Trypanosoma brucei: An evaluation of salicylhydroxamic acid as a trypanocidal drug

The chemotherapeutic potential of salicylhydroxamic acid (SHAM) was studied in adult rats infected with a strain of Trypanosoma brucei that kills the rats in about 100 hr. The median lethal dose, administered intraperitoneally in a carboxymethyl-cellulose suspension, is approximately 820 mg/kg body weight for male and 850 mg/kg for female rats. The apparent cause of death is severe depression of the central nervous system.Half-maximal inhibition of O₂ uptake by trypanosomes in vitro requires 15 μM SHAM, whereas 100 μM inhibits over 90%. This inhibitory effect on trypanosome respiration was used as a biological assay for the effective SHAM concentration in rat plasma. After administration of a sublethal SHAM dose to rats, the effective plasma SHAM concentration rose rapidly to about 500 μM and then fell to about 10 μM at 4 hr. Nevertheless, this dose did not significantly affect the survival time of rats infected with T. brucei. Even if, by repeated SHAM administration, the plasma SHAM concentration was kept at around 100 μM for more than 4 hr, no therapeutic effect was observed.These results show that O₂ uptake is not essential for the survival of trypanosomes in rats and they support the idea that bloodstream trypanosomes have an alternative pathway for glycolysis, allowing energy production in the absence of respiration.The possibility that SHAM or other inhibitors of trypanosome respiration could stilll be trypanocidal if used in conjunction with another inhibitor of glycolysis is discussed.     

Trypanosoma brucei: trypanocidal effect of salicylhydroxamic acid plus glycerol in infected rats.

Rats inoculated with Trypanosoma brucei brucei EATRO 427 and having a high degree of parasitemia were treated with a series of intra-peritoneal injections of Salicylhydroxamic acid (SHAM) plus glycerol. Permanent cures were obtained with 380 mg/kg SHAM plus 3.8 g/kg glycerol, a dosage regime which was just sublethal. Using a regime with which permanent cure was obtained, the SHAM concentration in the blood plasma remained above 2 mmole/liter for about 20 min, while the glycerol concentration remained above 22 mmole/liter for about 1 hr. The brain concentration of SHAM was close to the plasma concentration. The concentration of glycerol in the brain remained far below the plasma concentration, reaching 6 to 8 mmole/liter between 1 and 2 hr after the beginning of treatment. Treatment with glycerol did not affect the mobility of the trypanosomes nor the survival of infected rats after treatment with suramin.     

Micro-CT visualization of a promastigote secretory gel (PSG) and parasite plug in the digestive tract of the sand fly Lutzomyia longipalpis infected with Leishmania mexicana

Leishmaniasis is a debilitating disease of the tropics, subtropics and southern Europe caused by Leishmania parasites that are transmitted during blood feeding by phlebotomine sand flies (Diptera: Psychodidae). Using non-invasive micro-computed tomography, we were able to visualize the impact of the laboratory model infection of Lutzomyia longipalpis with Leishmania mexicana and its response to a second blood meal. For the first time we were able to show in 3D the plug of promastigote secretory gel (PSG) and parasites in the distended midgut of whole infected sand flies and measure its volume in relation to that of the midgut. We were also able to measure the degree of opening of the stomodeal valve and demonstrate the extension of the PSG and parasites into the pharynx. Although our pilot study could only examine a few flies, it supports the hypothesis that a second, non-infected, blood meal enhances parasite transmission as we showed that the thoracic PSG-parasite plug in infected flies after a second blood meal was, on average, more than twice the volume of the plug in infected flies that did not have a second blood meal.     

1,3-β-d-Glucan synthase ofNeurospora crassa: Partial purification and characterization of solubilized enzyme activity

1,3-β-d-Glucan synthase activity ofNeurospora crassa was rendered soluble by treatment of crude protoplast lysates with 0.1% 3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer containing 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, 1 mM dithiothreitol, 200 mM inorganic phosphate, 10 μM GTP, and 600 mM glycerol. Approximately 50% of enzyme activity was solubilized; soluble enzyme activity was purified 5.6-fold with a net 38% recovery by sucrose gradient density sedimentation. Partially purified enzyme activity had a half-life of 60 ± 10 h at 4°C, aK_m,app of 0.75 ± 0.05 mM, and a V_max,app of 35 ± 1 enzyme units/mg protein.     

Further studies of the effect of calcium on the time course of action of carbamylcholine at the neuromuscular junction

The rate at which the postjunctional membrane of muscle fibers becomes desensitized to the action of carbamylcholine is increased after the muscle has been soaked in solutions containing increased concentrations of calcium. Some further aspects of this effect of calcium were investigated by measuring changes in the input resistance of single fibers of the frog sartorius during local perfusion of the neuromuscular junction with 2.73 x 10^-3 M carbamylcholine in isolated muscles immersed in 165 mM potassium acetate. It was found that (a) sudden changes in the local concentration of calcium brought about by perfusing fibers with carbamylcholine solutions containing 20 mM calcium, 40 mM oxalate, or 40 mM EDTA were followed within 20 sec by marked changes in the rate of desensitization; (b) prior to 13 sec after the introduction of carbamylcholine, however, no effect on the input resistance could be detected even though the muscle had been presoaked in 10 mM calcium; (c) the ability of high concentrations of calcium to bring about rapid desensitization disappears when a lower concentration of carbamylcholine (0.137 x 10^-3 M) is applied to the muscle fiber. These findings suggest that calcium present in the extracellular fluid can act directly on the postjunctional membrane to promote the desensitization process and that an increased permeability of the membrane to calcium brought about by the presence of carbamylcholine is a factor which contributes to this action.     

Kinetic properties and regulation of glycerol-3-phosphate dehydrogenase from the overwintering, freezing-tolerant gall fly larva, Eurosta solidagenis

The kinetic properties of cytoplasmic glycerol-3-P dehydrogenase from the third instar larva of the gall fly, Eurosta solidaginis, were studied with emphasis on temperature effects on the enzyme and the regulation of enzyme activity during the synthesis of the cryoprotectant, glycerol. Isoelectrofocusing revealed one major and two minor forms of the enzyme with no alteration in the pI's or relative activities of the forms in larvae acclimated to 24 versus ?30 °C. Kinetic properties of the enzyme were also the same in larvae acclimated to high and low temperatures. Arrhenius plots were linear over a 30 to 0 °C range with an activation energy of 12,630 ± 185 cal/mol and a Q₁₀ of 2.16. The K_m for dihydroxyacetone-P was constant, at 50 μM, between 30 and 10 °C but increased by 75% at 0 °C; this increase may be a factor in the cessation of glycerol synthesis which occurs below 5 °C in this species. The K_m(NADH), by contrast, was higher (5–6 μM) at 30 °C but decreased (3 μM) at lower temperatures. In the reverse direction, K_m's were 340 μM for glycerol-3-P and 12 μM for NAD⁺. Effects of most inhibitors (of the forward reaction), glycerol-3-P (K_i = 2.4 mM), NAD⁺ (K_i = 0.2 mM), ATP, Mg·ATP, and P_i, were unaltered by assay temperature but ADP effects were potentiated by low temperature while citrate inhibition was greatest at high temperatures. Glycerol and sorbitol, which accumulate as cryoprotectants in E. solidaginis, had no significant effects on kinetic constants at any temperature but decreased the V_max activity of the enzyme. Thermal inactivation studies showed an increased thermal stability of the larval enzyme compared to the homologous enzyme from rabbit muscle while added polyols stabilized enzyme activity, decreasing the rate of enzyme inactivation at 50 °C.     

ATPase activity in plasmalemma-rich vesicles isolated by aqueous two-phase partitioning from Vicia faba mesophyll and epidermis: Characterization and influence of abscisic acid and fusicoccin

Plasmalemma-rich microsomal vesicles were prepared from whole leaf and acid-washed epidermal tissue of Vicia faba L. cv. Osnabrücker Markt by aqueous two-phase partitioning in dextran T-500 and polyethylenglycol 1350 aqueous phases. These vesicles were tightly sealed and predominantly right-side out, and contained a K⁺ -stimulated, mg²⁺-dependent and vanadate-sensitive ATPase. The enzyme from both tissues exhibited nearly identical properties: pH optimum 6.4, K_m for ATP 0.60 mM(whole leaf) and 0.67 mM (epidermis). V_max -480 nmol (mg protein)¹ min¹ (whole leaf) and 510 nmol (mg protein)¹ min¹ (epidermis), I₅₀ (Na₃,VO₄) 7.5 μM (whole leaf) and 15 μM (epidermis). The enzyme was not inhibited by NO₃(50 mM)or sodium azide (I mM). DCCD (20 μM) reduced enzyme activity to 50% (whole leaf) and 58% (epidermis), gramicidin S (20 μM) to 36% (whole leaf) and 41%(epidermis). Ca²⁺ inhibited the ATPase [I₅₀, C²⁺: 0.5 mM(whole leaf) and 0.8 mM(epidermis)]. Ca²⁺ inhibited the ATPase [I₅₀, C²⁺ 0.5 mM(whole leaf) und 0.8 (epidermis)]. The vanadate-sensitive ATPase from whole leaf and epidermal tissue was slightly but significantly stimulated by fusicoccin (FC) at a concentration (0.13 μM) promoting stomatal opening. The stimulation was not seen in the solubilized ATPase. Stomata of the cultivar used here were insensitive lo (±)ABA up to 2 μM level which is effective in most other cultivars and species. Likewise, at this concentration no effect of ABA on the activity of the epidermal ATPase was observed. The data are discussed with respect to the interaction of FC and ABA with the ATPase.     

Changes in sensitivity to anoxia of the cardiac action potential plateau during chick embryonic development.

The effects of anoxia on cardiac action potentials were studied at different stages of development of embryonic chick heart. The plateau phase of the action potential was markedly depressed by anoxia in old (15–16 days old) embryonic hearts without any significant change in other configurations of the action potential. Raising the concentration of glucose in the external fluid prevented the shortening of the action potential plateau by anoxia, and, conversely, a further reduction was observed in glucose-free media. In young (3–4 days old) embryonic hearts, the shortening of the action potential plateau was not produced by anoxia, but was produced by a combination of anoxia and glucose deprivation. When the action potential was shortened by anoxia in old hearts and by anoxia plus glucose deprivation in young hearts, isoproterenol (10^?5M), dibutyryl cyclic 3′,5′-adenosine monophosphate (dBcAMP: 1 mM) plus aminophylline (1 mM), and calcium ion (3–6 mM), partially reversed the shortened action potential in old hearts, but did not produce any prolongation in the young hearts. Therefore, the cation channels responsible for the action potential plateau in young hearts may be pharmacologically different from those in old hearts. The differences in action potential plateau between young and old hearts were discussed in relation to dependence upon energy.     

Methanol-dependent production of dihydroxyacetone and glycerol by mutants of the methylotrophic yeast Hansenula polymorpha blocked in dihydroxyacetone kinase and glycerol kinase

Summary Various factors controlling dihydroxyacetone (DHA) and glycerol production from methanol by resting cell suspensions of a mutant of Hansenula polymorpha, blocked in DHA kinase and glycerol kinase, were investigated. The presence of methanol (250mM) and an additional substrate (0.5%, w/v) to replenish the xylulose-5-phosphate required for the assimilation reaction (DHA synthase) was essential for significant triose production by this double mutant. A number of sugars were tested as additional substrates and C₅ sugars gave the highest triose accumulation (ca. 20mM after 45h). Glucose was the poorest additional substrate and triose production only started after its exhaustion, which occurred in the first few hours. Other sugars were metabolized at a much lower rate and accumulation of trioses began right at the start of the experiments and gradually increased with time. The production rate of total trioses increased, and the relative amount of glycerol diminished with higher oxygen supply rates. The data suggest that conversion of DHA into glycerol, catalysed by reduced nicotine adenine dinucleotide (NADH)-dependent DHA reductase, is partly regulated via intracellular NADH levels. Further support for this hypothesis was obtained in experiments with antimycin A, an inhibitor of the electron transport chain. Addition of higher amounts of methanol and xylose, either by increasing the initial concentrations or by repeated addition of these substrates, resulted in considerably enhanced productivity and a switch towards glycerol formation. After reaching a level of approximately 25mM the DHA concentration remained constant while the glycerol level gradually increased with time. After an incubation period of 350 h, a total of 3.9 M methanol and 0.62 M xylose had been converted, which resulted in accumulation of 0.76 M trioses, mostly glycerol.Offprint requests to: L. Dijkhuizen     

Determination of nitrate in blood by gas chromatography and gas chromatography–mass spectrometry

We devised a sensitive and simple method for determining nitrate in whole blood, using an extractive alkylation technique. Nitrate in whole blood was reduced to nitrite by hydrazine sulfate in the presence of Cu²⁺ and Zn²⁺ as catalysts, and alkylated with pentafluorobenzyl bromide using tetradecyldimethylbenzylammonium chloride as the phase-transfer catalyst. The obtained derivative was analyzed qualitatively by gas chromatography–mass spectrometry and quantitatively by gas chromatography with electron-capture detection. The detection limit of nitrate in whole blood was 0.01 mM. The calibration curve was linear over the concentration range from 0.02 to 1.0 mM for nitrate in whole blood. The accuracy and precision of the method were evaluated and the relative standard deviations were found to be within 10%. Using this method, the blood nitrate levels of two victims who committed suicide by inhaling automobile exhaust gas were determined.     

Optimization of Storage and Stability of Camel Liver Glutathione S-Transferase

Glutathione S-transferases (GSTs) are multifunctional enzymes and play an important role in cellular detoxification. Besides this, GSTs act as cytosolic carrier proteins that bind hydrophobic compounds such as heme, bilirubin, steroids, and polycyclic hydrocarbons. GST has great importance in biotechnology, as it is a target for vaccine and drug development and biosensors development for xenobiotics. Moreover, the GST tag has been extensively used for protein expression and purification. Until now, biophysical properties of camel liver GST have not been characterized. In the present study we have purified camel (Camelus dromedarius) liver GST to homogeneity in a single step by affinity chromatography with 23.4-fold purification and 60.6% yield. Our results showed that maximal activity of GST was at pH 6.5 and it was stable in the pH range of 5 to 10. The optimum temperature was 55°C and the T_m was 57°C. The chemical chaperone glycerol (3.3 M) was able to protect GST activity and aggregation against thermal denaturation by stabilizing the protein structure at 50 and 57°C, respectively. However, L-arginine (125 mM) did not protect GST against thermal stress. Far-ultraviolet circular dichroism (CD) spectra showed that glycerol protected the secondary structure of GST while L-arginine induced conformational changes under thermal stress. In conclusion, our studies on the GST stability suggest that glycerol works as a stabilizer and L-arginine acts as a destabilizer.     

Balance between Cyanide-Sensitive and -Insensitive Respiration in Wheat Root Mitochondria, as Influenced by Salt Concentration in the Plant Growth Medium

Mitochondria were isolated from 7-day-old wheat roots (Triticum vulgare Vill. cv. Svenno Spring Wheat) grown in either a full-strength culture medium (100%) or in the same medium diluted 100 times (1%). Outer membrane integrity was assayed using the cytochrome c reduction assay. This indicated about 20% damage. Using an oxygen electrode the respiration of the mitochondria was measured with either malate or succinate as the substrate (both 40 mM). KCN (3 mM) and salicylhydroxamic acid (SHAM, 1 mM) were used as inhibitors. The properties of the isolated mitochondria (STATE 3 rate, ADP/O ratio, and KCN-sensitivity) depend upon the ionic concentration of the growth medium of the roots. In the mitochondria isolated from roots grown in the 1% medium (1% mitochondria) there is a synergistic effect of KCN and SHAM. This means that electrons can be shifted from one pathway to the other when only one of the inhibitors is added. This flexibility between the electron pathways is almost nil in the mitochondria isolated from roots grown in the 100% medium (100% mitochondria). The maximal capacity of the alternative electron pathway (= rate in the presence of KCN) is higher in 1% (40 nmol O₂ min^?1 (mg protein)^?1) than in 100% mitochondria (20 nmol O₂ min^?1 (mg protein)^?1. In 100% mitochondria the alternative pathway seems to be operating at maximal capacity in the absence of KCN with both substrates and in both STATES 3 and 4. In 1% mitochondria the alternative pathway functions at >50% of its capacity in the absence of KCN.     

The activity and properties of poly(adenosine diphosphate ribose) polymerase in vitro during the embryonic development of the South African clawed toad Xenopus laevis

Characterization of poly(ADPribose) polymerase in isolated nuclei of Xenopus laevis embryos shows that maximum activity in vitro occurs at 25°C in 10 mM Tris-HCl buffer, pH 8.0, containing 20 mM MgCl₂, 1 mM dithiothreitol, and 3 mM NaF. Under these conditions the apparent K_m for NAD⁺ was 0.125 mM. The activity of the polymerase during embryogenesis was measured using both a whole embryo extract and isolated embryonic nuclei. Both of these sources of enzyme demonstrated an increase of approximately eightfold in the activity per cell, between early cleavage (stages 2–4; Nieuwkoop and Faber, 1956, “Normal Table of Xenopus laevis (Daudin),” North-Holland, Amsterdam) and late neurula (stages 23–24). From late neurula to early tadpole (stages 38–39) the activity of the extracted enzyme, calculated per cell, decreased by 64%. During the same period the activity of the enzyme in isolated nuclei increased by 40% to reach its maximum activity in early tail bud (stages 27–28), and thereafter decreased by 23%. These results indicate a possible involvement of poly(ADPribose) polymerase in these embryos in the cell differentiation processes, rather than in cell proliferation.     

Effect of Salicylhydroxamic Acid on Respiration, Photosynthesis, and Peroxidase Activity in Various Plant Tissues

Earlier reports from our laboratory described salicylhydroxamicacid (SHAM) stimulation of O₂ uptake by expanded soybean leavesor older green cotyledons. This stimulation could not be interpretedin terms of engagement or capacity of the cytochrome and alternativerespiratory pathways. In this report, we tested the possibilitythat a soluble peroxidase, which can be easily eluted from soybeanleaves and cotyledons, might be responsible for SHAM stimulationin whole tissue. The peroxidase catalyzes oxidation of NADHby O₂, is strongly stimulated by SHAM and benzhydroxamic acid(BHAM) and inhibited by KCN, propyl gallate and gentisic acid.This peroxidase, however, does not seem to be responsible forSHAM-stimulated O₂ uptake in whole, green tissue. In our earlier work reporting SHAM-stimulated respiration ingreen tissue, the samples had not been shielded from room light(10–20 µmol photons m^–2.s^–1). In thisreport, we show that O²-uptake rates of controls measured indarkness were always greater than those measured in room light.SHAM stimulation was not observed in the dark or in tissue withoutchlorophyll. We also found that CO₂ uptake of whole leafletsin saturating light was completely inhibited by SHAM fed throughthe transpiration stream. SHAM, therefore, is a potent inhibitorof photosynthesis. We conclude that the SHAM-stimulated respirationof green tissues we reported earlier likely was due to verylow rates of photosynthesis occurring under room light. ³Present address: SANDOZ Ltd., Agrobiological Research Station,4108 Witterswil, Switzerland ⁴Present address: WTC 1A3, Weyerhaeuser Co., Tacoma, WA 98477,U.S.A. (Received June 23, 1989; Accepted October 20, 1989)     

Calcium-dependent slow potassium conductance in rat skeletal myotubes

A prolonged hyperpolarizing afterpotential (amplitude 5–20 mV, half decay time about 400 msec at 25°C) follows the action potential in myotubes and myosacs cultured from rat skeletal muscle. This slow hyperpolarizing afterpotential (hap) is mediated by an increase in membrane K conductance, because its reversal potential follows the Nernst potential for K and is not affected by other ions. The conductance increase measured during the hap (up to four times the resting input conductance) correctly predicts the time course of the slow hap. The slow hap is Ca dependent. Its amplitude decreases when bath [Ca] is lowered, and both amplitude and duration increase when bath [Ca] is raised. The slow hap is blocked by intracellular injection of the calcium chelator, EGTA. It is inhibited by solutions containing 2–4 mM manganese or 1–5 mM barium, but is not blocked by 5–20 mM tetraethylammonium. Myotubes bathed in zero [Na], high [Ca] solutions show calcium action potentials, which are inhibited by 2–10 mM manganese, nickel or cobalt. Myotubes bathed in isotonic Ca salts (or in 2 mM Ca plus 5 mM caffeine) show long-lasting (up to 10 sec) spontaneous hyperpolarizations accompanied by prolonged contractions. These hyperpolarizations are associated with a large increase in input conductance, and they reverse in sign near the K equilibrium potential. They appear to reflect activation of the Ca-sensitive K conductance by Ca released from intracellular stores. The observation that spontaneous hyperpolarizations usually occur with no prior depolarization argues that at least a portion of the slow, Ca-sensitive K conductance system can be activated by internal Ca alone, with no requirement for plasma membrane depolarization. Cultured myotubes also have a faster K conductance system, which is inhibited by 5–20 mM tetraethylammonium or 1–5 mM barium, and is not dependent on Ca for its activation.     

Metabolism of biodiesel-derived glycerol in probiotic Lactobacillus strains

Three probiotic Lactobacillus strains, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus delbrueckii, were tested for their ability to assimilate and metabolize glycerol. Biodiesel-derived glycerol was used as the main carbon and energy source in batch microaerobic growth. Here, we show that the tested strains were able to assimilate glycerol, consuming between 38 and 48 % in approximately 24 h. L. acidophilus and L. delbrueckii showed a similar growth, higher than L. plantarum. The highest biomass reached was 2.11 g?L^?1 for L. acidophilus, with a cell mass yield (Y _X/S) of 0.37 g?g^?1. L. delbrueckii and L. plantarum reached a biomass of 2.06 and 1.36 g?L^?1. All strains catabolize glycerol mainly through glycerol kinase (EC 2.7.1.30). For these lactobacillus species, kinetic parameters for glycerol kinase showed Michaelis–Menten constant (K _m) ranging from 1.2 to 3.8 mM. The specific activities for glycerol kinase in these strains were in the range of 0.18 to 0.58 U?mg?protein^?1, with L. acidophilus ATCC 4356 showing the maximum specific activity after 24 h of cultivation. Glycerol dehydrogenase activity was also detected in all strains studied but only for the reduction of glyceraldehyde with NADPH (K _m for DL-glyceraldehyde ranging from 12.8 to 32.3 mM). This enzyme shows a very low oxidative activity with glycerol and NADP⁺ and, most likely, under physiological conditions, the oxidative reaction does not occur, supporting the assumption that the main metabolic flux concerning glycerol metabolism is through the glycerol kinase pathway.     

The Activity of Ribulose Diphosphate Carboxylase in Extracts of Gonyaulax polyedra in the Day and the Night Phases of the Circadian Rhythm of Photosynthesis

The ribulose 1,5-diphosphate carboxylase from Gonyaulax polyedra Stein. has a half-life of about four hours in buffer, but can be stabilized by the addition of 50% glycerol. The optimum pH is 7.8 to 8.0 and the optimum Mg²⁺ concentration is 3 mm. Heavy metal ions (Cu²⁺, Hg²⁺, Ni²⁺, Zn²⁺), EDTA, pyrophosphate, and adenosine triphosphate were strongly inhibitory. Ribulose 1,5-diphosphate carboxylase from Gonyaulax was not cold-sensitive or activated by light activation factor from tomato or Gonyaulax. No difference in the activity of this enzyme was detected when extracts prepared at the maximum and the minimum of the circadian rhythm of photosynthesis were compared. The Km of HCO₃⁻ was also the same (16 to 19 mm).     

Salicylic Acid induces cyanide-resistant respiration in tobacco cell-suspension cultures

Cyanide-resistant, alternative respiration in Nicotiana tabacum L. cv Xanthi-nc was analyzed in liquid suspension cultures using O₂ uptake and calorimetric measurements. In young cultures (4-8 d after transfer), cyanide inhibited O₂ uptake by up to 40% as compared to controls. Application of 20 μm salicylic acid (SA) to young cells increased cyanide-resistant O₂ uptake within 2 h. Development of KCN resistance did not affect total O₂ uptake, but was accompanied by a 60% increase in the rate of heat evolution from cells as measured by calorimetry. This stimulation of heat evolution by SA was not significantly affected by 1 mm cyanide, but was reduced by 10 mm salicylhydroxamic acid (SHAM), an inhibitor of cyanide-resistant respiration. Treatment of SA-induced or uninduced cells with a combination of cyanide and SHAM blocked most of the O₂ consumption and heat evolution. Fifty percent of the applied SA was taken up within 10 min, with most of the intracellular SA metabolized in 2 h. 2,6-Dihydroxybenzoic and 4-hydroxybenzoic acids also induced cyanide-resistant respiration. These data indicate that in tobacco cell-suspension culture, SA induces the activity and the capacity of cyanide-resistant respiration without affecting the capacity of the cytochrome c respiration pathway.     

17β‐estradiol supplementation attenuates ovariectomy‐induced increases in ATGL signaling and reduced perilipin expression in visceral adipose tissue

Adipocytes from post‐menopausal females have higher basal lipolytic rates than pre‐menopausal females, which contributes to increased risk of developing dyslipidemia following menopause. The purpose of this study was to delineate cellular mechanisms affecting adipose tissue function in the ovariectomized (OVX) mouse and also determine if physical activity or estrogen supplementation alter any detected changes. Female C57/Bl6 mice were placed into SHAM, OVX sedentary (OVX), OVX exercise (OVX‐Ex), and OVX sedentary + 17β‐estradiol (OVX + E₂) groups. Visceral fat mass, glycerol, and NEFA levels were significantly higher in OVX mice compared to SHAM animals, but were not elevated in the E₂‐treated animals. Voluntary running failed to change circulating levels of glycerol or NEFA in OVX mice, but did partially attenuate the increase in visceral fat mass. Adipose triglyceride lipase (ATGL) protein content was significantly elevated in visceral fat from OVX and OVX‐Ex groups compared to SHAM, while ATGL–CGI‐58 interaction was significantly higher in OVX than SHAM and OVX + E₂ mice. No significant differences in HSL phosphorylation were detected between groups, however, ERK1/2 phosphorylation was significantly elevated in the OVX mice. To determine if ERK1/2 function was critical for the increased glycerol levels, visceral fat was treated with MEK inhibitor PD98059, with no differences in glycerol release detected. Perilipin protein content was decreased significantly in OVX and OVX‐Ex mice compared to SHAM. Thus, these data suggest that increased ATGL signaling and reduced perilipin protein content may contribute to increased NEFA and glycerol levels in OVX mice, which are attenuated with E₂ treatment, but not by exercise. J. Cell. Biochem. 110: 420–427, 2010. © 2010 Wiley‐Liss, Inc.