Critical thermal maxima of early life stages of three tropical fishes: Effects of rearing temperature and experimental heating rate.

Marine ectotherms are often sensitive to thermal stress, and certain life stages can be particularly vulnerable (e.g., larvae or spawners). In this study, we investigated the critical thermal maxima (CTmax) of larval and early juvenile life stages of three tropical marine fishes (Acanthochromis polyacanthus, Amphiprion melanopus, and Lates calcarifer). We tested for potential effects of developmental acclimation, life stage, and experimental heating rates, and we measured metabolic enzyme activities from aerobic (citrate synthase, CS) and anaerobic pathways (lactate dehydrogenase, LDH). A slightly elevated rearing temperature neither influenced CTmax nor CS activity, which otherwise could have indicated thermal acclimation. However, we found CTmax to either remain stable (Acanthrochromis polyacanthus) or increase with body mass during early ontogeny (Amphiprion melanopus and Lates calcarifer). In all three species, faster heating rates lead to higher CTmax. Acute temperature stress did not change CS or LDH activities, suggesting that overall aerobic and anaerobic metabolism remained stable. Lates calcarifer, a catadromous species that migrates from oceanic to riverine habitats upon metamorphosis, had higher CTmax than the two coral reef fish species. We highlight that, for obtaining conservative estimates of a fish species' upper thermal limits, several developmental stages and body mass ranges should be examined. Moreover, upper thermal limits should be assessed using standardized heating rates. This will not only benefit comparative approaches but also aid in assessing geographic (re-) distributions and climate change sensitivity of marine fishes.

Egg activation in physiological polyspermy.

Fertilization is indispensable not only for restoring diploid genomes but also for the initiation of early embryonic cell cycles in sexual reproduction. While most animals exhibit monospermy, which is ensured by polyspermy blocks to prevent the entry of extra sperm into the egg at fertilization, several animals exhibit physiological polyspermy, in which the entry of several sperm is permitted but only one sperm nucleus participates in the formation of a zygote nucleus. Polyspermy requires that the sperm transmit the egg activation signal more slowly, thus allowing the egg to accept several sperm. An increase in intracellular Ca(2+) concentration induced by the fertilizing sperm is both necessary and sufficient for egg activation in polyspermy. Multiple small Ca(2+) waves induced by several fertilizing sperm result in a long-lasting Ca(2+) rise, which is a characteristic of polyspermic amphibian eggs. We introduced a novel soluble sperm factor for egg activation, sperm-specific citrate synthase, into polyspermic newt eggs to cause Ca(2+) waves. Citrate synthase may perform dual functions: as an enzyme in mitochondria and as a Ca(2+)-inducing factor in egg cytoplasm. We also discuss the close relationship between the mode of fertilization and the Ca(2+) rise at egg activation and consider changes in this process through evolution in vertebrates.

Low-volume interval training improves muscle oxidative capacity in sedentary adults.

INTRODUCTION: High-intensity interval training (HIT) increases skeletal muscle oxidative capacity similar to traditional endurance training, despite a low total exercise volume. Much of this work has focused on young active individuals, and it is unclear whether the results are applicable to older less active populations. In addition, many studies have used "all-out" variable-load exercise interventions (e.g., repeated Wingate tests) that may not be practical for all individuals. We therefore examined the effect of a more practical low-volume submaximal constant-load HIT protocol on skeletal muscle oxidative capacity and insulin sensitivity in middle-aged adults, who may be at a higher risk for inactivity-related disorders. METHODS: Seven sedentary but otherwise healthy individuals (three women) with a mean ± SD age, body mass index, and peak oxygen uptake (VO(2peak)) of 45 ± 5 yr, 27 ± 5 kg·m(-2), and 30 ± 3 mL·kg(-1)·min(-1) performed six training sessions during 2 wk. Each session involved 10 × 1-min cycling at ∼60% of peak power achieved during a ramp VO(2peak) test (eliciting ∼80%-95% of HR reserve) with 1 min of recovery between intervals. Needle biopsy samples (vastus lateralis) were obtained before training and ∼72 h after the final training session. RESULTS: Muscle oxidative capacity, as reflected by the protein content of citrate synthase and cytochrome c oxidase subunit IV, increased by ∼35% after training. The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α was increased by ∼56% after training, but the transcriptional corepressor receptor-interacting protein 140 remained unchanged. Glucose transporter protein content increased ∼260%, and insulin sensitivity, on the basis of the insulin sensitivity index homeostasis model assessment, improved by ∼35% after training. CONCLUSIONS: Constant-load low-volume HIT may be a practical time-efficient strategy to induce metabolic adaptations that reduce the risk for inactivity-related disorders in previously sedentary middle-aged adults.

The Ca2+ increase by the sperm factor in physiologically polyspermic newt fertilization: its signaling mechanism in egg cytoplasm and the species-specificity.

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca(2+) increase occurs as a Ca(2+) wave at each sperm entry site in the polyspermic egg. Some Ca(2+) waves are preceded by a transient spike-like Ca(2+) increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca(2+) wave was induced by a sperm factor derived from sperm cytoplasm after sperm-egg membrane fusion. The Ca(2+) increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca(2+) store for the Ca(2+) wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.

Effects of exercise training on vasodilatory protein expression and activity in rats.

Increased endothelium-dependent vasodilatation is associated with endurance exercise training. The purpose of this study was to test the hypothesis that increased endothelial nitric oxide synthase (eNOS) protein function, but not increased vascular smooth muscle sensitivity to NO, underlies augmented endothelium-dependent dilatation with training. To test these hypotheses, rats ran on a treadmill at 30 m/min (10% grade) for 60 min/day, 5 days/week, over 8-12 weeks (Trn). Training efficacy was demonstrated by greater (P < 0.05) hindlimb muscle citrate synthase activity and left ventricular mass-body mass ratio in Trn compared with sedentary control rats (Sed). Expression of eNOS protein in the aorta was increased with training (Sed, 1.00 ± 0.18 normalized units; Trn, 1.55 ± 0.23; P < 0.05). Aortic NOS activity was, however, unchanged by training (Sed, 1,505 ± 288 fmol/h/mg protein; Trn, 1,650 ± 247; n.s.). Expression of heat shock protein 90 and protein kinase B/Akt was not different between groups, nor was their association with eNOS. In follow-up series of rats, phosphorylated eNOS content (Serine 1177) was similar for Sed and Trn in both the aorta and gastrocnemius feed artery. Aortic NOS activity with eNOS phosphorylation status preserved was also similar between groups. Finally, cGMP concentration with a NO donor did not differ between groups (Sed, 73.0 ± 20.2 pmol/mg protein; Trn, 62.5 ± 12.9; n.s.). These findings indicate that training-induced increases in eNOS protein expression are not coupled to augmented function, illustrating the complexity of eNOS regulation. Further, they show that vascular sensitivity to NO is not altered by exercise training.

Genome-wide gene regulation of biosynthesis and energy generation by a novel transcriptional repressor in Geobacter species.

Geobacter species play important roles in bioremediation of contaminated environments and in electricity production from waste organic matter in microbial fuel cells. To better understand physiology of Geobacter species, expression and function of citrate synthase, a key enzyme in the TCA cycle that is important for organic acid oxidation in Geobacter species, was investigated. Geobacter sulfurreducens did not require citrate synthase for growth with hydrogen as the electron donor and fumarate as the electron acceptor. Expression of the citrate synthase gene, gltA, was repressed by a transcription factor under this growth condition. Functional and comparative genomics approaches, coupled with genetic and biochemical assays, identified a novel transcription factor termed HgtR that acts as a repressor for gltA. Further analysis revealed that HgtR is a global regulator for genes involved in biosynthesis and energy generation in Geobacter species. The hgtR gene was essential for growth with hydrogen, during which hgtR expression was induced. These findings provide important new insights into the mechanisms by which Geobacter species regulate their central metabolism under different environmental conditions.

Chronic activation of 5'-AMP-activated protein kinase changes myosin heavy chain expression in growing pigs.

The purpose of this study was to determine the effect of 5'-AMP-activated protein kinase (AMPK) on energy metabolism and myosin heavy chain (MyHC) isoform expression in growing pigs using chronic treatment with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) as a model. Four-week-old pigs were given daily injections of AICAR or 0.9% saline for 10 d. Treatment with AICAR increased (P < 0.05) AMPK activity in semitendinosus muscles (STM). Expression of skeletal muscle specific glucose transporter 4 (GLUT4) was also enhanced (P < 0.05) by AICAR treatment. Using real-time PCR, electrophoresis, and Western blot analyses, we confirmed that AICAR treatment caused a decrease (P < 0.05) in type IIa MyHC isoform mRNA and protein levels and a concomitant increase (P < 0.05) in type IIx MyHC containing fibers. Consistent with a MyHC isoform shift from IIa to IIx, muscles from pigs treated with AICAR had greater (P < 0.05) lactate dehydrogenase (LDH) activity. Moreover, muscle of treated pigs expressed greater (P < 0.05) message for LDH. Administration of AICAR, however, did not alter expression of PPAR-gamma coactivator-1alpha, fatty acid translocase, citrate synthase, or the activity of cytochrome c oxidase. Overall, these results indicate that activation of AMPK by AICAR causes muscle to assume a faster-contracting, more glycolytic nature. These data are in direct contrast to documented effects in rodent models, but these effects may be dependent on the time of administration and the overall growth status of the animal.

Investigation of carbon metabolism in "Dehalococcoides ethenogenes" strain 195 by use of isotopomer and transcriptomic analyses.

Members of the genus "Dehalococcoides" are the only known microorganisms that can completely dechlorinate tetrachloroethene and trichloroethene to the innocuous end product, ethene. This study examines the central metabolism in "Dehalococcoides ethenogenes" strain 195 via (13)C-labeled tracer experiments. Supported by the genome annotation and the transcript profile, isotopomer analysis of key metabolites clarifies ambiguities in the genome annotation and identifies an unusual biosynthetic pathway in strain 195. First, the (13)C-labeling studies revealed that strain 195 contains complete amino acid biosynthesis pathways, even though current genome annotation suggests that several of these pathways are incomplete. Second, the tricarboxylic acid cycle of strain 195 is confirmed to be branched, and the Wood-Ljungdahl carbon fixation pathway is shown to not be functionally active under our experimental conditions; rather, CO(2) is assimilated via two reactions, conversion of acetyl-coenzyme A (acetyl coenzyme A [acetyl-CoA]) to pyruvate catalyzed by pyruvate synthase (DET0724-0727) and pyruvate conversion to oxaloacetate via pyruvate carboxylase (DET0119-0120). Third, the (13)C-labeling studies also suggested that isoleucine is synthesized from acetyl-CoA and pyruvate via citramalate synthase (CimA, EC 2.3.1.182), rather than from the common pathway via threonine ammonia-lyase (EC 4.3.1.19). Finally, evidence is presented that strain 195 may contain an undocumented citrate synthase (>95% Re-type stereospecific), i.e., a novel Re-citrate synthase that is apparently different from the one recently reported in Clostridium kluyveri.

Cold adaptation of enzyme reaction rates.

A major issue for organisms living at extreme temperatures is to preserve both stability and activity of their enzymes. Cold-adapted enzymes generally have a reduced thermal stability, to counteract freezing, and show a lower enthalpy and a more negative entropy of activation compared to mesophilic and thermophilic homologues. Such a balance of thermodynamic activation parameters can make the reaction rate decrease more linearly, rather than exponentially, as the temperature is lowered, but the structural basis for rate optimization toward low working temperatures remains unclear. In order to computationally address this problem, it is clear that reaction simulations rather than standard molecular dynamics calculations are needed. We have thus carried out extensive computer simulations of the keto-enol(ate) isomerization steps in differently adapted citrate synthases to explore the structure-function relationships behind catalytic rate adaptation to different temperatures. The calculations reproduce the absolute rates of the psychrophilic and mesophilic enzymes at 300 K, as well as the lower enthalpy and more negative entropy of activation of the cold-adapted enzyme, where the latter simulation result is obtained from high-precision Arrhenius plots. The overall catalytic effect originates from electrostatic stabilization of the transition state and enolate and the reduction of reorganization free energy. The simulations, however, show psychrophilic, mesophilic, and hyperthermophilic citrate synthases to have increasingly stronger electrostatic stabilization of the transition state, while the energetic penalty in terms of internal protein interactions follows the reverse order with the cold-adapted enzyme having the most favorable energy term. The lower activation enthalpy and more negative activation entropy observed for cold-adapted enzymes are found to be associated with a decreased protein stiffness. The origin of this effect is, however, not localized to the active site but to other regions of the protein structure.

Methylcitrate synthase from Aspergillus fumigatus is essential for manifestation of invasive aspergillosis.

Invasive aspergillosis is a life-threatening disease mainly caused by the fungus Aspergillus fumigatus. In immunocompromised individuals conidia are not efficiently inactivated, which can end in invasive fungal growth. However, the metabolic requirements of the fungus are hardly known. Earlier investigations revealed an accumulation of toxic propionyl-CoA in a methylcitrate synthase mutant, when grown on propionyl-CoA-generating carbon sources. During invasive growth propionyl-CoA could derive from proteins, which are released from infected host tissues. We therefore assumed that a methylcitrate synthase mutant might display an attenuated virulence. Here we show that the addition of propionate to cell culture medium enhanced the ability of alveolar macrophages to kill methylcitrate synthase mutant but not wild-type conidia. When tested in a murine infection model, the methylcitrate synthase mutant displayed attenuated virulence and, furthermore, was cleared from tissues when mice survived the first phase of acute infection. The amplification of cDNA from infected mouse lungs confirmed the transcription of the methylcitrate synthase gene during invasion, which leads to the suggestion that amino acids indeed serve as growth-supporting nutrients during invasive growth of A. fumigatus. Thus, blocking of methylcitrate synthase activity abrogates fungal growth and provides a suitable target for new antifungals.

Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster.

Eggs of the newt, Cynops pyrrhogaster, arrested at the second meiotic metaphase are activated by sperm at fertilization and then complete meiosis to initiate development. We highly purified a sperm factor for egg activation from a sperm extract with several chromatographies. The purified fraction containing only a 45 kDa protein induced egg activation accompanied by an intracellular Ca2+ increase when injected into unfertilized eggs. Although injection of mouse phospholipase C (PLC) zeta-mRNA caused a Ca2+ increase and egg activation, partial amino acid sequences of the 45 kDa protein were homologous to those of Xenopus citrate synthase, but not to PLCs. An anti-porcine citrate synthase antibody recognized the 45 kDa protein both in the purified fraction and in the sperm extract. Treatment with the anti-citrate synthase antibody reduced the egg-activation activity in the sperm extract. Injection of porcine citrate synthase or mRNA of Xenopus citrate synthase induced a Ca2+ increase and caused egg activation. A large amount of the 45 kDa protein was localized in two lines elongated from the neck to the middle piece of sperm. These results indicate that the 45 kDa protein is a major component of the sperm factor for egg activation at newt fertilization.

Downhill treadmill running trains the rat spinotrapezius muscle.

There are currently no models of exercise that recruit and train muscles, such as the rat spinotrapezius, that are suitable for transmission intravital microscopic investigation of the microcirculation. Recent experimental evidence supports the concept that running downhill on a motorized treadmill recruits the spinotrapezius muscle of the rat. Based on these results, we tested the hypothesis that 6 wk of downhill running (-14 degrees grade) for 1 h/day, 5 days/wk, at a speed of up to 35 m/min, would 1) increase whole body peak oxygen uptake (Vo(2 peak)), 2) increase spinotrapezius citrate synthase activity, and 3) reduce the fatigability of the spinotrapezius during electrically induced 1-Hz submaximal tetanic contractions. Trained rats (n = 6) elicited a 24% higher Vo(2 peak) (in ml.min(-1).kg(-1): sedentary 58.5 +/- 2.0, trained 72.7 +/- 2.0; P < 0.001) and a 41% greater spinotrapezius citrate synthase activity (in mumol.min(-1).g(-1): sedentary 14.1 +/- 0.7, trained 19.9 +/- 0.9; P < 0.001) compared with sedentary controls (n = 6). In addition, at the end of 15 min of electrical stimulation, trained rats sustained a greater percentage of the initial tension than their sedentary counterparts (control 34.3 +/- 3.1%, trained 59.0 +/- 7.2%; P < 0.05). These results demonstrate that downhill running is successful in promoting training adaptations in the spinotrapezius muscle, including increased oxidative capacity and resistance to fatigue. Since the spinotrapezius muscle is commonly used in studies using intravital microscopy to examine microcirculatory function at rest and during contractions, our results suggest that downhill running is an effective training paradigm that can be used to investigate the mechanisms for improved microcirculatory function following exercise training in health and disease.

Effect of short-term sprint interval training on human skeletal muscle carbohydrate metabolism during exercise and time-trial performance.

Our laboratory recently showed that six sessions of sprint interval training (SIT) over 2 wk increased muscle oxidative potential and cycle endurance capacity (Burgomaster KA, Hughes SC, Heigenhauser GJF, Bradwell SN, and Gibala MJ. J Appl Physiol 98: 1895-1900, 2005). The present study tested the hypothesis that short-term SIT would reduce skeletal muscle glycogenolysis and lactate accumulation during exercise and increase the capacity for pyruvate oxidation via pyruvate dehydrogenase (PDH). Eight men [peak oxygen uptake (VO2 peak)=3.8+/-0.2 l/min] performed six sessions of SIT (4-7x30-s "all-out" cycling with 4 min of recovery) over 2 wk. Before and after SIT, biopsies (vastus lateralis) were obtained at rest and after each stage of a two-stage cycling test that consisted of 10 min at approximately 60% followed by 10 min at approximately 90% of VO2 peak. Subjects also performed a 250-kJ time trial (TT) before and after SIT to assess changes in cycling performance. SIT increased muscle glycogen content by approximately 50% (main effect, P=0.04) and the maximal activity of citrate synthase (posttraining: 7.8+/-0.4 vs. pretraining: 7.0+/-0.4 mol.kg protein -1.h-1; P=0.04), but the maximal activity of 3-hydroxyacyl-CoA dehydrogenase was unchanged (posttraining: 5.1+/-0.7 vs. pretraining: 4.9+/-0.6 mol.kg protein -1.h-1; P=0.76). The active form of PDH was higher after training (main effect, P=0.04), and net muscle glycogenolysis (posttraining: 100+/-16 vs. pretraining: 139+/-11 mmol/kg dry wt; P=0.03) and lactate accumulation (posttraining: 55+/-2 vs. pretraining: 63+/-1 mmol/kg dry wt; P=0.03) during exercise were reduced. TT performance improved by 9.6% after training (posttraining: 15.5+/-0.5 vs. pretraining: 17.2+/-1.0 min; P=0.006), and a control group (n=8, VO2 peak=3.9+/-0.2 l/min) showed no change in performance when tested 2 wk apart without SIT (posttraining: 18.8+/-1.2 vs. pretraining: 18.9+/-1.2 min; P=0.74). We conclude that short-term SIT improved cycling TT performance and resulted in a closer matching of glycogenolytic flux and pyruvate oxidation during submaximal exercise.

Disruption of the peroxisomal citrate synthase CshA affects cell growth and multicellular development in Dictyostelium discoideum.

Non-mitochondrial citrate synthase catalyses citrate synthesis in the glyoxylate cycle in gluconeogenesis. Screening Dictyostelium discoideum mutants generated by insertional mutagenesis isolated a poor-growing mutant that displayed aberrant developmental morphology on bacterial lawns. Axenically grown mutants developed normally and formed mature fruiting bodies on buffered agar. The affected locus encoded a novel protein (CshA) that was homologous to glyoxysomal citrate synthase. cshA was expressed maximally during vegetative growth and gradually decreased through subsequent developmental stages. An in vitro citrate synthase assay revealed that cshA disruption resulted in a 50% reduction in enzyme activity, implicating CshA as an active citrate synthase. The amino-terminus of CshA was found to have an atypical mitochondrial targeting signal, instead containing a unique nonapeptide sequence (RINILANHL) that was homologous to the conserved peroxisomal targeting signal 2 (PTS2). CshA protein was shown to be localized in the peroxisomes, and the RINILANHL sequence only efficiently targeted the peroxisomal green fluorescent protein. The growth defect of cshA(-) cells was associated with the impairment of phagocytosis and fluid-phase endocytosis, independent from cytokinesis. Disrupted multicellular development on bacterial lawns resulted from the abnormal susceptibility to the environmental conditions, perhaps because of citrate insufficiency. Taken together, these results provide new insights into the function of peroxisomal citrate synthase in cell growth and multicellular development.

Skeletal muscle enzymes as predictors of 24-h energy metabolism in reduced-obese persons.

BACKGROUND: Little is known about the effects of weight loss on the relation between skeletal muscle enzymes and energy metabolism. OBJECTIVE: This study was performed retrospectively to investigate the relation between skeletal muscle enzymes and 24-h energy metabolism in obese persons before and after weight loss. DESIGN: Ten women and 9 men [with body mass indexes (in kg/m(2)) > 30] underwent a 15-wk weight-loss program (-700 kcal/d). Body weight and composition, 24-h energy metabolism (whole-body indirect calorimetry), and maximal activities of phosphofructokinase (EC 2.7.1.11), creatine kinase (CK; EC 2.7.3.2), citrate synthase (CS; EC 4.1.3.7), 3-hydroxyacyl-CoA dehydrogenase (HADH; EC 1.1.1.35), and cytochrome-c oxidase (COX; EC 1.9.3.1) were determined from biopsy samples of the vastus lateralis taken before and after weight loss. RESULTS: Before weight loss, fat-free mass (FFM) was the only predictor of 24-h energy expenditure (R(2) = 0.70, P < 0.001), whereas the cumulative variance in sleeping metabolic rate explained by FFM and fat mass (FM) was 83% (P < 0.001). After weight loss, CS (r = 0.45, P = 0.05) and COX (r = 0.65, P < 0.01) were significantly associated with 24-h energy expenditure, whereas CK (r = 0.53, P < 0.05), CS (r = 0.45, P < 0.05), COX (r = 0.64, P < 0.01), and HADH (r = 0.45, P = 0.05) were all significant correlates of sleeping metabolic rate. After weight loss, FFM, FM, and COX explained 84% (P < 0.01) of the variance in 24-h energy expenditure, whereas FFM, FM, and CK all contributed to the cumulative variance in sleeping metabolic rate explained by this model (R(2) = 0.82, P < 0.05). CONCLUSION: Maximal activities of key skeletal muscle enzymes contribute to the variability in 24-h energy metabolism in reduced-obese persons.

Metabolic activities in Azospirillum lipoferum grown in the presence of NH4+.

The utilization of some agro-industrial wastes as soil conditioners to provide free-living nitrogen-fixing bacterial populations (e.g. Azospirillum spp.) with carbon and energy sources, may be an interesting perspective for agriculture. However, the presence of ammonium nitrogen in cultivated soils and/or various wastes could inhibit the growth of the nitrogen-fixing populations. The present investigation shows that growth of Azospirillum lipoferum was restricted at a dissolved oxygen (DO) concentration equal to 135 microM, when the initial NH4Cl concentration increased from 0.5 to 0.9 g/l. The activities of both citrate synthase (CS) and isocitrate dehydrogenase were significantly decreased in the presence of 0.9 g/l NH4Cl (e.g., 40% and 66%, respectively, in cells incubated for 95 h), while ammonium assimilation occurred via the glutamate dehydrogenase reaction. Furthermore, growth limitation occurred even in the presence of 0.5 g/l NH4Cl, when the DO concentration decreased from 135 to 30 microM. The activities of both CS and succinate dehydrogenase were dramatically decreased in cells grown at the lower DO concentration (e.g., 90% and 93% respectively, in a 95 h incubation), while ammonium assimilation was limited due to the low activities of both glutamate dehydrogenase and glutamate synthase. It is concluded that the threshold of ammonium concentration at which growth of A. lipoferum is limited, depends on the DO concentration in the medium.

Genetic analysis of iron citrate toxicity in yeast: implications for mammalian iron homeostasis.

Deletion of the yeast homologue of frataxin, YFH1, results in mitochondrial iron accumulation and respiratory deficiency (petite formation). We used a genetic screen to identify mutants that modify iron-associated defects in respiratory activity in Deltayfh1 cells. A deletion in the peroxisomal citrate synthase CIT2 in Deltayfh1 cells decreased the rate of petite formation. Conversely, overexpression of CIT2 in Deltayfh1 cells increased the rate of respiratory loss. Citrate toxicity in Deltayfh1 cells was dependent on iron but was independent of mitochondrial respiration. Citrate toxicity was not restricted to iron-laden mitochondria but also occurred when iron accumulated in cytosol because of impaired vacuolar iron storage. These results suggest that high levels of citrate may promote iron-mediated tissue damage.

Implications for altered glutamate and GABA metabolism in the dorsolateral prefrontal cortex of aged schizophrenic patients.

OBJECTIVE: Pharmacological, clinical, and postmortem studies suggest altered gamma-aminobutyric acid (GABA)-ergic and glutamatergic function in patients with schizophrenia. The dorsolateral prefrontal cortex is one key locus of abnormality. The precise neurochemical mechanisms underlying neurotransmitter alterations, such as hypoglutamatergia or GABA dysfunction, are not well understood. This study investigated key biochemical elements of GABA and glutamate metabolism in brain specimens from schizophrenic patients. The activities of nine principal GABA and glutamate-associated metabolic enzymes were measured concurrently in the dorsolateral prefrontal cortex of antemortem-assessed and neuropathologically characterized schizophrenic and comparison subjects. METHOD: Postmortem dorsolateral prefrontal cortex specimens from schizophrenia, Alzheimer's disease, and normal nonpsychiatric comparison subjects were assayed to determine activities of the principal glutamate and GABA-metabolizing enzymes glutamine synthetase, glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, phosphate-activated glutaminase, alanine aminotransferase, aspartate aminotransferase, glutamic acid decarboxylase, GABA-transaminase, and succinic semialdehyde dehydrogenase. RESULTS: Glutamic acid decarboxylase activities were twofold greater and phosphate-activated glutaminase activities were fourfold greater in the schizophrenic group than in the comparison group. Differences in postmortem interval, tissue pH, inhibition of phosphate-activated glutaminase, and medication effects could not account for the differences. Differences in phosphate-activated glutaminase and glutamic acid decarboxylase activities in equivalent specimens from Alzheimer's patients were not observed. The activities of the remaining enzymes were unchanged. CONCLUSIONS: Greater phosphate-activated glutaminase and glutamic acid decarboxylase activities, specific to schizophrenia patients, provide additional biochemical evidence that dorsolateral prefrontal cortex glutamate and GABA metabolism is altered in schizophrenic subjects. These greater activities are consistent with models of a dysregulated glutamatergic/GABA-ergic state in schizophrenia.

Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. A case study of endosymbiotic gene transfer.

The citric acid or tricarboxylic acid cycle is a central element of higher-plant carbon metabolism which provides, among other things, electrons for oxidative phosphorylation in the inner mitochondrial membrane, intermediates for amino-acid biosynthesis, and oxaloacetate for gluconeogenesis from succinate derived from fatty acids via the glyoxylate cycle in glyoxysomes. The tricarboxylic acid cycle is a typical mitochondrial pathway and is widespread among alpha-proteobacteria, the group of eubacteria as defined under rRNA systematics from which mitochondria arose. Most of the enzymes of the tricarboxylic acid cycle are encoded in the nucleus in higher eukaryotes, and several have been previously shown to branch with their homologues from alpha-proteobacteria, indicating that the eukaryotic nuclear genes were acquired from the mitochondrial genome during the course of evolution. Here, we investigate the individual evolutionary histories of all of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle using protein maximum likelihood phylogenies, focusing on the evolutionary origin of the nuclear-encoded proteins in higher plants. The results indicate that about half of the proteins involved in this eukaryotic pathway are most similar to their alpha-proteobacterial homologues, whereas the remainder are most similar to eubacterial, but not specifically alpha-proteobacterial, homologues. A consideration of (a) the process of lateral gene transfer among free-living prokaryotes and (b) the mechanistics of endosymbiotic (symbiont-to-host) gene transfer reveals that it is unrealistic to expect all nuclear genes that were acquired from the alpha-proteobacterial ancestor of mitochondria to branch specifically with their homologues encoded in the genomes of contemporary alpha-proteobacteria. Rather, even if molecular phylogenetics were to work perfectly (which it does not), then some nuclear-encoded proteins that were acquired from the alpha-proteobacterial ancestor of mitochondria should, in phylogenetic trees, branch with homologues that are no longer found in most alpha-proteobacterial genomes, and some should reside on long branches that reveal affinity to eubacterial rather than archaebacterial homologues, but no particular affinity for any specific eubacterial donor.