Citrate modulates the regulation by Zn2+ of N-methyl-D-aspartate receptor-mediated channel current and neurotransmitter release.

The effect of the two metal-ion chelators EDTA and citrate on the action of N-methyl-D-aspartate (NMDA) receptors was investigated by use of cultured mouse cerebellar granule neurons and Xenopus oocytes, respectively, to monitor either NMDA-evoked transmitter release or membrane currents. Transmitter release from the glutamatergic neurons was determined by superfusion of the cells after preloading with the glutamate analogue D-[3H]aspartate. The oocytes were injected with mRNA isolated from mouse cerebellum and, after incubation to allow translation to occur, currents mediated by NMDA were recorded electrophysiologically by voltage clamp at a holding potential of -80 mV. It was found that citrate as well as EDTA could attenuate the inhibitory action of Zn2+ on NMDA receptor-mediated transmitter release from the neurons and membrane currents in the oocytes. These effects were specifically related to the NMDA receptor, since the NMDA receptor antagonist MK-801 abolished the action and no effects of Zn2+ and its chelators were observed when kainate was used to selectively activate non-NMDA receptors. Since it was additionally demonstrated that citrate (and EDTA) preferentially chelated Zn2+ rather than Ca2+, the present findings strongly suggest that endogenous citrate released specifically from astrocytes into the extracellular space in the brain may function as a modulator of NMDA receptor activity. This is yet another example of astrocytic influence on neuronal activity.

Two-component kinase-like activity of nm23 correlates with its motility-suppressing activity

Nm23 genes, which encode nucleoside diphosphate kinases, have been implicated in suppressing tumor metastasis. The motility of human breast carcinoma cells can be suppressed by transfection with wild-type nm23-H1, but not by transfections with two nm23-H1 mutants, nm23-H1S12OG and nm23-H1P96S. Here we report that nm23-H1 can transfer a phosphate from its catalytic histidine to aspartate or glutamate residues on 43-kDa membrane proteins. One of the 43-kDa membrane proteins was not phosphorylated by either nm23-H1P96S or nm23-H1S120G, and another was phosphorylated much more slowly by nm23-H1P96S and by nm23-H1S120G than by wild-type nm23-H1. Nm23-H1 also can transfer phosphate from its catalytic histidine to histidines on ATP-citrate lyase and succinic thiokinase. The rates of phosphorylation of ATP-citrate lyase by nm23-H1S120G and nm23-H1P96S were similar to that by wild-type nm23-H1. The rate of phosphorylation of succinic thiokinase by nm23-H1S120 was similar to that by wild-type nm23-H1, and the rate of phosphorylation of succinic thiokinase by nm23-H1P96S was about half that by wild-type nm23-H1. Thus, the transfer of phosphate from nm23-H1 to aspartates or glutamates on other proteins appears to correlate better with the suppression of motility than does the transfer to histidines.

The Caenorhabditis elegans seven-transmembrane protein ODR-10 functions as an odorant receptor in mammalian cells

The nematode Caenorhabditis elegans exhibits behavioral responses to many volatile odorants. Chemotaxis toward one such odorant, diacetyl (butanedione), requires the function of a seven-transmembrane receptor protein encoded by the odr-10 gene. To determine directly whether ODR-10 protein is an odorant receptor, it is necessary to express the protein in a heterologous system and show that it responds to diacetyl by activation of a G protein signaling pathway. Here we demonstrate that human cells expressing ODR-10 on their surfaces exhibit a transient elevation in intracellular Ca2+ levels after diacetyl application. Volatile compounds that differ from diacetyl only by the addition of a methyl group (2,3-pentanedione) or the absence of a keto group (butanone) are not ODR-10 agonists. Behavioral responses to these compounds are not dependent on odr-10 function, so ODR-10 specificity in human cells resembles in vivo specificity. The apparent affinity of ODR-10 for diacetyl observed in human cells is consistent with the diacetyl concentration ranges that allow efficient nematode chemotaxis. ODR-10 expressed in human cells also responds to two anionic compounds, pyruvate and citrate, which are metabolic precursors used for diacetyl production by certain bacterial species. Ca2+ elevation in response to ODR-10 activation is due to release from intracellular stores.

Genetically determined chloride-sensitive hypertension and stroke

The stroke-prone spontaneously hypertensive rat (SHRSP) is a genetically determined model of “salt-sensitive” stroke and hypertension whose full phenotypic expression is said to require a diet high in Na+ and low in K+. We tested the hypothesis that dietary Cl− determines the phenotypic expression of the SHRSP. In the SHRSP fed a normal NaCl diet, supplementing dietary K+ with KCl exacerbated hypertension, whereas supplementing either KHCO3 or potassium citrate (KB/C) attenuated hypertension, when blood pressure (BP) was measured radiotelemetrically, directly and continually. Supplemental KCl, but not KB/C, induced strokes, which occurred in all and only those rats in the highest quartiles of both BP and plasma renin activity (PRA). PRA was higher with KCl than with KB/C. These observations demonstrate that with respect to both severity of hypertension and frequency of stroke the phenotypic expression of the SHRSP is (i) either increased or decreased, depending on whether the anionic component of the potassium salt supplemented is, or is not, Cl−; (ii) increased by supplementing Cl− without supplementing Na+, and despite supplementing K+; and hence (iii) both selectively Cl−-sensitive and Cl−-determined. The observations suggest that in the SHRSP selectively supplemented with Cl− the likelihood of stroke depends on the extent to which both BP and PRA increase.

Selective disruption of protein aggregation by cyclodextrin dimers

β-Cyclodextrin (CD) dimers (n = 11) were synthesized and tested against eight enzymes, seven of which were dimeric or tetrameric, for inhibitor activity. Initial screening showed that only l-lactate dehydrogenase and citrate synthase were inhibited but only by two specific CD dimers in which two β-CDs were linked on the secondary face by a pyridine-2,6-dicarboxylic group. Further investigation suggested that these CD dimers inhibit the activity of l-lactate dehydrogenase and citrate synthase at least in part by disruption of protein–protein aggregation.

Effect of age on in vivo rates of mitochondrial protein synthesis in human skeletal muscle

A progressive decline in muscle performance in the rapidly expanding aging population is causing a dramatic increase in disability and health care costs. A decrease in muscle endurance capacity due to mitochondrial decay likely contributes to this decline in muscle performance. We developed a novel stable isotope technique to measure in vivo rates of mitochondrial protein synthesis in human skeletal muscle using needle biopsy samples and applied this technique to elucidate a potential mechanism for the age-related decline in the mitochondrial content and function of skeletal muscle. The fractional rate of muscle mitochondrial protein synthesis in young humans (24 ± 1 year) was 0.081 ± 0.004%·h−1, and this rate declined to 0.047 ± 0.005%·h−1 by middle age (54 ± 1 year; P < 0.01). No further decline in the rate of mitochondrial protein synthesis (0.051 ± 0.004%·h−1) occurred with advancing age (73 ± 2 years). The mitochondrial synthesis rate was about 95% higher than that of mixed protein in the young, whereas it was approximately 35% higher in the middle-aged and elderly subjects. In addition, decreasing activities of mitochondrial enzymes were observed in muscle homogenates (cytochrome c oxidase and citrate synthase) and in isolated mitochondria (citrate synthase) with increasing age, indicating declines in muscle oxidative capacity and mitochondrial function, respectively. The decrease in the rates of mitochondrial protein synthesis is likely to be responsible for this decline in muscle oxidative capacity and mitochondrial function. These changes in muscle mitochondrial protein metabolism may contribute to the age-related decline in aerobic capacity and muscle performance.

Manufacturing DNA microarrays of high spot homogeneity and reduced background signal

Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-l-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface.

Oxaloacetate Transport into Plant Mitochondria1

The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake of 14C-labeled OA into liposomes with incorporated mitochondrial membrane proteins preloaded with various dicarboxylates or citrate. OA was found to be transported in an obligatory counterexchange with malate, 2-oxoglutarate, succinate, citrate, or aspartate. Phtalonate inhibited all of these countertransports. OA-malate countertransport was inhibited by 4,4′-dithiocyanostilbene-2,2′-disulfonate and pyridoxal phosphate, and also by p-chloromercuribenzene sulfonate and mersalyl, indicating that a lysine and a cysteine residue of the translocator protein are involved in the transport. From these and other inhibition studies, we concluded that plant mitochondria contain an OA translocator that differs from all other known mitochondrial translocators. Major functions of this translocator are the export of reducing equivalents from the mitochondria via the malate-OA shuttle and the export of citrate via the citrate-OA shuttle. In the cytosol, citrate can then be converted either into 2-oxoglutarate for use as a carbon skeleton for nitrate assimilation or into acetyl-coenzyme A for use as a precursor for fatty acid elongation or isoprenoid biosynthesis.

Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii1

NAD-isocitrate dehydrogenase (NAD-IDH) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by successive chromatography steps on Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to be an octamer composed of 45-kD subunits. The presence of isocitrate plus Mn2+ protected the enzyme against thermal inactivation or inhibition by specific reagents for arginine or lysine. NADH was a competitive inhibitor (Ki, 0.14 mm) and NADPH was a noncompetitive inhibitor (Ki, 0.42 mm) with respect to NAD+. Citrate and adenine nucleotides at concentrations less than 1 mm had no effect on the activity, but 10 mm citrate, ATP, or ADP had an inhibitory effect. In addition, NAD-IDH was inhibited by inorganic monovalent anions, but l-amino acids and intermediates of glycolysis and the tricarboxylic acid cycle had no significant effect. These data support the idea that NAD-IDH from photosynthetic organisms may be a key regulatory enzyme within the tricarboxylic acid cycle.

Aluminum-Resistant Arabidopsis Mutants That Exhibit Altered Patterns of Aluminum Accumulation and Organic Acid Release from Roots1

Al-resistant (alr) mutants of Arabidopsis thaliana were isolated and characterized to gain a better understanding of the genetic and physiological mechanisms of Al resistance. alr mutants were identified on the basis of enhanced root growth in the presence of levels of Al that strongly inhibited root growth in wild-type seedlings. Genetic analysis of the alr mutants showed that Al resistance was semidominant, and chromosome mapping of the mutants with microsatellite and random amplified polymorphic DNA markers indicated that the mutants mapped to two sites in the Arabidopsis genome: one locus on chromosome 1 (alr-108, alr-128, alr-131, and alr-139) and another on chromosome 4 (alr-104). Al accumulation in roots of mutant seedlings was studied by staining with the fluorescent Al-indicator dye morin and quantified via inductively coupled argon plasma mass spectrometry. It was found that the alr mutants accumulated lower levels of Al in the root tips compared with wild type. The possibility that the mutants released Al-chelating organic acids was examined. The mutants that mapped together on chromosome 1 released greater amounts of citrate or malate (as well as pyruvate) compared with wild type, suggesting that Al exclusion from roots of these alr mutants results from enhanced organic acid exudation. Roots of alr-104, on the other hand, did not exhibit increased release of malate or citrate, but did alkalinize the rhizosphere to a greater extent than wild-type roots. A detailed examination of Al resistance in this mutant is described in an accompanying paper (J. Degenhardt, P.B. Larsen, S.H. Howell, L.V. Kochian [1998] Plant Physiol 117: 19–27).

Energy metabolism, enzymatic flux capacities, and metabolic flux rates in flying honeybees.

Honeybees rely primarily on the oxidation of hexose sugars to provide the energy required for flight. Measurement of VCO2 (equal to VO2, because VCO2/VO2 = 1.0 during carbohydrate oxidation) during flight allowed estimation of steady-state flux rates through pathways of flight muscle energy metabolism. Comparison of Vmax values for flight muscle hexokinase, phosphofructokinase, citrate synthase, and cytochrome c oxidase with rates of carbon and O2 flux during flight reveal that these enzymes operate closer to Vmax in the flight muscles of flying honeybees than in other muscles previously studied. Possible mechanistic and evolutionary implications of these findings are discussed.

Daidzin suppresses ethanol consumption by Syrian golden hamsters without blocking acetaldehyde metabolism.

Daidzin is a potent, selective, and reversible inhibitor of human mitochondrial aldehyde dehydrogenase (ALDH) that suppresses free-choice ethanol intake by Syrian golden hamsters. Other ALDH inhibitors, such as disulfiram (Antabuse) and calcium citrate carbimide (Temposil), have also been shown to suppress ethanol intake of laboratory animals and are thought to act by inhibiting the metabolism of acetaldehyde produced from ingested ethanol. To determine whether or not daidzin inhibits acetaldehyde metabolism in vivo, plasma acetaldehyde in daidzin-treated hamsters was measured after the administration of a test dose of ethanol. Daidzin treatment (150 mg/kg per day i.p. for 6 days) significantly suppresses (> 70%) hamster ethanol intake but does not affect overall acetaldehyde metabolism. In contrast, after administration of the same ethanol dose, plasma acetaldehyde concentration in disulfiram-treated hamsters reaches 0.9 mM, 70 times higher than that of the control. In vitro, daidzin suppresses hamster liver mitochondria-catalyzed acetaldehyde oxidation very potently with an IC50 value of 0.4 microM, which is substantially lower than the daidzin concentration (70 microM) found in the liver mitochondria of daidzin-treated hamsters. These results indicate that (i) the action of daidzin differs from that proposed for the classic, broad-acting ALDH inhibitors (e.g., disulfiram), and (ii) the daidzin-sensitive mitochondrial ALDH is not the one and only enzyme that is essential for acetaldehyde metabolism in golden hamsters.