lewis Lung carcinoma and human HT29 colon carcinoma were generated to uncover the molecular mechanisms between diminished ability of metabolizing pyruvate to lactate and tumor cell growth in vitro and in vivo. In this study, a significant correlation between tumor weight and serum LDH in in vivo tumor models was found, which additionally correlated with the in vitro LDH secretion. In vitro, suppression of LDH-A led to anti-proliferative effects in Lewis Lung and B16F10 tumor cells, while having no effect on HT29 cells. Interestingly, the consequence of limiting LDH activity in vitro did not show a direct correlation to the in vivo anti-tumor effects in LDH-deficient tumors: B16F10 tumor growth was unaffected by silencing LDH-A, while Lewis Lung and HT29 demonstrated a drastic reduction in tumor growth in vivo. B16F10 tumors were found to have increased VEGF expression upon LDH knockdown, and therefore were able to compensate the tumor growth inhibition-driven by LDH deficiency through increased tumor vascularization in vivo. In contrast, LDH-V suppressed Lewis Lung cells demonstrated an impeded glucose uptake ability under normoxic and hypoxic conditions. Subsequently, a genome-wide gene expression analysis and functional assays of LDH-A deficient and control HT29 clones revealed potential new oncogenic features of LDH-A in tumor migration and invasion as well as a feedback loop to HIF1alpha. In summary, the collective data presented in this thesis provide new insights of LDH-V in tumor progression and therapeutic strategies for the treatment of cancer.

No description available.

Rainbow trout lactate dehydrogenase (LDH) B2' B2' , B2' B2" and B2" B2" phenotypes were tested under a variety of conditions for swimming endurance, oxygen consumption rates, and blood pH, p02, and lactate. The B2" B2" phenotype exhibited a lower swimming endurance, under a limited oxygen supply, than the B2' B2' and B2' B2" phenotypes. Blood pH at fatigue did not differ among the LOH phenotypes that swam under low oxygen conditions (2 mg/1). Blood p02 at fatigue was significantly higher for females than for males under low oxygen conditions. Oxygen consumption rates were significantly higher for males than for females at dissolved oxygen concentrations of 3 mg/1 to 8 mg/1. The depression in oxygen consumption rates at low oxygen levels was greater for males than for females. The increase in blood lactate with swimming under low oxygen concentrations and static exposure to low oxygen depended on LOH phenotype. The adaptive significance of the LDH B2' and B2" alleles was discussed. Thirteen populations of Intermountain cutthroat trout were sampled for biochemical genetic variation. Variation was observed in muscle aspartate aminotransferase for all populations which would fit a genetic model of two loci and three alleles. Unique muscle A group lactate dehydrogenase variation was observed in Snake Valley cutthroat trout which suggested the presence of several variant alleles. Allele frequencies for tetrazolium oxidase changed during three consecutive years in Bear Lake cutthroat trout that were trapped and spawned artificially. The possible causes of those changes were discussed. No variation was observed in alphaglycerophosphate dehydrogenase, isocitrate dehydrogenase, eye lactate dehydrogenase or muscle malate dehydrogenase in cutthroat trout.

The lens of the eye needs to be transparent with a high refractive index to focus images on the retina. In cataracts the lens becomes opaque, eventually leading to blindness. There are many possible causes of cataract but a lot of evidence implicates oxidative damage as contributing to opacification. This includes epidemiological studies showing that diets rich in antioxidants lowered the prevalence of cataract. This research tested the hypothesis that if cataracts were at least partially caused by oxidative damage then their progression would be slowed by application of antioxidants. The antioxidants used were two plant compounds found in the diet, resveratrol and quercetin. The system used was sheep lenses cultured in Eagles Minimal Essential Medium (EMEM). Lenses remained transparent for up to 7 days in EMEM but became opaque within 24 h when exposed to 1 mM hydrogen peroxide (H2O2). The lens is exposed to H2O2 in vivo as it is found in the aqueous humor. Prior Lenses pre-treated with quercetin reduced but did not prevent opacification. Lens cell death, as determined by measurement of leakage of lactate dehydrogenase, was found to increase with H2O2 and the increase was prevented by pre-treatment with antioxidants. The role of the endogenous antioxidant glutathione was also investigated. It was found that H2O2 decreased the amount of reduced glutathione in the lens cortex and increased the levels of oxidised glutathione but only at levels of 2 mM and above. Thus the results of this research indicate that H2O2 at low concentration (1 mM) is able to damage lens cells and cause opacification without affecting the reduced glutathione levels and that the exogenous antioxidants have some ability to protect the lens.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Ebselen is a seleno compound whose antioxidant properties have been attributed to its thiol-peroxidase and thioredoxin-like activity: it decomposes peroxides at the expense of reduced thiols. However, the excessive oxidation of thiols can be potentially toxic when it is not associated with peroxides degradation. Thus, this work investigated if LDH can be a possible in vitro target to toxicity of ebselen in comparison with diphenyl diselenide and diphenyl ditelluride, two antioxidant organochalcogens that can easily interact with thiol in exchange reaction. LDH inhibition was tested in homogenates from rat liver and heart, and in purified LDH from rabbit muscle. Ebselen was the most potent inhibitor of LDH. A maximal inhibitory effect was obtained at 2 μM to LDH purified and at 20 μM to LDH from heart and liver homogenates. Moreover, diphenyl diselenide followed by diphenyl ditelluride also presented a significant inhibitory effect on LDH activity. In addition, we observe that DTT was able to revert the inhibition of LDH induced by all compounds tested, confirming the involvement of essential thiol groups on LDH inhibition by organocalchogens. In conclusion, our results show that liver and heart LDH may be a possible target for toxicity of organochalcogens at relative low concentrations. However, the protection afforded by substrates may hide this potential molecular target of organochalcogenides. Our results also indicate that the use of LDH as a marker of cell viability may be biased by a direct inhibitory effect of ebselen or other chalcogenides on LDH, resulting in false protection in in vitro system. / O Ebselen é um composto de selênio o qual tem suas propriedades antioxidantes atribuídas à sua atividade mimética da tiorredoxina e tiolperoxidase: ele decompõe peróxidos à custa de tióis reduzidos. Contudo, a oxidação excessiva de tióis pode ser potencialmente tóxica quando não esta associada com a degradação de peróxidos. Assim, este trabalho investiga se a LDH pode ser um possível alvo à toxicidade do ebselen, em comparação com o disseleneto de difenila e o ditelureto de difenila, dois organocalcogênios antioxidantes que podem facilmente interagir com grupos tiol. A inibição da LDH foi testada em homogeneizados de fígado e coração de ratos, e em LDH purificada de músculo de coelhos. O Ebselen foi o mais potente inibidor da LDH. O seu efeito inibitório máximo foi obtido com 2 μM para a LDH purificada e 20 μM para a LDH de homogeneizados de fígado e coração de ratos. Além disso, o disseleneto de difenila, seguido do ditelureto de difenila, também apresentaram efeito inibitório significativo sobre a atividade da LDH. Em adição, observou-se que o DTT foi capaz de reverter a inibição da LDH induzida pelos compostos testados, confirmando o envolvimento de grupos tiol essenciais da LDH no processo de inibição pelos organocalcogênios. Em conclusão, estes resultados mostram que a LDH de fígado e coração pode ser um possível alvo para a toxicologia de organocalcogênios a doses relativamente baixas. Nossos resultados também indicam que o uso da LDH como um marcador de viabilidade celular pode ser mascarada por um efeito inibitório direto do ebselen, ou outros calcogênios, sobre a LDH, resultando em uma falsa proteção em um sistema in vitro.