Addition Of Malate Research Articles

The sources of carbon and reducing power for fatty acid synthesis in the heterotrophic plastids of developing sunflower (Helianthus annuus L.) embryos

The provision of carbon substrates and reducing power for fatty acid synthesis in the heterotrophic plastids of developing embryos of sunflower (Helianthus annuus L.) has been investigated. Profiles of oil and storage protein accumulation were determined and embryos at 17 and 24 days after anthesis (DAA) were selected to represent early and late periods of oil accumulation. Plastids isolated from either 17 or 24 DAA embryos did not incorporate label from [1-(14)C]glucose 6-phosphate (Glc6P) into fatty acids. Malate, when supplied alone, supported the highest rates of fatty acid synthesis by the isolated plastids at both stages. Pyruvate supported rates of fatty acid synthesis at 17 DAA that were comparable to those supported by malate, but only when incubations also included Glc6P. The stimulatory effect of Glc6P on pyruvate utilization at 17 DAA was related to the rapid utilization of Glc6P through the oxidative pentose phosphate pathway (OPPP) at this stage. Addition of pyruvate to incubations containing [1-(14)C]Glc6P increased OPPP activity (measured as (14)CO(2) release), while the addition of malate suppressed it. Observations of the interactions between the rate of metabolite utilization for fatty acid synthesis and the rate of the OPPP are consistent with regulation of the OPPP by redox control of the plastidial glucose 6-phosphate dehydrogenase activity through the demand for NADPH. During pyruvate utilization for fatty acid synthesis, flux through the OPPP increases as NADPH is consumed, whereas during malate utilization, in which NADPH is produced by NADP-malic enzyme, flux through the OPPP is decreased.

Repetitive Transient Depolarizations of the Inner Mitochondrial Membrane Induced by Proton Pumping

Single mitochondria show the spontaneous fluctuations of ΔΨm. In this study, to examine the mechanism of the fluctuations, we observed ΔΨm in single isolated heart mitochondria using time-resolved fluorescence microscopy. Addition of malate, succinate, or ascorbate plus TMPD to mitochondria induced polarization of the inner membrane followed by repeated cycles of rapid depolarizations and immediate repolarizations. ADP significantly decreased the frequency of the rapid depolarizations, but the ADP effect was counteracted by oligomycin. On the other hand, the rapid depolarizations did not occur when mitochondria were polarized by the efflux of K+ from the matrix. The rapid depolarizations became frequent with the increase in the substrate concentration or pH of the buffer. These results suggest that the rapid depolarizations depend on the net translocation of protons from the matrix. The frequency of the rapid depolarizations was not affected by ROS scavengers, Ca2+, CsA, or BA. In addition, the obvious increase in the permeability of the inner membrane to calcein (MW 623) that was entrapped in the matrix was not observed upon the transient depolarization. The mechanisms of the spontaneous oscillations of ΔΨm are discussed in relation to the matrix pH and the permeability transitions.

Direct Transfer of NADH From Malate Dehydrogenase to Complex I in <I>Escherichia coli</I>

During aerobic growth of Escherichia coli, nicotinamide adenine dinucleotide (NADH) can initiate electron transport at either of two sites: Complex I (NDH-1 or NADH:ubiquinone oxidoreductase) or a single-subunit NADH dehydrogenase (NDH-2). We report evidence for the specific coupling of malate dehydrogenase to Complex I. Membrane vesicles prepared from wild type cultures retain malate dehydrogenase and are capable of proton translocation driven by the addition of malate + NAD. This activity was inhibited by capsaicin, an inhibitor specific to Complex I, and it proceeded with deamino-NAD, a substrate utilized by Complex I, but not by NDH-2. The concentration of free NADH produced by membrane vesicles supplemented with malate + NAD was estimated to be 1 microM, while the rate of proton translocation due to Complex I was consistent with a somewhat higher concentration, suggesting a direct transfer mechanism. This interpretation was supported by competition assays in which inactive mutant forms of malate dehydrogenase were able to inhibit Complex I activity. These two lines of evidence indicate that the direct transfer of NADH from malate dehydrogenase to Complex I can occur in the E. coli system.

Effects of aluminum on organic acid metabolism and secretion by red spruce cell suspension cultures and the reversal of Al effects on growth and polyamine metabolism by exogenous organic acids.

In the absence of added Al, the concentration of succinate in cultured red spruce (Picea rubens Sarg.) cells was 15-20 times higher (> 600 nmol g-1FW) than that of citrate or oxalate and 4-6 times higher than that of malate. Addition of AlCl3 (effective monomeric Al concentrations of 0.23 and 0.48 mM) to 3-day-old suspension cultures significantly increased cellular succinate concentrations with a concomitant decrease in cellular oxalate concentrations. However, in the medium of Al-treated cell cultures, both succinate and oxalate concentrations were significantly higher than in the medium of cell cultures without added Al, and oxalate concentrations were several times higher than succinate concentrations. Aluminum did not significantly affect the cellular concentrations of malate, ascorbate and citrate, and none of these organic acids was present in detectable quantities in the medium. Exogenous succinate alone or with Al had no effect on cellular free polyamine concentrations or cell mass. Aluminum caused a significant increase in cellular putrescine concentrations. Addition of malate had a positive effect on growth and completely reversed the effects of Al on cell physiology. In contrast, the addition of oxalate and citrate only partly reversed the effects of Al.

Mechanism of superoxide anion generation in intact mitochondria in the presence of lucigenin and cyanide.

In the presence of cyanide and various respiratory substrates (succinate or pyruvate + malate) addition of high concentrations of lucigenin (400 microM; Luc2+) to rat liver mitochondria can induce a short-term flash of high amplitude lucigenin-dependent chemiluminescence (LDCL). Under conditions of cytochrome oxidase inhibition by cyanide the lucigenin-induced cyanide-resistant respiration (with succinate as substrate) was not inhibited by uncouplers (FCCP) and oligomycin. Increase in transmembrane potential (Deltaphi) value by stimulating F0F1-ATPase functioning (induced by addition of MgATP to the incubation medium) caused potent stimulation of the rate of cyanide-resistant respiration. At high Deltaphi values (in the presence of MgATP) cyanide resistant respiration of mitochondria in the presence of succinate or malate with pyruvate was insensitive to tenoyltrifluoroacetone (TTFA) or rotenone, respectively. However, in both cases respiration was effectively inhibited by myxothiazol or antimycin A. Mechanisms responsible for induction of LDCL and cyanide resistant mitochondrial respiration differ. In contrast to cyanide-resistant respiration, generation of LDCL signal, that was suppressed only by combined addition of Complex III inhibitors, antimycin A and myxothiazol, is a strictly potential-dependent process. It is observed only under conditions of high Deltaphi value generated by F0F1-ATPase functioning. The data suggest lucigenin-induced intensive generation of superoxide anion in mitochondria. Based on results of inhibitor analysis of cyanide-resistant respiration and LDCL, a two-stage mechanism of autooxidizable lucigenin cation-radical (Luc*+) formation in the respiratory chain is proposed. The first stage involves two-electron Luc2+ reduction by Complexes I and II. The second stage includes one-electron oxidation of reduced lucigenin (Luc(2e)). Reactions of Luc(2e) oxidation involve coenzyme Q-binding sites of Complex III. This results in formation of autooxidizable Luc*+ and superoxide anion generation. A new scheme for lucigenin-dependent electron pathways is proposed. It includes formation of fully reduced form of lucigenin and two-electron-transferring shunts of the respiratory chain. Lucigenin-induced activation of superoxide anion formation in mitochondria is accompanied by increase in ion permeability of the inner mitochondrial membrane.

Effect of the addition of malate on in vitro rumen fermentation of cereal grains.

Batch cultures of mixed rumen micro-organisms were used to study the effects of different concentrations of malate (Rumalato(R); Norel & Nature S.A., Barcelona, Spain; composed of disodium malate-calcium malate (0.16:0.84, w/w)) on the fermentation of four cereal grains (maize, barley, wheat and sorghum). Rumen contents were collected from four Merino sheep fed lucerne hay ad libitum and supplemented with 300 g concentrate/d. Rumalato(R) was added to the incubation bottles to achieve final concentrations of 0, 4, 7 and 10 mm-malate. Gas production was measured at regular intervals up to 120 h. Malate increased (P<0.01) the average fermentation rate of all substrates, and the lag time decreased (P<0.05) linearly with increasing concentrations of malate for all substrates, with the exception of sorghum. In 17 h incubations, the final pH and total volatile fatty acid production increased (P<0.001) linearly for all substrates as malate concentration increased from 0 to 10 mm. Propionate and butyrate production increased (P<0.05), while the value of the acetate : propionate ratio and l-lactate concentrations decreased (P<0.05) linearly with increasing doses of malate. Malate treatment increased (P<0.05) the CO2 production and decreased the production of CH4, although this effect was not significant (P>0.05) for maize. Malate at 4 and 7 mm increased (P<0.05) optical density of the cultures measured at 600 nm for maize, with no differences for the other substrates. The results indicate that malate may be used as a feed additive for ruminant animals fed high proportions of cereal grains, because it increased pH and propionate production and decreased CH4 production and l-lactate concentrations; however, in general, no beneficial effects of 10 compared with 7 mm-malate were observed.

Role of sugars and organic acids in regulating the concentration and activity of the alternative oxidase in Poa annua roots.

Detached roots of Poa annua were used to study alternative oxidase protein expression upon the addition of sucrose, glucose, fructose, inositol, mannitol, citrate or malate, at a concentration of 1 or 10 mM for 24 h. After 24 h the capacity of cytochrome c oxidase was decreased equally in all treatments. Only citrate induced the expression of the alternative oxidase, especially at a concentration of 1 mM (15-fold). The activity of the alternative pathway (measured with the (18)O-fractionation technique) was not affected by the addition of sucrose for 24 h as compared with time zero. However, after the addition of citrate or mannitol the activity of the alternative pathway decreased to almost zero. The discrepancy between the large increase in alternative oxidase protein concentration when citrate was applied and the concomitant decrease in alternative pathway activity is discussed.

Fluorescence Imaging of Metabolic Responses in Single Mitochondria

The membrane potentials, rates of NAD(P)H formation, and rates of flavoprotein reduction have been measured for single mitochondria isolated from porcine hearts. These metabolic responses were elicited by the addition of malate and measured using fluorescence microscopy. For the measurements of mitochondrial membrane potential, mitochondria were stained with tetramethylrhodamine ethyl ester, and the membrane potentials of single mitochondria were determined. Individual mitochondria maintained the membrane potential at around −80 mV before addition of malate. Upon the addition of malate, each mitochondrion was rapidly polarized to around −100 ∼ −140 mV and underwent repeated cycles of polarization and depolarization, which were probably caused by openings and closings of permeability transition pores. NAD(P)+ and flavoprotein were reduced immediately after addition of malate and then slowly became reoxidized. Thus, single mitochondria can undergo rapid and repetitive changes in membrane potential, but not in the redox state of NAD(P)H and flavoprotein.

Regulation of the transport system for C4-dicarboxylic acids in Bacillus subtilis.

Transport systems for C4-dicarboxylates, such as malate, fumarate and succinate, are poorly understood in Gram-positive bacteria. The whole genome sequence of Bacillus subtilis revealed two genes, ydbE and ydbH, whose deduced products are highly homologous to binding proteins and transporters for C4-dicarboxylates in Gram-negative bacteria. Between ydbE and ydbH, genes ydbF and ydbG encoding a sensor-regulator pair, were located. Inactivation of each one of the ydbEFGH genes caused a deficiency in utilization of fumarate or succinate but not of malate. Expression of ydbH, encoding a putative transporter, was stimulated in a minimal salt medium containing 0-05% yeast extract but repressed by the addition of malate to the medium. Inactivation of the putative sensor-regulator pair or solute-binding protein, ydbFG or ydbE, caused complete loss of ydbH expression. The utilization of fumarate and stimulation of ydbH expression resumed in a ydbE null mutant in which ydbFGH were overproduced. Based on these observations, together with analysis of the sequence similarities of the deduced product, we conclude that YdbH is a C4-dicarboxylate-transport protein and its expression is regulated by a C4-dicarboxylate sensor kinase-regulator pair, YdbF and YdbG. Furthermore, it is suggested that YdbE does not directly participate in transport of C4-dicarboxylates, but plays a sensory role in the ydbF-ydbG two-component system, giving rise to specificity or increased efficiency to the system. Deletion analysis of the promoter region of ydbH revealed that a direct repeat sequence was required for the activation of ydbH expression. A catabolite-responsive element (CRE) was also found in the -10 region of the promoter, suggesting negative regulation by a CRE-binding protein.

Respiratory state and phosphatidylserine import in brain mitochondria in vitro.

The mechanism of phosphatidylserine (PS) movement from donor membranes into rat brain mitochondria was investigated. Mitochondria were incubated with liposomes and subjected to density gradient centrifugation. The energized state was monitored by flow cytometry measuring the fluorescence of membrane-potential-sensitive rhodamine-123 dye. Mitochondria density decreased upon increase of the respiratory rate, as a consequence of their association with liposomes. After interaction of mitochondria with (14)C-PS containing liposomes, (14)C-PS became a substrate of PS decarboxylase, as monitored by the formation of (14)C-phosphatidylethanolamine (PE), indicating translocation of (14)C-PS to the inner membrane. The kinetics of (14)C-PE formation showed a high rate upon addition of ADP, malate and pyruvate (state 3) compared to control (state 1). In state 3, (14)C-PE formation decreased in the presence of NaN(3). Mitochondria-associated membranes (MAM) are the major site of PS synthesis. However, their role in the translocation of PS to mitochondria has not been completely elucidated. A crude mitochondrial fraction (P(2)) containing MAM, synaptosomes and myelin was prelabeled with (14)C-PS and incubated in different respiratory states. At a high respiratory rate, low-density labeled mitochondria, whose band overlaps that of synaptosomes, were obtained by centrifugation. A parallel decrease of both radioactivity and protein in MAM fraction was observed, indicating that the association of MAM and mitochondria had occurred. Synthesis and translocation of (14)C-PS in P(2) membranes were also studied by incubating P(2) with (14)C-serine. In the resting state (14)C-PS accumulated in MAM, indicating that the transfer to mitochondria was a limiting step. In state 3 both the transfer rate of (14)C-PS and its conversion to (14)C-PE increased. Respiratory mitochondrial activity modulated the association of MAM and mitochondria, triggering a mechanism that allowed the transport of PS across the outer mitochondrial membrane.

Abnormal Mitochondrial Function in Myocardium of Dogs with Chronic Heart Failure

Chronic heart failure (HF) is associated with morphologic abnormalities of cardiac mitochondria that include hyperplasia, reduced organelle size and compromised structural integrity. In the present study, we examined mitochondrial respiration in myocardium of 10 normal dogs and 10 dogs with chronic HF (LV ejection fraction 24±2%) produced by intracoronary micro-embolizations. Mitochondrial respiratory rates were determined using a Clark electrode in an oxygraph cell containing saponin-skinned muscle bundles. Basal respiratory rate (VO), respiratory rate after addition of substrates, glutamate and malate (VSUB) and state 3 respiratory rate (VADP, after addition of ADP), were measured in tissue samples from the subendocardial and subepicardial LV free wall, interventricular septum and right-ventricular free wall. No differences were observed in basal respiratory rates between normal and HF tissue, while VSUBwas significantly lower in HF compared to normal. VADPwas 50–60% lower in HF compared to normal tissue (P<0.001). The results indicate abnormal mitochondrial respiratory activity in myocardium of dogs with chronic HF. These findings support the concept of low myocardial energy production in HF that can contribute to the global cardiac dysfunction.

Factors affecting lactate and malate utilization by Selenomonas ruminantium.

Lactate utilization by Selenomonas ruminantium is stimulated in the presence of malate. Because little information is available describing lactate-plus-malate utilization by this organism, the objective of this study was to evaluate factors affecting utilization of these two organic acids by two strains of S. ruminantium. When S. ruminantium HD4 and H18 were grown in batch culture on DL-lactate and DL-malate, both strains coutilized both organic acids for the initial 20 to 24 h of incubation and acetate, propionate, and succinate accumulated. However, when malate and succinate concentrations reached 7 mM, malate utilization ceased, and with strain H18, there was a complete cessation of DL-lactate utilization. Malate utilization by both strains was also inhibited in the presence of glucose. S. ruminantium HD4 was unable to grow on 6 mM DL-lactate at extracellular pH 5.5 in continuous culture (dilution rate, 0.05 h-1) and washed out of the culture vessel. Addition of 8 mM DL-malate to the medium prevented washout on 6 mM DL-lactate at pH 5.5 and resulted in succinate accumulation. Addition of malate also increased bacterial protein, acetate, and propionate concentrations in continuous culture. These results suggest that 8 mM DL-malate enhances the ability of strain HD4 to grow on 6 mM DL-lactate at extracellular pH 5.5.

High rates of extracellular superoxide generation by cultured human fibroblasts: involvement of a lipid-metabolizing enzyme.

Expression of NADPH oxidase and low superoxide generation (approx. 0.06 nmol/min per 10(6) cells) by cytokine- or ionophore-stimulated human fibroblasts is known. However, we here show that these cells also contain an ectoplasmic enzyme, distinct from NADPH oxidase, which can generate superoxide (2.19 +/- 0.14 nmol/min per 10(6) cells) at levels similar to phorbol ester-stimulated monocytes on exogenous NADH addition. Superoxide generation was temperature-dependent, insensitive to chelation (desferal), and had a K(m) (app)(NADH) of 11.5 microM. Inhibitor studies showed that there was no involvement of NADPH oxidase (diphenylene iodonium, diphenyl iodonium), prostaglandin H synthase (indomethacin), xanthine oxidase (allopurinol), cytochrome P-450 (metyrapone) or mitochondrial respiration (rotenone, antimycin A). NAD+ was a competitive inhibitor, whereas NADPH supported 40% of the rate seen with NADH. No luminescence was observed after the addition of lactate, malate, pyruvate, GSH or L-cysteine. NADH-stimulated superoxide generation was enhanced by the addition of (3-30 microM) arachidonic acid, linoleic acid or (5S)-hydroxyeicosatetraenoic acid [(5S)-HETE] but not palmitic acid, (15S)-hydroperoxyeicosatetraenoic acid [(15S)-HPETE], (15S)-HETE or (12S)-HETE. Several features suggest involvement of an enzyme related to 15-lipoxygenase, and, in support of this, we show superoxide generation and NADH oxidation by recombinant rabbit reticulocyte 15-lipoxygenase. The large amounts of superoxide measured suggest that the fibroblast extracellular enzyme could be a major source of reactive oxygen species after tissue damage.

Soil pH increase due to biological decarboxylation of organic anions

Organic matter incorporated into soils may influence soil pH. Until now the detailed processes responsible for a change in soil pH after the incorporation of organic matter were not completely understood. We studied the effects of organic anions, glycine as a representative of amino acids, and glucose as a representative of carbohydrates on soil pH in aerobic incubation experiments. Addition of malate and citrate resulted in an immediate soil pH increase associated with additional CO 2 release. Addition of acetate had no major effect on soil pH but if applied at higher rates decreased soil CO 2 release. Malate added together with ammonium resulted in an immediate soil pH increase, which was followed by a pH decrease associated by an increase in nitrate after 10 days. Also glycine gave an immediate pH increase which was associated with an equivalent decrease of soil organic N extractable with CaCl 2 solution. Addition of glucose gave a slight pH decrease in the first days of incubation followed by an increase to the initial pH value. The results are interpreted in terms of decarboxylation of organic anions, a process which requires one proton per carboxylic group decarboxylated. Under aerobic soil conditions decarboxylation is a major process in organic matter decomposition. The decomposition of carbohydrates in the glycolytic pathway produces carboxylic groups which, after dissociation, may decrease soil pH. As soon as these groups are decarboxylated in the citrate cycle an equivalent amount of protons is required inducing a rise in soil pH. For this reason addition of glucose brought about only a transient soil pH decrease.

Oxidation of coniferyl alcohol to lignin-like products by tobacco xylem cell walls

Cell walls, isolated from the xylem of tobacco ( Nicotiana tabacum cy Samsun) stems, oxidized coniferyl alcohol both in the absence of exogenous hydrogen peroxide and in the presence of catalase. The oxidation was monitored by the reduction of A of coniferyl alcohol at 260 nm and confirmed by the decline of coniferyl alcohol measured by reverse-phase HPLC. Although some water-soluble products were noted in the supernatant during incubation, the products appeared to have greater affinity for the cell walls. After 168 hr incubation, several products were extracted from the walls with methanol. These were retained on the reverse-phase column and were probably more hydrophobic than coniferyl alcohol. The major methanol-soluble product, which may be the dilignol, dehydrodiconiferyl alcohol, was purified by reverse-phase HPLC. The cell walls after complete oxidation of coniferyl alcohol were yellow/orange and had an elevated lignin content. The increased lignin content was probably the result of the deposition of guaiacyl-type ‘lignin-like’ material on to or into the cell walls. The presence of 500 units ml −1 catalase or superoxide dismutase did not prevent the deposition of this ‘lignin-like’ material. The addition of malate, nicotinamide adenine dinucleotides (NAD) and Mn 2+ ions did not enhance the deposition of lignin-like material. This suggests a non-peroxidative polymerization of coniferyl alcohol by the cell walls and supports the theory that cell-wall-associated oxidases participate in lignin formation.

Aluminum Resistance in Triticum aestivum Associated with Enhanced Exudation of Malate

Two aluminum (Al)-resistant (Atlas 66, Maringa) and two Al-sensitive (Roblin, Katepwa) cultivars of Triticum aestivum (wheat) were grown under aseptic conditions in the presence and absence of Al to evaluate the potential role of organic anion exudates in conferring resistance to Al. Five organic anions, α-ketoglutarate, citrate, malate, succinate and fumarate, were commonly detected in the root exudates, but only malate and succinate were consistently exuded in all cultivars under all treatments. Under control conditions, malate was exuded in higher quantities from roots of Al-resistant cultivars (Atlas 66 and Maringa), compared with the Al-sensitive cultivars. Exposure to 100 μM Al increased exudation of malate from roots of Al-resistant cultivars by 100–120 %, while in the Al-sensitive cultivars, exudation of malate was reduced. A decrease in exudation of succinate was observed in Atlas 66 and Maringa with 100 μM Al, while no effect was observed in Roblin and Katepwa. Differences between cultivars in the effect of Al on malate accumulation were detected as early as 24 h after exposure. Addition of exogenous malate (250 μM to 500 μM) to nutrient media containing 100 μM Al restored root elongation in Al-sensitive cultivars, Roblin and Katepwa, to control levels. To determine whether exudation of malate from roots reflected de novo synthesis arising from activity of the TCA cycle, plants were labeled with 14C-acetate. With the exception of acetate itself, malate was the only organic anion in which 14C was detected. In Al-resistant cultivars, treatment with Al increased exudation of 14C into malate by 48 to 54 % when expressed as a percent of total label in root exudates. In Al-sensitive cultivars, incorporation of 14C into malate declined by 22 to 29 % with exposure to Al. The unique pattern of 14C labeling and enhanced exudation of malate in the Al-resistant cultivars, Atlas 66 and Marïnga, provides strong although indirect evidence for a role of malate in Al-resistance.

Malate‐sensitive anion channels enable guard cells to sense changes in the ambient CO2 concentration

SummaryMalate is a characteristic metabolite in the photosynthesis of C4 and CAM plants. Furthermore, changes in the intracellular concentration of this organic acid provide part of the osmotic motor for guard cells. Since alterations in the malate concentration influence the photosynthetic capacity on one side and stomatal action on the other, it was studied whether the extracellular malate level represents an indicator of changes in the ambient CO2 concentration and a key regulator of ion transport in guard cells.Here it is demonstrated that alterations in the ambient CO2 level modify the extracellular malate concentration of Vicia faba leaves. Elevated external malate caused stomatal closure in a concentration‐dependent manner (Kmmal = 0.3 mM). Slight variations in the external malate concentration strongly regulate the voltage‐dependent properties of GCAC1, an anion‐release channel in the plasma membrane of guard cells. Superfusion of guard cell protoplasts with malate levels in the physiological range (Kmmal = 0.4 mM) caused the voltage gate to shift towards the resting potential of the cell‐activating GCAC1. Single‐channel conductance was dependent on the extracellular chloride concentration (KmCl = 3 mM). In the absence of extracellular chloride the plasma membrane lacked anion conductance until the addition of malate induced channel opening. Isophthalate was a powerful agonist in both malate‐induced processes, channel regulation and stomatal closure, indicating that modulation of GCAC1 is a key step in stomatal action. It was thus concluded that feedback regulation of volume and turgor with respect to the ambient CO2 concentration via malate‐sensitive anion channels may provide a CO2 sensor to guard cells.

The Influence of Cryopreservation on Parameters of Energetic Metabolism and Motility of Fowl Spermatozoa

The objective of this study was to estimate the effect of cryopreservation on the main pathways of energetic metabolism and motility of fowl spermatozoa. Sperm diluted 1:5 with the cryoprotective medium containing ethylene glycol (1.4 M final concentration) was frozen at the rate of 2-3°C/min to -25°C with a pause on the plateau of crystallization and then at an exponentially increasing rate to - 196°C. The frozen sperm was thawed in two successive water baths at 0 and at 41°C. After cryopreservation, the rate of radioactive glucose oxidation to 14CO2 slightly decreased, the rate of labeled glutamate oxidation remained unchanged, and the rate of labeled succinate oxidation increased twofold. After freeze-thawing, the rates of endogenous respiration with and without 2,4-dinitrophenol decreased; the oxidation rate of exogenous succinate in the presence of 2,4-dinitrophenol, rotenone, and digitonin slightly decreased; and the rate of respiration in the presence of ascorhate, N,N,N′,N′ tetramethyl-p-phenylenediamine, antimycin A, 2,4-dinitrophenol, and digitonin did not differ from that seen in control. Sperm respiration was highly sensitive to rotenone; antimycin A and cyanide blocked oxygen consumption completely. Succinate, added after 2,4-dinitrophenol and rotenone, stimulated respiration of thawed spermatozoa, which indicated plasma membrane damage. The addition of exogenous malate in the presence of 2,4-dinitrophenol and digitonin restored the respiration rate of thawed spermatozoa to that of unfrozen cells. The rate of respiration of thawed spermatozoa with oligomycin was higher than that of control cells. 2,4-Dinitrophenol stimulated respiration under these conditions. Cryopreservation caused a decrease in the percentage of progressively moving cells and the percentage of beating flagella before and after the successive treatment with the medium containing Triton X-100 and ATP. The data obtained suggest that cryopreservation decreased the overall flow through glycolysis, the citric acid cycle, and the respiratory chain, affected oxidative phosphorylation, injured plasma membranes, and damaged the motility apparatus of spermatozoa; however, the respiratory chain and tricarboxylic acid cycle retained a high degree of functional integrity.

Hepatic ketogenesis in newborn pigs is limited by low mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity.

In newborn-pig hepatocytes, the rate of oleate oxidation is extremely low, despite a very low malonyl-CoA concentration. By contrast, the sensitivity of carnitine palmitoyltransferase (CPT) I to malonyl-CoA inhibition is high, as suggested by the very low concentration of malonyl-CoA required for 50% inhibition of CPT I (IC50). The rates of oleate oxidation and ketogenesis are respectively 70 and 80% lower in mitochondria isolated from newborn-pig liver than from starved-adult-rat liver mitochondria. Using polarographic measurements, we showed that the oxidation of oleoyl-CoA and palmitoyl-L-carnitine is very low when the acetyl-CoA produced is channelled into the hydroxymethylglutaryl-CoA (HMG-CoA) pathway by addition of malonate. In contrast, the oxidation of the same substrates is high when the acetyl-CoA produced is directed towards the citric acid cycle by addition of malate. We demonstrate that the limitation of ketogenesis in newborn-pig liver is due to a very low amount and activity of mitochondrial HMG-CoA synthase as compared with rat liver mitochondria, and suggest that this could promote the accumulation of acetyl-CoA and/or beta-oxidation products that in turn would decrease the overall rate of fatty acid oxidation in newborn- and adult-pig livers.

Effect of water and nitrogen additions on free-living nitrogen fixer populations in desert grass root zones.

In this study we measured changes in population levels of free-living N2-fixing bacteria in the root zones of potted Bouteloua eriopoda and Sporobolus flexuosus plants as well as the photosynthetic indices of the plants in response to added nitrogen, added water, and added water plus nitrogen treatments. In addition, N2 fixer population changes in response to added carbon source and nitrogen were measured in plant-free soil columns. There were significant increases in the numbers of N2 fixers associated with both plant species in the water and the water plus nitrogen treatments. Both treatments increased the photosynthetic index, suggesting that plant exudates were driving N2 fixer population changes. Population increases were greatest in the water plus nitrogen treatments, indicating that added nitrogen was synergistic with added water and suggesting that nitrogen addition spared bacteria the metabolic cost of N2 fixation, allowing greater reproduction. Plant-free column studies demonstrated a synergistic carbon-nitrogen effect when carbon levels were limiting (low malate addition) but not when carbon was abundant (high malate), further supporting this hypothesis. The results of this study indicate the presence of N2 fixer populations which interact with plants and which may play a role in the nitrogen balance of desert grasslands.