A novel metabolic network leads to enhanced citrate biogenesis in <Emphasis Type="Italic">Pseudomonas </Emphasis><Emphasis Type="Italic">fluorescens</Emphasis> exposed to aluminum toxicity

Aluminum (Al), an environmental toxin, is known to have a negative impact on various biological systems. However, some microbes have devised intricate mechanisms to combat the toxic influence of this trivalent metal. In this study, Pseudomonas fluorescens grown in malate invoked a unique metabolic shift to promote the synthesis of citrate, a metabolite involved in the sequestration of Al. Electrophoretic and spectrophotometric assays revealed several malate-metabolizing enzymes including malate dehydrogenase (MDH) and malic enzyme (ME) displayed increases in activity and expression in the Al-treated cells. Whereas pyruvate dehydrogenase (PDH) also showed increased activity and expression in the Al-stressed cultures, phosphoenolpyruvate carboxykinase (PEPCK) displayed a marked diminution in the Al-treated cells. The upregulation of citrate synthase (CS) coupled with the diminished activities of aconitase (ACN) and NAD-isocitrate dehydrogenase (NAD-ICDH) appeared to be instrumental in the accumulation of citrate. HPLC experiments revealed high levels of citrate in the Al-stressed cultures. Thus, an Al-enriched environment provoked a metabolic shift in P. fluorescens dedicated to the conversion of malate to citrate.     

Overexpression of <Emphasis Type="Italic">Citrus junos</Emphasis> mitochondrial citrate synthase gene in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> confers aluminum tolerance

Aluminum (Al) toxicity is one of the major factors that limit plant growth in acid soils. Al-induced release of organic acids into rhizosphere from the root apex has been identified as a major Al-tolerance mechanism in many plant species. In this study, Al tolerance of Yuzu (Citrus Junos Sieb. ex Tanaka) was tested on the basis of root elongation and the results demonstrated that Yuzu was Al tolerant compared with other plant species. Exposure to Al triggered the exudation of citrate from the Yuzu root. Thus, the mechanism of Al tolerance in Yuzu involved an Al-inducible increase in citrate release. Aluminum also elicited an increase of citrate content and increased the expression level of mitochondrial citrate synthase (CjCS) gene and enzyme activity in Yuzu. The CjCS gene was cloned from Yuzu and overexpressed in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated methods. Increased expression level of the CjCS gene and enhanced enzyme activity were observed in transgenic plants compared with the wild-type plants. Root growth experiments showed that transgenic plants have enhanced levels of Al tolerance. The transgenic Nicotiana plants showed increased levels of citrate in roots compared to wild-type plants. The exudation of citrate from roots of the transgenic plants significantly increased when exposed to Al. The results with transgenic plants suggest that overexpression of mitochondrial CS can be a useful tool to achieve Al tolerance.     

Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis: cloning and expression in Saccharomyces cerevisiae

Citrate synthase, an essential enzyme of the tricarboxylic acid cycle in mitochondria, was purified from acetate-grown Candida tropicalis. Results from SDS-PAGE and gel filtration showed that this enzyme was a dimer composed of 45-kDa subunits. A citrate synthase cDNA fragment was amplified by the 5′-RACE method. Nucleotide sequence analysis of this cDNA fragment revealed that the deduced amino acid sequence contained an extended leader sequence which is suggested to be a mitochondrial targeting signal, as judged from helical wheel analysis. Using this cDNA probe, one genomic citrate synthase clone was isolated from a yeast λEMBL3 library. The nucleotide sequence of the gene encoding C. tropicalis citrate synthase, CtCIT, revealed the presence of a 79-bp intron in the N-terminal region. Sequences essential as yeast splicing motifs were present in this intron. When the CtCIT gene including its intron was introduced into Saccharomyces cerevisiae using the promoter UPR-ICL, citrate synthase activity was highly induced, which strongly indicated that this intron was correctly spliced in S. cerevisiae. Received: 20 November 1996 / Accepted: 25 February 1997     

The effect of thyroid hormones on the aerobic development of locomotor and cardiac muscles in the barnacle goose

The role of thyroid hormones in the development of the locomotor and cardiac muscles of the barnacle goose (Branta leucopsis) was investigated. From 2 weeks of age, goslings were treated with thyroxine, triiodothyronine, or methimazole (a thyroid inhibitor). Birds were killed at 6 weeks (n = 5) or 9 weeks (n = 4) and various locomotor and cardiac muscle masses recorded and tissue samples taken for analysis of citrate synthase activity. The effects of thyroxine and triiodothyronine were not significantly different from each other, except in the case of the iliofibularis at 9 weeks. The mass-specific citrate synthase activity of the iliofibularis, semimembranosus muscles and liver were significantly increased by thyroid hormone treatment. Cardiac muscle showed a significant hypertrophy at 9 weeks of age following treatment with thyroxine. Hypothyroidism induced by treatment with methimazole exhibited the greatest effect on the pectoralis muscle, reducing citrate synthase activity by 33%. Mass-specific citrate synthase activity of the pectoralis and pectoralis mass (% body wt.) were found to be highly correlated (r ² = 0.74) at 6 weeks of age. It is suggested that thyroid hormones may be involved in controlling the tissue-specific timing of the maturation of locomotor and cardiac muscles. Accepted: 16 September 1996     

Comparative studies of coupled assays for phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) from higher plants is usually assayed by using malate dehydrogenase (MDH) as a coupling enzyme. To avoid erroneous readings caused by metal ions, which convert oxaloacetate (OAA) to pyruvate, lactic dehydrogenase can be included. Reporting the total NADH used by both coupling enzymes gives the total OAA production. Microbial PEPC has been assayed by employing citrate synthase (CS) as a coupling enzyme which detects the reaction of CoA with Ellman's reagent. Comparable K_m values for MgPEP are found with the two assays. When MDH alone is used as the coupling system, the V_max value is about 60% larger than the one found with the CS assay. However, when MDH is added to the CS assay without the NADH cofactor, V_max is brought back to the same level as that with the NADH-coupled enzyme. Malate inhibition of PEPC assayed with the CS coupling system is blocked by low concentrations of citrate in the range produced in the assay. High concentrations of citrate inhibit PEPC. Glucose-6-phosphate in concentrations higher than 1 m M blocks the response of PEPC to added MDH in the CS assay.     

Substrate and metal complexes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae provide new insights into the catalytic mechanism

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthases are metal-dependent enzymes that catalyse the first committed step in the biosynthesis of aromatic amino acids in microorganisms and plants, the condensation of 2-phophoenolpyruvate (PEP) and d-erythrose 4-phosphate (E4P) to DAHP. The DAHP synthases are possible targets for fungicides and represent a model system for feedback regulation in metabolic pathways. To gain further insight into the role of the metal ion and the catalytic mechanism in general, the crystal structures of several complexes between the tyrosine-regulated form of DAHP synthase from Saccharomyces cerevisiae and different metal ions and ligands have been determined. The crystal structures provide evidence that the simultaneous presence of a metal ion and PEP result in an ordering of the protein into a conformation that is prepared for binding the second substrate E4P. The site and binding mode of E4P was derived from the 1.5A resolution crystal structure of DAHP synthase in complex with PEP, Co2+, and the E4P analogue glyceraldehyde 3-phosphate. Our data suggest that the oxygen atom of the reactive carbonyl group of E4P replaces a water molecule coordinated to the metal ion, strongly favouring a reaction mechanism where the initial step is a nucleophilic attack of the double bond of PEP on the metal-activated carbonyl group of E4P. Mutagenesis experiments substituting specific amino acids coordinating PEP, the divalent metal ion or the second substrate E4P, result in stable but inactive Aro4p-derivatives and show the importance of these residues for the catalytic mechanism.     

Effect of aging on the response of biochemical markers in the rabbit subjected to short-term partial bladder obstruction

Purpose Partial bladder outlet obstruction (PBOO) results in marked biochemical alterations in the bladder. In this study, we focused on comparison of thapsigargin sensitive sarco/endoplasmic reticulum Ca²⁺ ATPase activity (SERCA) and Citrate Synthase after short term PBOO in young versus old rabbits. Materials and methods A total of 20 young and 20 mature male rabbits were divided into 4 sub-groups of 5 rabbits each (4 obstructed and 1 sham-control rabbit). The rabbits in the groups were evaluated after 1, 3, 7, and 14 days of obstruction, respectively. The activities of SERCA and citrate synthase were examined as markers for sarcoplasmic reticular calcium storage and release and mitochondrial function, respectively. Results The SERCA activity of bladder body smooth muscle in the young animals increased at 7 and 14 days. For the old rabbits, the SERCA activity decreased significantly by 1 day and remained this level throughout the course of obstruction, and was significantly lower than young at all time periods. The citrate synthase activity in the young animals decreased over the 1–7 days, and then returned toward control level by 14 days following obstruction. In the old animals, citrate synthase activity of bladder body smooth muscle progressively decreased over the course of the study, and was significantly lower in the old than the young animals after 14 days obstructed. Conclusion The urinary bladders of the young rabbits have a considerable greater ability to adapt to PBOO than do those of the old rabbits. The deterioration of mitochondrial and SR function may be important mechanisms underlying geriatric voiding dysfunction.     

Evaluation of muscle oxidative potential by 31P-MRS during incremental exercise in old and young humans

The purpose of this study was to compare muscle oxidative capacity between moderately active young and old humans by measuring intracellular threshold (IT) during exercise with ³¹P-magnetic resonance spectroscopy (³¹P-MRS). Changes in phosphocreatine, inorganic phosphate, and intracellular pH were measured by ³¹P-MRS during a progressive unilateral ankle plantar flexion exercise protocol in groups of moderately active old (n=12, mean age 66.7 years) and young (n=13, mean age 26.2 years) individuals. From muscle biopsy samples of the lateral gastrocnemius, citrate synthase (CS) activity was determined in six subjects from each group, and fibre type composition was determined in nine old and ten young subjects. The old group had a lower IT for pH, as a percentage of peak work rate (P<0.05), despite a similar CS activity compared to the young. IT was significantly correlated with CS activity (R=0.59; P<0.05), but not with fibre type composition. It was concluded that metabolic responses to exercise are affected by ageing, as indicated by a lower IT in old compared to young individuals. Accepted: 7 May 1998     

Cloning and overexpression in Escherichia coli of the gene encoding citrate synthase from the hyperthermophilic Archaeon Sulfolobus solfataricus

The citrate synthase (CS) gene from the hyperthermophilic Archaeon Sulfolobus solfataricus has been cloned and sequenced. The gene encodes a polypeptide of 378 amino acids with a calculated polypeptide molecular mass of 42 679. High-level expression was achieved in Escherichia coli and the recombinant citrate synthase was purified to homogeneity using a heat step and dye-ligand affinity chromatography. This procedure yielded approximately 26 mg of pure CS per liter of culture, with a specific activity of 41 U/mg. The enzyme exhibited a half-life of 8 min at 95°C. A homology-modelled structure of the S. solfataricus CS has been generated using the crystal structure of the enzyme from the thermoacidophilic Archaeon Thermoplasma acidophilum with which it displays 58% sequence identity. The modelled structure is discussed with respect to the thermostability properties of the enzyme. Received: August 10, 1997 / Accepted: October 23, 1997     

The Ca 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 Ca²⁺ increase occurs as a Ca²⁺ wave at each sperm entry site in the polyspermic egg. Some Ca²⁺ waves are preceded by a transient spike-like Ca²⁺ increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca²⁺ wave was induced by a sperm factor derived from sperm cytoplasm after sperm–egg membrane fusion. The Ca²⁺ increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca²⁺ store for the Ca²⁺ 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.     

Novel sites in the p65 subunit of NF-kappaB interact with TFIIB to facilitate NF-kappaB induced transcription

The citM gene from Lactococcus lactis CRL264 was demonstrated to encode for an oxaloacetate decarboxylase. The enzyme exhibits high levels of similarity to malic enzymes (MEs) from other organisms. CitM was expressed in Escherichia coli, purified and its oxaloacetate decarboxylase activity was demonstrated by biochemical and genetic studies. The highest oxaloacetate decarboxylation activity was found at low pH in the presence of manganese, and the K_m value for oxaloacetate was 0.52 ± 0.03 mM. However, no malic activity was found for this enzyme. Our studies clearly show a new group of oxaloacetate decarboxylases associated with the citrate fermentation pathway in gram-positive bacteria. Furthermore, the essential catalytic residues were found to be conserved in all members of the ME family, suggesting a common mechanism for oxaloacetate decarboxylation.     

Interactive effect of exercise training and growth hormone administration on glucose tolerance and muscle GLUT4 protein expression in rats

The insulin-resistance effect of growth hormone (GH) administration has been frequently reported. The present study investigated the effect of GH administration on glucose tolerance and muscle GLUT4 protein expression in exercise-trained and untrained rats. Forty-eight rats were weight-matched and assigned to the following 4 groups: control, GH, exercise training, and exercise training + GH groups. After 2 weeks of GH injections (65 µg/kg/day) and exercise training, the glucose tolerance and insulin response were measured in these rats. The GLUT4 protein level, glycogen storage, and citrate synthase activity were determined in red gastrocnemius and plantaris muscles. Daily GH administration elevated the curves of the oral glucose tolerance test and insulin response compared with those of saline-injected control rats. Furthermore, exercise training completely eliminated this GH-induced insulin resistance as determined 18 h after the last bout of exercise training. Additionally, exercise training significantly increased muscle glycogen storage and GLUT4 protein levels. GH administration did not affect the GLUT4 protein and glycogen storage increases induced by exercise training, but the citrate synthase activity in the plantaris muscle was further elevated by GH administration to a level above that induced by training. In conclusion, this is the first study that demonstrates that regular exercise training prevents GH-induced insulin-resistance side effect in rats.     

Intense Exercise Induces Mitochondrial Dysfunction in Mice Brain

There are conflicts between the effects of free radical over-production induced by exercise on neurotrophins and brain oxidative metabolism. The objective of this study was to investigate the effects of intense physical training on brain-derived neurotrophic factor (BDNF) levels, COX activity, and lipoperoxidation levels in mice brain cortex. Twenty-seven adult male CF1 mice were assigned to three groups: control untrained, intermittent treadmill exercise (3 × 15 min/day) and continuous treadmill exercise (45 min/day). Training significantly (P < 0.05) increased citrate synthase activity when compared to untrained control. Blood lactate levels classified the exercise as high intensity. The intermittent training significantly (P < 0.05) reduced in 6.5% the brain cortex COX activity when compared to the control group. BDNF levels significantly (P < 0.05) decreased in both exercise groups. Besides, continuous and intermittent exercise groups significantly (P < 0.05) increased thiobarbituric acid reactive species levels in the brain cortex. In summary, intense exercise promoted brain mitochondrial dysfunction due to decreased BDNF levels in the frontal cortex of mice.     

Tannic acid inhibits in vitro iron-dependent free radical formation

The antioxidant activity of tannic acid (TA), a plant polyphenol claimed to possess antimutagenic and anticarcinogenic activities, was studied by monitoring (i) 2-deoxyribose degradation (a technique for OH detection), (ii) ascorbate oxidation, (iii) ascorbate radical formation (determined by EPR analysis) and (iv) oxygen uptake induced by the system, which comprised Fe(III) complexes (EDTA, nitrilotriacetic acid (NTA) or citrate as co-chelators), ascorbate and oxygen. TA removes Fe(III) from the co-chelators (in the case of EDTA, this removal is slower than with NTA or citrate), forming an iron-TA complex less capable of oxidizing ascorbate into ascorbate radical or mediating 2-deoxyribose degradation. The effectiveness of TA against 2-deoxyribose degradation, ascorbate oxidation and ascorbate radical formation was substantially higher in the presence of iron-NTA (or iron-citrate) than with iron-EDTA, which is consistent with the known formation constants of the iron complexes with the co-chelators. Oxygen uptake and 2-deoxyribose degradation induced by Fe(II) autoxidation were also inhibited by TA. These results indicate that TA inhibits OH formation induced by Fe(III)/ascorbate/O(2) mainly by arresting Fe(III)-induced ascorbate oxidation and Fe(II) autoxidation (which generates Fe(II) and H(2)O(2), respectively), thus limiting the production of Fenton reagents and OH formation. We also hypothesize that the Fe(II) complex with TA exhibits an OH trapping activity, which explains the effect of TA on the Fenton reaction.     

Aluminium induces changes in organic acids metabolism in Coffea arabica suspension cells with differential Al-tolerance

The primary Al-tolerance mechanism in plants involves exudation and/or accumulation of specific organic acid species, which form non-phytotoxic complexes with Al³⁺ under physiological conditions. An evaluation was done of the role of organic acids in the tolerance mechanism of a cell suspension line of coffee Coffea arabica that exhibits Al-tolerance (LAMt) but for which the metabolic tolerance mechanism remains unknown. Significant differences existed in malate dehydrogenase and citrate synthase activities (key enzymes in organic acids metabolism) between protein extracts (day 7 of culture cycle) of the L2 (Al-sensitive) and LAMt (Al-tolerant) cells when cell suspensions were treated with 100 μM AlCl₃. HPLC analysis showed that the suspension cells of both lines exudate malate when incubated in a minimal solution but that exudation was not enhanced by treatment with AlCl₃ (100 μM). This is the first study demonstrating that plant Al-tolerance may be associated with down-regulation of malate dehydrogenase and citrate synthase activities.     

A New Form of Structural Peptidoglycan in Selenomonas ruminantium: Existence of Polyamine in Peptidoglycan

The chemical constituents of calluses of Thujopsis dolabrata (Hiba), Chamaecyparis obtusa (Hinoki), Chamaecyparis pisifera (Sawara), and Platycladus orientalis (Konotegashiwa), all of which belong to Cupressaceae, were examined by GC analysis. The main components of these calluses were diterpenoids of an abietane-type and sitosterol in common, while the chemical constituents of these parent plants were different from each other.     

Maintenance of myoglobin concentration in human skeletal muscle after heavy resistance training

The aim of this study was to determine the effects of 8 weeks of resistance training (RT) on the myoglobin concentration ([Mb]) in human skeletal muscle, and to compare the change in the [Mb] in two different RT protocols. The two types of protocol used were interval RT (IRT) of moderate to low intensity with a high number of repetitions and a short recovery time, and repetition RT (RRT) of high intensity with a low number of repetitions and a long recovery time. A group of 11 healthy male adults voluntarily participated in this study and were divided into IRT (n = 6) and RRT (n = 5) groups. Both training protocols were carried out twice a week for 8 weeks. At the completion of the training period, the one-repetition maximal force values and isometric force were increased significantly in all the subjects, by about 38.8% and 26.0%, respectively (P < 0.01). The muscle fibre composition was unchanged by the 8 weeks of training. The muscle fibre cross-sectional areas were increased significantly by both types of training in all fibre types (I, IIa and IIb, mean + 16.1 %, P < 0.05). The [Mb] showed no significant changes at the completion of the training [IRT from 4.63 (SD 0.63) to 4.48 (SD 0.72), RRT from 4.47 (SD 0.75) to 4.24 (SD 0.80) mg x g(-1) wet tissue] despite a significant decrease in citrate synthase activity [IRT from 5.27 (SD 1.45) to 4.49 (SD 1.48), RRT from 5.33 (SD 2.09) to 4.85 (SD 1.87) micromol x min(-1) x g(-1) wet tissue; P < 0.05] observed after both protocols. These results suggested that myoglobin and mitochondria enzymes were regulated by different mechanisms in response to either type of RT. Moreover, the maintained [Mb] in hypertrophied muscle should preserve oxygen transport from capillaries to mitochondria even when diffusion distance is increased.     

Biochemical controls of citrate synthase in chickpea bacteroids

Bacteroids formed by Mesorhizobium ciceri CC 1192 in symbiosis with chickpea plants (Cicer arietinum L.) contained a single form of citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating) enzyme; EC 4.1.3.7], which had the same electrophoretic mobility as the enzyme from the free-living cells. The citrate synthase from CC 1192 bacteroids had a native molecular mass of 228 ± 32 kDa and was activated by KCl, which also enhanced stability. Double reciprocal plots of initial velocity against acetyl-CoA concentration were linear, whereas the corresponding plots with oxaloacetate were nonlinear. The K _m value for acetyl-CoA was 174 μM in the absence of added KCl, and 88 μM when the concentration of KCl in reaction mixtures was 100 mM. The concentrations of oxaloacetate for 50% of maximal activity were 27 μM without added KCl and 14 μM in the presence of 100 mM KCl. Activity of citrate synthase was inhibited 50% by 80 μM NADH and more than 90% by 200 μM NADH. Inhibition by NADH was linear competitive with respect to acetyl-CoA (K _is = 23.1 ± 3 μM) and linear noncompetitive with respect to oxaloacetate (K _is = 56 ± 3.8 μM and K _ii = 115 ± 15.4 μM). NADH inhibition was relieved by NAD⁺ and by micromolar concentrations of 5′-AMP. In the presence of 50 or 100 mM KCl, inhibition by NADH was apparent only when the proportion of NADH in the nicotinamide adenine dinucleotide pool was greater than 0.6. In the microaerobic environment of bacteroids, NADH may be at concentrations that are inhibitory for citrate synthase. However, this inhibition is likely to be relieved by NAD⁺ and 5′-AMP, allowing carbon to enter the tricarboxylic acid cycle. Received: 14 July 1999 / Accepted: 20 September 1999     

One Novel Mitochondrial Citrate Synthase from <Emphasis Type="Italic">Oryza sativa</Emphasis> L. can Enhance Aluminum Tolerance in Transgenic Tobacco

Rice exhibits the greatest aluminum (Al) tolerance compared with other cereals such as wheat, barley, maize, etc. A full-length gene, OsCS1, encoding citrate synthase, which is highly induced by aluminum toxicity in rice (Oryza sativa L.), was isolated. Sequence analysis and the sub-cellular localization of OsCS1 in yeast revealed that it is a mitochondrial citrate synthase. OsCS1 was induced by Al toxicity. Several independent transgenic tobacco lines expressing OsCS 1 exhibitted increased citrate efflux and extraordinary Al tolerance. Possible outlook for OsCS1 to be applied to enhance plant tolerance to Al toxicity was also discussed.     

Effects of space flight on GLUT-4 content in rat plantaris muscle

 The effects of 14 days of space flight on the glucose transporter protein (GLUT-4) were studied in the plantaris muscle of growing 9-week-old, male Sprague Dawley rats. The rats were randomly separated into five groups: pre-flight vivarium ground controls (PF-VC) sacrificed approximately 2 h after launch; flight groups sacrificed either approximately 5 h (F-R0) or 9 days (F-R9) after the return from space; and synchronous ground controls (SC-R0 and SC-R9) sacrificed at the same time as the respective flight groups. The flight groups F-R0 and F-R9 were exposed to micro-gravity for 14 days in the Spacelab module located in the cargo bay of the shuttle transport system – 58 of the manned Space Shuttle for the NASA mission named ”Spacelab Life Sciences 2”. Body weight and plantaris weight of SC-R0 and F-R0 were significantly higher than those of PF-VC. Neither body weight nor plantaris muscle weight in either group had changed 9 days after the return from space. As a result, body weight and plantaris muscle weight did not differ between the flight and synchronous control groups at any of the time points investigated. The GLUT-4 content (cpm/μg membrane protein) in the plantaris muscle did not show any significant change in response to 14 days of space flight or 9 days after return. Similarly, citrate synthase activity did not change during the course of the space flight or the recovery period. These results suggest that 14 days of space flight does not affect muscle mass or GLUT-4 content of the fast-twitch plantaris muscle in the rat. Received: 25 March 1997 / Accepted: 18 August 1997