Lactate Medium Research Articles

Intestinal Resident Yeast Candida glabrata Requires Cyb2p-Mediated Lactate Assimilation to Adapt in Mouse Intestine

The intestinal resident Candida glabrata opportunistically infects humans. However few genetic factors for adaptation in the intestine are identified in this fungus. Here we describe the C. glabrata CYB2 gene encoding lactate dehydrogenase as an adaptation factor for survival in the intestine. CYB2 was identified as a virulence factor by a silkworm infection study. To determine the function of CYB2, we analysed in vitro phenotypes of the mutant Δcyb2. The Δcyb2 mutant grew well in glucose medium under aerobic and anaerobic conditions, was not supersensitive to nitric oxide which has fungicidal-effect in phagocytes, and had normal levels of general virulence factors protease, lipase and adherence activities. A previous report suggested that Cyb2p is responsible for lactate assimilation. Additionally, it was speculated that lactate assimilation was required for Candida virulence because Candida must synthesize glucose via gluconeogenesis under glucose-limited conditions such as in the host. Indeed, the Δcyb2 mutant could not grow on lactate medium in which lactate is the sole carbon source in the absence of glucose, indicating that Cyb2p plays a role in lactate assimilation. We hypothesized that Cyb2p-mediated lactate assimilation is necessary for proliferation in the intestinal tract, as the intestine is rich in lactate produced by bacteria flora, but not glucose. The Δcyb2 mutant showed 100-fold decreased adaptation and few cells of Saccharomyces cerevisiae can adapt in mouse ceca. Interestingly, C. glabrata could assimilate lactate under hypoxic conditions, dependent on CYB2, but not yeast S. cerevisiae. Because accessible oxygen is limited in the intestine, the ability for lactate assimilation in hypoxic conditions may provide an advantage for a pathogenic yeast. From those results, we conclude that Cyb2p-mediated lactate assimilation is an intestinal adaptation factor of C. glabrata.

Abstract 993: Enhanced EGFR activity of prostate cancer cells by low pH, low glucose environment

Abstract Background: Hypoxia, lack of nutrients and high aerobic glycolysis creates a microenvironment of high lactate, low glucose and low pH conditions within a solid tumor. The purpose of the current research is to study how the cancer cells adapt to a microenvironment with low pH, low glucose and high lactate levels with selective growth advantages. Methods: We simulated solid tumor microenvironment conditions by making DMEM media with low glucose (2mM), high lactate (10mM) and low pH (6.5). To study the growth advantages of this microenvironment, we determined the effect of low pH, low glucose and lactate medium on EGFR activation. Prostate cancer (PC3) cells were grown in six-well plates in 10% serum containing DMEM and serum starved overnight before they were changed to experimental medium. Control cells were grown in normal DMEM. We treated PC3 cells growing in normal DMEM and experimental medium with EGF at 1ng/ml. Protein samples were collected at different time intervals and subjected to western blotting to observe the activation of EGFR by using phospho EGFR antibody. EGFR and actin antibodies were used as control and loading controls, respectively. Results: Robust and sustained phosphorylation of EGFR for longer time was observed with EGF treatment when the cells were grown in low pH, low glucose and high lactate medium when compared with the cells grown in normal DMEM medium. Conclusion: The robust activation of EGFR with low pH, low glucose and high lactate medium suggest that the microenvironment containing high lactate provides growth or survival advantages to the cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 993. doi:10.1158/1538-7445.AM2011-993

An Analysis of Performance in Elite Young Cross-Country Skiing: Classical Style Vs Skate Skiing

Problem statement: The aim of this study was to assess the differences in the skiing performance of two skiing styles (classical style Vs. skate skiing), in a group of young high-level skiers. Approach: Four high-level male junior cross-country skiers from the Centre of Winter Sports of Aragon (Spain), were selected for this study that was developed during two competitions: 10 km free technique and 10 km classical technique performed in the same circuit and under the same conditions (snow quality, temperature, wind and relative humidity). Physiological variables, maximal and medium heart rate, blood lactate concentration (basal, final and during the recovery, after 2, 4, 6, 8 and 10 min) and medium skiing speed, were analyzed with GPS Garmin Forerruner 305® Heart Rate Pulse and Lactate Dr. Lange analyzer®. Normally distributed data (Kolmogorov-Smirnov Test and Shapiro-Wilk) were statistically analyzed with one way ANOVA and t-Test for post-hoc comparisons (p<0.05). No-normally distributed data were analyzed with the Friedman and Wilcoxon Tests (p<0.05). Results: No significant differences have been recorded in the medium skiing speed, maximal and medium heart rate between the two styles. The peak heart rate was the same in both styles (188 bpm), although medium heart rate was lower in skate skiing vs. classical skiing (165 bpm Vs. 175 bpm). Blood lactate concentrations recorded significant differences during 4, 6, 8 and 10 min of recovery, being higher in free technique (12.7±1.7; 11.4±1.8; 10.4±1.4 and 8.9±1.6 mmol L−1 Vs. 8.7±1.3; 7.5±1.6; 6.3±1.3 and 5.6±1.1 mmol L−1, respectively). Conclusion: The results of this study suggest that skate skiing generates a higher impact on the cross-country skier than classical skiing, due to higher blood lactate concentrations during 4, 6, 8 and 10 min of recovery. The own characteristics of the free technique may be the reason of the lower mean heart rate.

Bioelectrochemical Mn(II) Leaching from Manganese Ore by Lactococcus lactis SK071115

L. lactis sk071115 has been shown to grow more actively and generate lower levels of lactate in glucose-defined medium with nitrate than in medium with Mn(IV). By adding Mn(IV) to a L. lactis culture, lactate production was relatively reduced in combination with Mn(II) production, but cell mass production levels did not increase. Both cell-free extract and intact L. lactis cells reacted electrochemically with Mn(IV) but did not react with Mn(II) upon cyclic voltammetry using neutral red (NR) as an electron mediator. A modified graphite felt cathode with NR (NR-cathode) was employed to induce electrochemical reducing equivalence for bacterial metabolism. Cell-free L. lactis extract catalyzed the reduction of Mn(IV) to Mn(II) under both control and electrochemical reduction conditions; however, the levels of Mn(II) generated under electrochemical reduction conditions were approximately 4 times those generated under control conditions. The levels of Mn(II) generated by the catalysis of L. lactis immobilized in the NR-cathode (L-NR-cathode) under electrochemical reduction conditions were more than 4 times that generated under control conditions. Mn(II) production levels were increased by approximately 2.5 and 4.5 times by the addition of citrate to the reactant under control and electrochemical reduction conditions, respectively. The cumulative Mn(II) produced from manganese ore by catalysis of the L-NR-cathode for 30 days reached levels of approximately 3,800 and 16,000 mg/l under control and electrochemical reduction conditions, respectively. In conclusion, the electrochemical reduction reaction generated by the NR-cathode activated the biochemical reduction of Mn(IV) to Mn(II) by L. lactis.

Large-scale production of magnetosomes by chemostat culture of Magnetospirillum gryphiswaldense at high cell density

BackgroundMagnetotactic bacteria have long intrigued researchers because they synthesize intracellular nano-scale (40-100 nm) magnetic particles composed of Fe3O4, termed magnetosomes. Current research focuses on the molecular mechanisms of bacterial magnetosome formation and its practical applications in biotechnology and medicine. Practical applications of magnetosomes are based on their ferrimagnetism, nanoscale size, narrow size distribution, dispersal ability, and membrane-bound structure. However, the applications of magnetosomes have not yet been developed commercially, mainly because magnetotactic bacteria are difficult to cultivate and consistent, high yields of magnetosomes have not yet been achieved.ResultsWe report a chemostat culture technique based on pH-stat feeding that yields a high cell density of Magnetospirillum gryphiswaldense strain MSR-1 in an auto-fermentor. In a large-scale fermentor, the magnetosome yield was significantly increased by adjusting the stirring rate and airflow which regulates the level of dissolved oxygen (DO). Low concentration of sodium lactate (2.3 mmol l-1) in the culture medium resulted in more rapid cell growth and higher magnetosome yield than high concentration of lactate (20 mmol l-1). The optical density of M. gryphiswaldense cells reached 12 OD565 nm after 36 hr culture in a 42 L fermentor. Magnetosome yield and productivity were 83.23 ± 5.36 mg l-1 (dry weight) and 55.49 mg l-1 day-1, respectively, which were 1.99 and 3.32 times higher than the corresponding values in our previous study.ConclusionsCompared to previously reported methods, our culture technique with the MSR-1 strain significantly increased cell density, cell yield, and magnetosome yield in a shorter time window and thus reduced the cost of production. The cell density and magnetosome yield reported here are the highest so far achieved with a magnetotactic bacteria. Refinement of this technique will enable further increase of cell density and magnetosome yield.

In vitro effects of simulated microgravity on Sertoli cell function

With the advent of space flights questions concerning the effects of microgravity (0×G) on human reproductive physiology have received great attention. The aim of this study was to evaluate the influence of 0×G on Sertoli cells. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal and Sex Hormone Binding Globilin (SHBG) changes by immunohistochemistry, for antioxidant content by RT-PCR and for culture medium lactate concentrations by protein chemistry. Cells were cultured for 6, 24 and 48 h on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1×G) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or lysed and RNA-extracted or used for culture medium lactate measurements as needed. At 0×G, Sertoli cytoskeleton became disorganized, microtubules fragmented and SHBG undetectable already after 24 h, with alterations worsening by 48 h. It was evident that various antioxidant systems appreciably increased during the first 24 h but significantly decreased at 48 h. No changes occurred in the 1×G samples. Initially, 0×G seemed to disturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate production at 0×G slightly decreased after 24 h. Further experiments are needed in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out male infertility as a possible consequence, which could be a problem, as life expectancy increases.

Products of glucose and lactate fermentation, and utilization of amino acids by Clostridium estertheticum subspp. laramiense and estertheticum growing in meat juice medium.

The type strains of Clostridium estertheticum subsp. laramiense and C. estertheticum subsp. estertheticum both utilized glucose and glycogen when growing in meat juice medium and fermented lactate, but ceased growth when glucose was exhausted. The fermentation products from glucose were butyrate, acetate, and formate; those from lactate were 1-butanol, ethanol, butyrate, and formate. Both organisms utilized several amino acids (not containing sulfur) during their cultivation in meat juice medium and did not produce H(2)S. The optimum and maximum temperatures for growth of C. estertheticum subsp. laramiense were 10 degrees C, and 20 to 22 degrees C, respectively. Those same optimum and maximum temperatures have previously been determined for C. estertheticum subsp. estertheticum. The pH range for growth of the two organisms, 5.5 to 7.5, was also the same. Both organisms were beta-hemolytic and formed subterminal spores. Thus, the organisms did not display the difference in fermentation products, optimum and maximum temperatures, hemolysis, and spore position that were reported to be the differentiating characteristics of the subspecies. The findings indicate that vacuum-packaged meat should be spoiled similarly by the two type strains.

Toward understanding the thermodynamics of TALSPEAK process. Medium effects on actinide complexation

The ingenious combination of lactate and diethylenetriamine-N,N,N',N",N"-pentaacetic acid (DTPA) as an aqueous actinide-complexing medium forms the basis of the successful separation of americium and curium from lanthanides known as the TALSPEAK process. While numerous reports in the prior literature have focused on the optimization of this solvent extraction system, considerably less attention has been devoted to the understanding of the basic thermodynamic features of the complex fluids responsible for the separation. The available thermochemical information for both lactate and DTPA protonation and metal complexation reactions are representative of the behavior of these ions under idealized conditions. Significant departures from the speciation predicted based on reported thermodynamic values may be present in the TALSPEAK aqueous environment. Thus, thermodynamic parameters describing the separation chemistry of this process require further examination at conditions significantly removed from conventional ideal systems commonly employed in fundamental solution chemistry. In this report, calorimetric studies of lactate protonation equilibrium illustrate a distinct influence of nitrate, perchlorate, methylsulfonate and triflate on the thermodynamic parameters associated with TALSPEAK aqueous chemistry. Triflate is identified a closer non-complexing representative of lactate solution chemistry. The reported initial two stability constants for the complexation of americium and neodymium by lactate in triflate medium suggest that the identified medium effects do not significantly affect the speciation.

Estudo eletroquímico comparativo do aço ISO 5832-9 em diferentes meios de interesse biológico

Foi estudado o comportamento eletroquímico a 37°C do aço inoxidável ISO 5832-9, em meios de NaCl 0,9 %, de Ringer Lactato e meio mínimo de Eagle (MEM), por voltametria linear e análises da superfície por microscopia eletrônica de varredura (MEV) e por espectroscopia por dispersão de energia (EDS). Foram feitos ensaios mecânicos e testes de toxicidade. O aço ISO 5832-9 se encontra passivado no potencial de corrosão e não apresenta corrosão por pite nos três meios estudados em toda faixa de potencial investigada, desde o potencial de corrosão até 50 mV acima do potencial de transpassivação. Em meio de MEM, no entanto, as análises por MEV e EDS mostraram que o referido aço, nesse valor mais elevado de potencial, apresentou um comportamento diferente, com perda das inclusões de óxido de manganês. Os potenciais de corrosão, Ecorr (potencial de circuito aberto estacionário) bem como os valores de densidade de corrente de passivação, variaram na seguinte ordem: Ecorr, RL &lt; Ecorr, NaCl &lt; Ecorr, MEM. e jMEM &lt;&lt; jRL ≅ jNaCl. No ensaio de citotoxicidade, o aço foi caracterizado como não-tóxico.

ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1

The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Delta hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.

Protective Effect of Combination of Sulforaphane and Riluzole on Glutamate-Mediated Excitotoxicity

Threohydroxyaspartate (THA) causes glutamate excitotoxicity in motor neurons in organotypic culture of rat spinal cord. Some drugs, including sulforaphane (SF) and riluzole, can protect motor neuron against excitotoxicity. It has been demonstrated that SF is a potent inducer of Phase II enzymes, while riluzole is a classic anti-glutamate agent. The objective of the current study is to investigate whether the combination of SF and riluzole is superior to either one used alone. In our study, the combination of SF with riluzole not only stimulates the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), reduced nicotinamide adenine dinucleotide phosphate (NADPH): quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1), but also reduces the extracellular accumulation of glutamate. When used at optimal doses, SF (10 microM) and riluzole (5 microM), either alone or in combination, all exert significant and similar neuroprotection, as measured by the number of motor neuron, medium malondialdehyde (MDA) level and lactate dehydrogenase (LDH) level. When used at low doses, the combination is better than each agent used alone. In conclusion, these results suggest the potential utility of combination use of SF and riluzole for protection of motor neuron against excitotoxicity.

Abstract B97: Dichloroacetate (DCA) enhances activities of sorafenib against hepatocellular carcinoma (HCC) via modulation of aberrant cellular metabolism of HCC cells

Abstract To meet high energy demand for proliferation, cancer cells rely more on aerobic glycolysis than on oxidative phosphorylation (OXPHOS). This aberrant cellular metabolism of cancer, well-known as the “Warburg effect”, may confer survival advantages and drug resistance to cancer cells. Pyruvate dehydrogenase (PDH) is the gatekeeper of OXPHOS, and is inhibited by pyruvate dehyrogenase kinase (PDK) through phosphorylation. Dichloroacetate (DCA), a synthetic PDK inhibitor, can reverse glycosis to OXPHOS through PDH activation, and has been used for the treatment of congenital lactic acidosis for more than 30 years. The present study explored whether modulating the Warburg effect by DCA can improve the efficacy of sorafenib (a multi-kinase inhibitor of Raf, VEGFR and PDGFR), the current standard systemic therapy for HCC. Modulation of the Warburg effect was evaluated by measurement of lactate in culture medium and PDH activity. In vitro growth-inhibitory effects were measured by median-effect analyses in a panel of human HCC cell-lines (Hep3B, Huh-7, Sk-hep1, HCC36 and HA22T) cultured in normoxic condition. In vivo growth-inhibitory effects were measured in both subcutaneous and orthotopic murine HCC xenograft models. Apoptosis was evaluated by flow cytometry (annexin V staining and subG1 fraction analysis) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Expression of apoptosis-related proteins was analyzed by Western blotting. We found that DCA reduced lactate production and increased PDH activity in HCC cells in a dose-dependent manner, indicating switching glucose metabolism from glycolysis to OXPHOS. DCA (10–60 mM) induced synergistic growth-inhibitory and apoptotic effects with sorafenib in most HCC cell-lines tested. Nude mice carrying subcutaneously or intrahepatically implanted Hep3B cells were treated with vehicle, DCA 100 mg/kg /day (equivalent to the dosage used to treat lactic acidosis in human), sorafenib 10 mg/kg /day, and DCA plus sorafenib, via gavage for 21 days. DCA-sorafenib combination synergistically suppressed the tumor growth without advertently affecting the body weight of mice. Synergistic induction of apoptosis was shown by TUNEL assay. Our data suggest that modulation of aberrant cellular metabolism of HCC cells by DCA may improve the clinical efficacy of sorafenib. (Supported by grants NSC97-3112-B-002-012, NSC98-3112-B-002-007, and NSC98-3112-B-002-037) Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B97.

Aup1-mediated Regulation of Rtg3 during Mitophagy

Mitophagy is an autophagic process that degrades mitochondria by an intracellular engulfment that leads to their delivery into the lumen of the cell's hydrolytic compartment, such as the lysosome in animal cells or the vacuole in yeast. It is hypothesized that such processes serve a quality control function to prevent or slow the accumulation of malfunctioning mitochondria, which are thought in turn to underlie central aspects of the aging process in eukaryotic organisms. We recently identified a conserved mitochondrial protein phosphatase homolog, Aup1, which is required for efficient stationary phase mitophagy in yeast. In the present report, we demonstrate that the retrograde signaling pathway (RTG) is defective in aup1Delta mutants. In agreement with a role for Aup1 in the regulation of the RTG pathway, we find that deletion of RTG3, a transcription factor that mediates the RTG response, causes a defect in stationary phase mitophagy and that deletion of AUP1 leads to changes in Rtg3 phosphorylation patterns under these conditions. In addition, we find that mitophagic conditions lead to induction of RTG pathway target genes in an Aup1-dependent fashion. Thus, our results suggest that the function of Aup1 in mitophagy could be explained through its regulation of Rtg3-dependent transcription.

Engulfment of Axon Debris by Microglia Requires p38 MAPK Activity

The clearance of debris after injuries to the nervous system is a critical step for restoration of the injured neural network. Microglia are thought to be involved in elimination of degenerating neurons and axons in the central nervous system (CNS), presumably restoring a favorable environment after CNS injuries. However, the mechanism underlying debris clearance remains elusive. Here, we establish an in vitro assay system to estimate phagocytosis of axon debris. We employed a Wallerian degeneration model by cutting axons of the cortical explants. The cortical explants were co-cultured with primary microglia or the MG5 microglial cell line. The cortical neurites were then transected. MG5 cells efficiently phagocytosed the debris, whereas primary microglia showed phagocytic activity only when they were activated by lipopolysaccharide or interferon-beta. When MG5 cells or primary microglia were co-cultured with degenerated axons, p38 mitogen-activated protein kinase (MAPK) was activated in these cells. Engulfment of axon debris was blocked by the p38 MAPK inhibitor SB203580, indicating that p38 MAPK is required for phagocytic activity. Receptors that recognize dying cells appeared not to be involved in the process of phagocytosis of the axon debris. In addition, the axons undergoing Wallerian degeneration did not release lactate dehydrogenase, suggesting that degeneration of the severed axons and apoptosis may represent two distinct self-destruction programs. We observed regrowth of the severed neurites after axon debris was removed. This finding suggests that axon debris, in addition to myelin debris, is an inhibitory factor for axon regeneration.

Culture pH and osmolality influence proliferation and embryoid body yields of murine embryonic stem cells

The realization of many stem cell based regenerative therapies will depend on culture production. Murine embryonic stem cells (mESC) provide a practical model for stem cell process research as they can be readily obtained at relatively high numbers and purities. However, an understanding of their environmental tolerance ranges is still lacking. These tolerance ranges were explored using an embryoid body (EB) formation assay as a functional measure of mESC maintenance. The mESC were exposed to wide ranges of initial medium glucose, glutamine, ammonium, lactate, pH and osmolality conditions. Within the common ranges of conventional maintenance cultures, the only environmental variables that significantly influenced EB yields were pH and osmolality. Dose–response experiments with these two variables revealed that, within 48h, the EB yield was, for example, ∼3-fold decreased (p<0.05) when mESC were cultured either at an initial pH of 7.0 or an osmolality of 400mOsm/kg compared to a medium at pH 7.3 and 300mOsm/kg. This decline was due to decreases in both the growth rate and in the fraction of EB-forming cells. More extreme pH (6.7 or 7.75) as well as osmolality (200 or 500mOsm/kg) conditions reduced the EB formation potential to even lower levels. The decreased growth rates and EB forming potential of mESC cultured at pH 6.7 or 7.75 for 24 or 48h, when returned to pH 7.3 medium, recovered to control levels within 96–144h. These studies provide guidance in developing optimal environmental tolerance ranges within which both the mESC output from maintenance cultures as well as the subsequent EB yields can be maximized.

Response of blood vessels in vitro to hyperbaric oxygen (HBO): Modulation of VEGF and NOx release by external lactate or arginine

Hyperbaric oxygen therapy (HBO) is suggested to promote angiogenesis during wound healing, but the mechanisms involved are not understood. This study used a novel isolated blood vessel preparation to explore the effects of air, normobaric oxygen or hyperbaric oxygen (2.2 ATA for 90 min) on the angiogenesis factor, vascular endothelial growth factor (VEGF), nitrite and nitrate (NOx), lactate dehydrogenase (LDH) and lactate release from the tissue in normal Krebs Ringer, and the Ringer supplemented with either l-arginine, or 15 mM lactate to mimic a wound environment, or both (l-arginine+lactate). The in vitro blood vessel preparation remained viable during all experiments. There were no effects of HBO treatment on any of the parameters measured in normal Krebs Ringer, but some treatment-dependent effects were observed in supplemented Krebs Ringer. In the lactate supplemented Krebs Ringer, medium LDH levels increased in response to either normobaric oxygen (NBO) or HBO, compared to air alone. There were also small, but statistically significant increases in total glutathione due to HBO treatment, compared to NBO or air in the lactate supplemented medium, and in the combined supplement. There were no effects of HBO on NOx, changes in external medium lactate levels, or tissue VEGF in any of the Krebs Ringers tested. However, post treatment increases in VEGF were observed in the lactate supplemented medium, and for lactate release into the medium for the combined supplement. We conclude that HBO does not cause NO or VEGF production from the blood vessel in normal Krebs Ringer, but the data from supplemented medium show that the response of the tissue is subtly affected by the chemical environment around the blood vessel, and the tissue is more responsive to HBO when wound conditions are mimicked.

Interaction of Tim23 with Tim50 Is Essential for Protein Translocation by the Mitochondrial TIM23 Complex

The TIM23 complex is the major translocase of the mitochondrial inner membrane responsible for the import of essentially all matrix proteins and a number of inner membrane proteins. Tim23 and Tim50, two essential proteins of the complex, expose conserved domains into the intermembrane space that interact with each other. Here, we describe in vitro reconstitution of this interaction using recombinantly expressed and purified intermembrane space domains of Tim50 and Tim23. We established two independent methods, chemical cross-linking and surface plasmon resonance, to track their interaction. In addition, we identified mutations in Tim23 that abolish its interaction with Tim50 in vitro. These mutations also destabilized the interaction between the two proteins in vivo, leading to defective import of preproteins via the TIM23 complex and to cell death at higher temperatures. This is the first study to describe the reconstitution of the Tim50-Tim23 interaction in vitro and to identify specific residues of Tim23 that are vital for the interaction with Tim50.

Effects of Chromium Picolinate on Oxidative Damage in Primary Piglet Hepatocytes

Chromium picolinate is a popular nutritional supplement whose safety has been questioned because of the potential risk of oxidative DNA damage. To investigate this possibility, a dose-dependent study was performed in piglet hepatocyte cultures in which low (8 microM), medium (200 microM), and high (400 microM) doses of chromium picolinate were tested and compared to untreated controls. After 48 h incubation, there were no significant differences in the levels of intracellular reactive oxygen species, medium lactate dehydrogenase activity, and comet indicators between the three experimental groups and controls (p > 0.05). In the 8 microM-treated group, the intracellular malondialdehyde content was significantly decreased relative to controls (p < 0.05). All of the studied parameters showed a dose-dependent increase that was statistically significant between the low and high doses (p < 0.05). These results suggest that: (1) chromium picolinate may affect the oxidative status of piglet hepatocytes; (2) the appropriate dose (approximately physiological concentration) of chromium picolinate can inhibit lipid peroxidation, and (3) high doses of chromium picolinate have no significant effects on oxidative damage in piglet hepatocytes, but the existing evidence also imply that exposure to a higher dose appears to be unwarranted.

The Warburg Effect in Leukemia-Stroma Cocultures Is Mediated by Mitochondrial Uncoupling Associated with Uncoupling Protein 2 Activation

In 1956, Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that bypassed the Pasteur effect (i.e., the inhibition of anaerobic fermentation by oxygen). Since then, permanent defects in oxygen consumption that could explain the dependence of cancer cells on aerobic glycolysis have not been identified. Here, we show that under normoxic conditions exposure of leukemia cells to bone marrow-derived mesenchymal stromal cells (MSC) promotes accumulation of lactate in the culture medium and reduces mitochondrial membrane potential (DeltaPsiM) in both cell types. Notably, the consumption of glucose was not altered in cocultures, suggesting that the accumulation of lactate was the result of reduced pyruvate metabolism. Interestingly, the decrease in DeltaPsiM was mediated by mitochondrial uncoupling in leukemia cells and was accompanied by increased expression of uncoupling protein 2 (UCP2). HL60 cells fail to increase UCP2 expression, are not uncoupled after coculture, and do not exhibit increased aerobic glycolysis, whereas small interfering RNA-mediated suppression of UCP2 in OCI-AML3 cells reversed mitochondrial uncoupling and aerobic glycolysis elicited by MSC. Taken together, these data suggest that microenvironment activation of highly conserved mammalian UCPs may facilitate the Warburg effect in the absence of permanent respiratory impairment.

Follicular fluid concentrations of glucose, lactate and pyruvate in buffalo and sheep, and their effects on cultured oocytes, granulosa and cumulus cells

The objective was to determine ovarian follicular fluid concentrations of glucose, lactate, and pyruvate in relation to follicle size in buffalo and sheep. The effect of varying concentrations of these substances on in vitro oocyte maturation, oocyte protein content, and granulosa and cumulus cell growth was also investigated. Follicular fluid was aspirated from various sizes of follicles (from ovaries without a dominant follicle) collected from adult, cycling nonpregnant buffalo (Bubalus bubalis) and sheep (Ovis aries) during the breeding season. Overall, mean (+/-S.E.M.) concentrations (mM) were glucose 2.42+/-0.31 and 1.40+/-0.22, lactate 7.56+/-2.61 and 10.42+/-1.64, and pyruvate 0.02+/-0.01 and 0.002+/-0.00, in buffalo and sheep, respectively. In both species, as follicles became larger, concentrations of glucose significantly increased, lactate significantly decreased, but pyruvate was not affected. Oocyte maturation was higher (P<0.05) in medium containing supra-physiological concentrations of either glucose (5 mM), or pyruvate (10 mM) alone, or physiological concentrations of glucose, lactate and pyruvate in combination, compared to supra-physiological concentrations of lactate (15 mM) alone, or sub- or supra-physiological concentrations of glucose, lactate and pyruvate in combination (both species). The protein content of oocytes was not significantly affected by the concentration of glucose, lactate, and pyruvate in the maturation medium. However, growth of granulosa and cumulus cells was higher (P<0.05) in medium containing supra-physiological concentrations of glucose (5 mM) alone, or pyruvate (10 mM) alone, or physiological, or supra-physiological concentrations of glucose, lactate and pyruvate in combination, compared to supra-physiological concentrations of lactate (15 mM) alone, or sub-physiological concentrations of glucose, lactate and pyruvate in combination (both species). In conclusion, concentrations of glucose, pyruvate and lactate in the medium had cell type-specific effects on oocyte maturation, and on growth of granulosa and cumulus cells. Furthermore, glucose and pyruvate were the principal energy sources for oocytes and follicular somatic cells in buffalo and sheep.