Intracellular redox state and control of gluconeogenesis in perfused chicken liver

The role of the cellular redox state in the control of gluconeogenesis was studied in hemoglobin-free perfused chicken liver, by fluorimetric measurement of the redox states of intracellular pyridine nucleotides. The aminotransferase inhibitor, aminooxyacetate, completely inhibited gluconeogenesis from lactate in the perfused rat liver and to a small extent in the perfused chicken liver. In chicken liver, the highest rate of glucose production was seen with lactate, followed by fructose, pyruvate, and glycerol. When compared at 5 mM, the rate of glucose production from pyruvate was only 10% of that from lactate. Glucose production from a pyruvate/lactate mixture decreased with increasing proportions of pyruvate, together with redox changes of pyridine nucleotides to a more oxidized state. Increased reduction of pyridine nucleotides upon infusion of ethanol was associated with an increased glucose production from pyruvate, and the increase was abolished during octanoate infusion. This abolishment was accompanied by an increase in the acetoacetate to beta-hydroxybutyrate ratio with an oxidation of pyridine nucleotides. The octanoate-inhibited gluconeogenesis occurred at the higher lactate concentration (10 mM) with a transient oxidation of pyridine nucleotides. No significant inhibition was observed at 1 mM lactate, although an instant reduction of pyridine nucleotides was taking place. The rate of beta-hydroxybutyrate generation during octanoate infusion was 2.2 times higher at 1 mM than at 10 mM lactate. The inhibitory effect of octanoate on glyconeogenesis was completely relieved by the addition of NH4Cl. The results demonstrate that the regeneration of NADH in the cytosol is limited in chicken liver, and that gluconeogenesis is regulated, in part, by alteration in the redox states of mitochondria and cytosol.     

Level of redox potential as a possible contributing influence in the pathogenicity of oral anaerobes

Dental plaque anaerobes may be associated with the etiology of periodontal disease. This has created an interest in the potential pathogenicity of oral anaerobes. We compared the metabolic activity of anaerobic corynebacteria (C. parvum, C. anaerobium) and corresponding aerobic species (C. diphtheriae, C. xerosis). The anaerobes exhibited lower levels of RNA synthesis, ranging from 5 to 10 fold over the aerobes. We further examined these anaerobes, plus Actinomyces naeslundi N16 (isolated from the anaerobic region of periodontally-diseased tissues), for the influence of redox potential on RNA level and antigenic function. Notable increases in RNA were found at specific Eh levels; the extent and direction of the changes varied with the different organisms. This environmental feature appeared to effect corresponding changes in agglutinability and PCA reactivity with antisera against the anaerobes cultured at different redox potentials. For example, while antisera against certain organisms (C. parvum, A. naeslundi) cultured under the most reuced conditions showed an intense PCA reaction, other antisera against the same organism cultured under less reduced conditions were non-reactive. Hence, alterations in redox potential may lead to alteredetabolism and to altered antigencity. Our results imply such a microbial response to environmental stress.     

Mitochondrial citrate synthase is immobilized in vivo

The enzymes of the tricarboxylic acid cycle in the mitochondrial matrix are proposed to form a multienzyme complex, in which there is channeling of substrates between enzyme active sites. However no direct evidence has been obtained in vivo for the involvement of these enzymes in such a complex. We have labeled the tricarboxylic acid cycle enzyme, citrate synthase 1, in the yeast Saccharomyces cerevisiae, by biosynthetic incorporation of 5-fluorotryptophan. Comparison of the 19F NMR resonance intensities from the labeled enzyme in the intact cell and in cell-free lysates indicated that the enzyme is motionally restricted in vivo, consistent with its participation in a multienzyme complex.     

Mitochondrial redox change in gerbil hippocampus before and after transient ischemia

Athletes who need high endurance capacity often use training at moderately high altitude (1500-3000 m) to improve oxygen delivery and utilization because of a hypoxia-induced increase of the red blood cell volume and adaptations at the muscular level. As maximal heart rates decrease at high altitude and plasma lactate levels for a given workload change during prolonged exposure to high altitude, it can be difficult to control and adapt the intensity and duration of the work-outs. Furthermore, maximal performance capacity decreases and therefore training intensity at high altitude is usually reduced compared to training at sea level. To avoid these disadvantages at high altitude a concept of living at moderately high altitude and training at lower elevations, termed "live high-train low" evolved, opposing the conventional concept of "live high-train high". A third option using a hypobaric chamber ("live low-train low") is hardly used anymore for training athletes. Studies on the effects of conventional high-altitude training for the improvement of athletic performance often lack a rigorous controlled design and yield controversial results. Regarding the new concept of "live high-train low" there is only one controlled study on college athletes and it shows a minor advantage of this new approach compared to conventional high-altitude training. However, training concepts are especially important for elite competitive athletes, and controlled studies with such individuals are very difficult to perform. Therefore, it appears that today we cannot answer the question of whether altitude-specific physiologic factors or non-altitude-related benefits of training camps account for the success of individual athletes.     

Beyond global oxygen supply-demand relations: in search of measures of dysoxia

Variations in serum molecular forms of prolactin (PRL) from an adolescent woman presenting amenorrhea-galactorrhea are reported. Persistent hyperprolactinemia and hypoestrogenism were demonstrated as well as the presence of a pituitary tumor with suprasellar extension. Bromocriptine was given at progressive doses up to 37 mg daily, decreasing the hyperprolactinemia and galactorrhea. After 2 years of treatment the patient noticed symptoms of gastric intolerance, bromocriptine was discontinued and a rebound of hyperprolactinemia was observed. Lisuride was administered instead resulting in a new decrease in PRL serum levels, disappearance of galactorrhea and beginning of regular menses. Serum gel chromatographic analysis was carried out before and during lisuride treatment. The first chromatographic analysis showed a predominance of high molecular weight (approximately 66 KD) PRL, accounting for more than 90% of the immunoreactive PRL. The second chromatography showed the major peak of immunoreactive PRL displaced to the right (molecular weight of 22 KD), which was eluted near the PRL standard. With these chromatographic patterns it is concluded that the pituitary macroprolactinoma secreted different molecular forms of PRL and treatment with lisuride appeared to exert some effect on the PRL molecular size secreted by the pituitary.     

Ring hydroxylation of [o-3H]methoxychlor as a probe for liver microsomal CYP2B activity: potential for in vivo CYP2B assay

An in vitro radiometric assay selective for inducible CYP2B activity is described. The assay is based on the quantification of 3H2O release that occurs during o-ring hydroxylation of [o-3H]methoxychlor by liver microsomes in the presence of NADPH. 3H2O is isolated by removing > 99.9% of the parent compound and organic metabolites by facile charcoal extraction and filtration. There was no evidence for an NIH shift during ring hydroxylation, and there was little or no isotope effect. Selectivity for CYP2B was demonstrated using liver microsomes prepared from rats and mice treated with inducers of different CYP isoforms. Ring hydroxylation of [o-3H]methoxychlor was elevated 11.4-fold over control values in liver microsomes from male rats treated with phenobarbital. With mice, phenobarbital treatment elevated liver microsomal ring hydroxylation 7.1-fold. Clofibrate, 3-methylcholanthrene, or beta-naphthoflavone treatment of male rats or pyridine treatment of female rats did not elevate liver microsomal ring-hydroxylase activity, indicating that CYP4A, 1A, and 2E1 do not support this reaction. In female rats, dexamethasone and pregnenolone-16 alpha-carbonitrile treatment elevated ring hydroxylation up to 5.5- and 3.2-fold, respectively, an activity that may be attributed to CYP2B induction in those animals. Incubation of liver microsomes from phenobarbital-treated males with monospecific anti-CYP2B monoclonal antibodies (Mab) inhibited ring-hydroxylase activity up to 86%, demonstrating predominantly CYP2B-mediated catalysis. An 86% inhibition by these Mabs was also observed using liver microsomes from male mice treated with phenobarbital, indicating the assay is not limited to rats. The CYP2B mechanism-based inhibitor orphenadrine caused a 76% decline in activity, providing further evidence for CYP2B involvement. Unlike other CYP2B-selective assays, this method may be readily adapted to in vivo studies, by measuring urinary excretion of 3H2O as an indication of total body CYP2B activity.     

Evaluation of high density DRAMs as a nuclear radiation detector

The research is based on the nuclear radiation induced soft error phenomenon associated with dynamic random access memory devices (DRAMs). Samples of 256 kbit and 1 Mbit decapped DRAMs from several manufactures were exposed to standard alpha sources and showed a linear response with an intrinsic detection efficiency approaching 10%. Sensitivity studies were performed to evaluate the effects of DRAM operating voltage, refresh frequency and the data pattern stored prior to irradiation. The associated mechanism of soft error phenomenon is discussed. Samples were also exposed to gamma rays up to 10(5) rad to examine the total dose effect. The annealing phenomenon after gamma exposure is also presented.     

Mitochondrial respiration and the state of adenine nucleotide phosphorylation in rat liver following multiple administration of 3-methylcholanthrene and phenobarbital]

Five infertile males, ages 25 to 35, with oligospermia and varicocele had following gonadotropin-releasing hormone (LRF) infusion a rise of serum follicle-stimulating hormone, luteinizing hormone, and testosterone levels which was not different from that of normal fertile males. The response of these hormones to LRF infusion was unaltered by spermatic vein ligation, but a significant elevation of the sperm count occurred. Thus, improvement in sperm count following spermatic vein ligation is not mediated via changes in peripheral gonadotropin or testosterone concentrations.     

Mitochondrial DNA synthesis studied autoradiographically in various cell types in vivo

An accelerated weight gain is noted in the heart of Ca-deficient, hypertensive chick embryos maintained in a shell-less culture in vitro. We previously observed that the Ca handling property of cardiomyocytes isolated from the shell-less embryo is altered, i.e., faster Ca uptake, suggesting a requirement for adequate Ca supply and/or proper Ca handling in embryonic cardiac development. In this study, we have examined the function of Ca on cardiomyocytes by analyzing the effects of 1) various Ca concentration in the culture medium (NCa, 1.8 mmol/ L; HCa, 2.8 mmol/L; LCa, 0.9 mmol/L), and 2) various modulators of Ca handling on cell proliferation and phenotype regulation in chick embryonic cardiomyocytes. The analytical parameters included cell number, DNA content, expression of cell cycle-specific and cardiomyocyte-specific proteins, and creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) enzyme activities. Cell number and total DNA were significantly larger (P < 0.01) in LCa cultures compared with those in NCa. The level of LDH was elevated (P < 0.01), but that of CPK was lowered in LCa. Expression of the G1-S-specific protein PCNA was raised, but that of the contractile proteins myosin and tropomyosin was substantially suppressed in LCa; in HCa, the cells did not proliferate as well, whereas the level of contractile proteins was higher. Thapsigargin, a sarcoplasmic reticulum (SR)-specific, Ca-ATPase inhibitor, simulated the effects of LCa by enhancing cell proliferation and lowering the expression of tropomyosin. These results suggest that culturing in low Ca concentration and inhibition of SR Ca pumping enhance myocardial cell proliferation and suppress sarcomeric protein expression, perhaps by inducing cellular de-differentiation. The in vitro effects of medium Ca concentration and Ca handling modulators on cardiomyocytes also suggest that the in vivo cardiomegaly of the SL embryos is a direct result of Ca-deficiency, and that Ca is important in the phenotype regulation of cardiomyocytes.     

Mitochondrial transmembrane potential and free radical production in excitotoxic neurodegeneration

OBJECTIVE: To evaluate quality-of-life (QOL) parameters in patients undergoing esophagectomy, curative or palliative, for carcinoma. DESIGN: Nonconsecutive case series. PATIENTS: Eighty-eight patients who underwent esophagectomy for cancer (curative, n=49 [56%]; palliative, n=39 [44%]) provided QOL assessments over an 18-month period. SETTING: Procedures for referral care were performed by a single team of clinicians in a tertiary referral center. Evaluations of QOL were made by 1 independent trained investigator. OUTCOME MEASURES: Data were documented by questionnaire at interview and parameters evaluated included an esophageal module for the type and quantity of food intake, severity of related symptoms on eating, Eastern Cooperative Oncology Groups (ECOG) performance status, sleep, pain, leisure activity, working capacity, outlook on life, general well-being, and support from family and friends. A summation of selected parameters was used to calculate a total score. RESULTS: Significant improvements were recorded in both the curative and palliative groups for at least 1 year following surgery in the type (P<.03) and quantity (P<.03) of food intake and severity of diet-related symptoms (P<.02), when compared with preoperative considerations. Findings were comparable between the groups with regard to dietary intake. Pain status and total scores were worse in the palliative group at 9 months postoperatively but no significant differences between the groups were evident at any time for sleep, leisure activity, and ECOG performance status. CONCLUSIONS: To our knowledge, there are no previous data regarding a comparison of QOL considerations in patients who have undergone either potentially curative or palliative esophagectomy for malignant disease. Data analysis revealed that palliative esophagectomy provided enhanced QOL with marked symptomatic benefits and enjoyment of daily living comparable to that observed following curative resection.     

Metabolic abnormalities of mitochondrial redox potential in postburn multiple system organ failure

Forty-five burn patients underwent sequential assays for plasma acetoacetate and beta-hydroxybutyrate concentrations as well as plasma amino acid levels. Those patients who went on to develop multiple system organ failure were noted to have a decrease in their acetoacetate concentration with time, whereas there was no change in those patients who failed to develop multiple system organ failure. The plasma concentration of beta-hydroxybutyrate was not altered by multiple system organ failure. In addition, the plasma acetoacetate/beta-hydroxybutyrate ratio was found to be directly related to the plasma concentration of branched chain amino acids and inversely related to the concentration of aromatic amino acids.     

The use of lidocaine as a test of liver function in liver transplantation

The hepatic metabolism of lidocaine to monoethyl-glycinexylidide (MEGX) is the basis of a dynamic test of liver function. To understand its potential value in liver transplantation, the latter has been considered in the following three separate stages: pretransplantation assessment of potential candidates, potential liver donors, and the transplant recipient. In pretransplantation patients, data support its role in assessing risk of morbidity and mortality. In assessment of the liver transplant donor, there are differences concerning apparent usefulness, and these must be resolved. In the liver transplant recipient, serial measurements are useful to measure real-time hepatic metabolic activity. Low MEGX values reflect the clinical condition of the patient, and the importance of entirely assessing the patient, not just noting the test result, is paramount. This review has considered the role of the MEGX test in liver transplantation.     

Modulation of GDP-dissociation inhibitor protein membrane retention by the cellular redox state in adipocytes

Genetic screens in zebrafish have provided mutations in hundreds of genes with essential functions in the developing embryo. To investigate the possible uses of chromosomal rearrangements in the analysis of these mutations, we genetically characterized three gamma-ray induced alleles of cyclops (cyc), a gene required for development of midline structures. We show that cyc maps near one end of Linkage Group 12 (LG 12) and that this region is involved in a reciprocal translocation with LG 2 in one gamma-ray induced mutation, cyc(b213). The translocated segments together cover approximately 5% of the genetic map, and we show that this rearrangement is useful for mapping cloned genes that reside in the affected chromosomal regions. The other two alleles, cyc(b16) and cyc(b229), have deletions in the distal region of LG 12. Interestingly, both of these mutations suppress recombination between genetic markers in LG 12, including markers at a distance from the deletion. This observation raises the possibility that these deletions affect a site required for meiotic recombination on the LG 12 chromosome. The cyc(b16) and cyc(b229) mutations may be useful for balancing other lethal mutations located in the distal region of LG 12. These results show that chromosomal rearrangements can provide useful resources for mapping and genetic analyses in zebrafish.