Gradual Inhibition Research Articles

Evidence That Prestin Has at Least Two Voltage-dependent Steps

Prestin is a voltage-dependent membrane-spanning motor protein that confers electromotility on mammalian cochlear outer hair cells, which is essential for normal hearing of mammals. Voltage-induced charge movement in the prestin molecule is converted into mechanical work; however, little is known about the molecular mechanism of this process. For understanding the electromechanical coupling mechanism of prestin, we simultaneously measured voltage-dependent charge movement and electromotility under conditions in which the magnitudes of both charge movement and electromotility are gradually manipulated by the prestin inhibitor, salicylate. We show that the observed relationships of the charge movement and the physical displacement (q-d relations) are well represented by a three-state Boltzmann model but not by a two-state model or its previously proposed variant. Here, we suggest a molecular mechanism of prestin with at least two voltage-dependent conformational transition steps having distinct electromechanical coupling efficiencies.

Activation of guanine-β- d-arabinofuranoside and deoxyguanosine to triphosphates by a common pathway blocks T lymphoblasts at different checkpoints

The deoxyguanosine (GdR) analog guanine-ß- d-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.

The Effects of Anti-A and Anti-B On Platelet Function: An in Vitro Model of ABO Non-Identical Transfusion.

Abstract 2120Poster Board II-97Background: Transfusion of ABO non-identical red blood cells (RBCs) can cause immune mediated hemolytic transfusion reactions. Therefore, only ABO identical RBCs are transfused, except in emergencies, when group O RBCs are transfused. Use of exclusively ABO identical plasma and platelet (PLT) transfusions is not uniformly practiced nor always feasible despite reports of hemolytic reactions. Since PLTs and soluble plasma proteins possess A and B antigens, ABO non-identical PLTs could, theoretically, be activated and/or rendered hypofunctional by anti-A and anti-B antibodies (Abs) in transfused or recipient plasma. Recent findings demonstrate that transfusion of ABO non-identical PLTs is associated with increased bleeding in surgical patients and patients with leukemia. Blunt trauma patients who received at least one ABO non-identical blood product transfusion demonstrated a significantly higher RBC usage (12.3 ± 6.9 SD versus 8.4 ± 9.9 SD, p-value 0.0011) compared to those patients who received only ABO identical transfusions (Transfusion. 2007;47:192A). ABO identical PLT transfusions in leukemia patients were a significant predictor of survival (Leukemia. 2008;22:631-5). In a multi center retrospective analysis of more than one million cancer patients over a period of 9 years, Khorana et al. demonstrated an overall venous thromboembolism (VTE) rate of 4.1%. In multivariate risk factor analysis, the association between blood transfusions and VTE had an odds ratio of 1.35 (1.31-1.39, 95% CI) with a p value of < 0.001 (Arch Intern Med. 2008;168:2377-81). We hypothesized that PLTs activated by ABO Abs might have altered function. Methods and Materials: PLT function was evaluated by testing aggregation in platelet rich plasma (PRP). Aggregation was performed with PRP from 7 type A and 6 type B normal blood donors following a 10 min incubation period at 37°C with either normal saline, group O or AB plasma. PLTs were activated by 20 mM ADP and aggregation quantitated from the maximum change in OD. Similar experiments were repeated utilizing different titration of the commercial anti-A and anti-B anti-sera. Results: Following incubation with O plasma, PLT aggregation was inhibited by a mean of 38% and 18% for group A and B PLTs, respectively (P ≤ 0.005) (Figure). A trend toward inhibition was observed when type A PLTs were incubated with control AB plasma (average of 14%, P = 0.187), whereas type B PLT showed no inhibition when incubated with AB plasma (P = 0.939) (Table 1). PLT aggregation with the anti-sera showed gradual inhibition correlated with the antibody titer (Table 2). Conclusion: Mediators in group O plasma, most likely anti-A and anti-B Abs, cause impaired PLT aggregation in ABO non-identical PLTs. These in vitro findings may explain, at least in part, clinical observations that patients receiving ABO non-identical PLT transfusions experience more bleeding than recipients of ABO identical PLT transfusions.Table 1:PLT aggregation of A and B PRP with saline, O and AB plasma.Blood Donor TypeNAverage Percentage of Platelet Aggregation (SD)Normal Saline“O” PlasmaP value*“AB” PlasmaP valueA792 (7.4)54 (9.9)< 0.00578 (2.9)0.187B685 (6.8)67 (9.8)0.00585.3 (7.9)0.939P value < 0.05 is considered statistically significant. [Display omitted] Table 2:PLT aggregation of A and B PRP with different titration of the commercial anti-A and anti-B anti-sera.Anti-sera/PlasmaType A PRPP valueType B PRPP valueBaseline93.7 (3.1)—83.4 (11)—1:102448.7 (8.5)0.00636.3 (7.8)0.00051:51257.3 (2.5)0.000147.7 (7.5)0.0021:25659.5 (3.5)0.00859.5 (0.7)0.0021:12855.5 (3.5)0.00667 (2.8)0.027AB plasma87.7 (3.2)0.0881.2 (16)0.88 Disclosures:No relevant conflicts of interest to declare.

Wheat sprout extract-induced apoptosis in human cancer cells by proteasomes modulation

Natural occurring modulators of proteasome functionality are extensively investigated for their implication in cancer therapy. On the basis of our previous evidences both on proteasomal inhibition by monomeric polyphenols, and on the characterization of wheat sprout hydroalcoholic extract, herein we thoroughly report on a comparative study of the effect of wheat sprout extract on both normal and tumour cells. Treatment of isolated 20S proteasomes with wheat sprout extracts induced a gradual inhibition of all proteasome activities. Next, two wheat sprout extract components were separated: a polyphenol and a protein fraction. Both components exerted an in vitro inhibitory effect on proteasome activity. HeLa tumour cells and FHs 74 Int normal cells were exposed to both fractions, resulting in different rates of proteasome inhibition, with tumour cells showing a significantly higher degree of proteasome impairment and apoptosis induction. Furthermore, a decrease in proteasome activities and in cell survival of the human plasmacytoma RPMI 8226 cell line, upon the same treatments, was observed. Collectively, our results provide additional evidences supporting the possible use of natural extracts as coadjuvants in cancer treatments.

Modification by Single Ubiquitin Moieties Rather Than Polyubiquitination Is Sufficient for Proteasomal Processing of the p105 NF-κB Precursor

Activation of NF-kappaB is regulated via numerous ubiquitin- and proteasome-mediated steps; an important one is processing of the precursor p105 to the p50 active subunit. The mechanisms involved are largely unknown, because this is an exceptional case where the ubiquitin system does not destroy its substrate completely. Here, we demonstrate that proteasomal processing of p105 requires ubiquitin but not generation of polyubiquitin chains. In vitro, ubiquitin species that cannot polymerize mediate processing. In yeasts that express nonpolymerizable ubiquitins, processing proceeds normally, whereas degradation of substrates that are dependent on polyubiquitination is inhibited. Similar results were obtained in mammalian cells. Interestingly, processing requires multiple monoubiquitinations, because progressive elimination of lysines in p105 is accompanied by gradual inhibition of p50 generation. Finally, the proteasome recognizes the multiply monoubiquitinated p105. These findings suggest that a proteolytic signal can be composed of a cluster of single ubiquitins, not necessarily a chain.

Enigmatic effect of cellular ATP on fatty acid biosynthesis: Stimulation by moderate decrease and inhibition by increase of cellular ATP

The cellular ATP concentration was tested for its effect on fatty acid biosynthesis from glucose in hepatocytes. ATP was manipulated by adding increasing concentrations of cycloheximide, amytal, atractyloside, 2,4-dinitrophenol or adenosine. A slight decrease in cellular ATP coincided with a stimulation of fatty acid biosynthesis whereas a further lowering of cellular ATP resulted in a gradual inhibition. Increasing the cellular ATP level by titration with adenosine had the opposite effect. These results are in line with the suggestion that fatty acid biosynthesis from glucose is an energy-yielding process which is stimulated by a moderate drop in cellular ATP.

Insulin, cGMP, and TGF-β Signals Regulate Food Intake and Quiescence in C. elegans: A Model for Satiety

Despite the prevalence of obesity and its related diseases, the signaling pathways for appetite control and satiety are not clearly understood. Here we report C. elegans quiescence behavior, a cessation of food intake and movement that is possibly a result of satiety. C. elegans quiescence shares several characteristics of satiety in mammals. It is induced by high-quality food, it requires nutritional signals from the intestine, and it depends on prior feeding history: fasting enhances quiescence after refeeding. During refeeding after fasting, quiescence is evoked, causing gradual inhibition of food intake and movement, mimicking the behavioral sequence of satiety in mammals. Based on these similarities, we propose that quiescence results from satiety. This hypothesized satiety-induced quiescence is regulated by peptide signals such as insulin and TGF-beta. The EGL-4 cGMP-dependent protein kinase functions downstream of insulin and TGF-beta in sensory neurons including ASI to control quiescence in response to food intake.

Extinction learning: neurological features, therapeutic applications and the effect of aging

Extinction learning consists of the usually gradual inhibition of the retrieval of a previously learned response or behavior. It is widely used for the treatment of syndromes of learned fear, such as phobias and post-traumatic stress disorder. It relies on well-identified molecular processes in the hippocampus, basolateral amygdala, ventromedial prefrontal cortex (vmPFC) and entorhinal cortex. In humans, thickness of the orbital cortex, vmPFC and the anterior cingulate cortex correlates with the capacity to extinguish. The three regions are functionally inter-related (see below). The development of learned fear syndromes in humans is viewed by many as being due to a deficit of extinction, and so of the capacity to deal with fear. Blockade of NMDA receptors, inhibition of protein synthesis in the vmPFC or blockade of protein synthesis or of various molecular signaling cascades in the hippocampus, amygdala or entorhinal cortex impairs extinction. d-cycloserine, a partial agonist at NMDA receptors, enhances extinction in animals and humans and may help extinction to exert its therapeutic effect. Cannabinoids also enhance extinction, acting through CB1 receptors, but their therapeutic use is not warranted.

Wheat sprout extract induces changes on 20S proteasomes functionality

Wheat sprouts contain a very high level of organic phosphates and a powerful cocktail of different molecules such as enzymes, reducing glycosides and polyphenols. The antioxidant properties of wheat sprouts have been widely documented and it has been shown that they are able to protect DNA against free-radicals mediated oxidative damage. Furthermore, we have recently reported on the effects of several polyphenols on 20S proteasomes, underlying the dual role of epigallocatechin-3-gallate as an antioxidant and a proteasome effector in cancer cells. The aim of this study was to investigate the effects of wheat sprout extracts on 20S proteasome functionality. Wheat sprout extracts have been analysed and characterized for their polyphenolic content using the Folin-Ciocalteau reagent and RP-HPLC technique. Comparing our data with a polyphenol standard mixture we identified five different polyphenols: gallic acid, epigallocatechin-3-gallate, epigallocatechin, epicatechin and catechin. The treatment of isolated 20S proteasomes with the extract induced a gradual inhibition of all the tested components, ChT-L, T-L, PGPH and BrAAP, in both the complexes. At low extract concentration a slight activation of the enzyme was evident only for the BrAAP component of the constitutive enzyme and the ChT-L activity of the immunoproteasome. β-casein degradation rate decreased, particularly with the immunoproteasome. Human Colon adenocarcinoma (Caco) cells, stimulated with 12- O-tetradecanoylphorbol-13-acetate, showed activation of the 20S proteasome activities at short incubation times and an increase in intracellular oxidative proteins. Cells treatment with wheat sprout extract led to proteasome inhibition in unstimulated cells and attenuated the effects mediated by TPA. Finally, exposure to the extract affected the expression levels of pro-apoptotic proteins.

Redox-dependent change of nucleotide affinity to the active site of the mammalian complex I.

A very potent and specific inhibitor of mitochondrial NADH:ubiquinone oxidoreductase (complex I), a derivative of NADH (NADH-OH) has recently been discovered (Kotlyar, A. B., Karliner, J. S., and Cecchini, G. (2005) FEBS Lett. 579, 4861-4866). Here we present a quantitative analysis of the interaction of NADH-OH and other nucleotides with oxidized and reduced complex I in tightly coupled submitochondrial particles. Both the rate of the NADH-OH binding and its affinity to complex I are strongly decreased in the presence of succinate. The effect of succinate is completely reversed by rotenone, antimycin A, and uncoupler. The relative affinity of ADP-ribose, a competitive inhibitor of NADH oxidation, is also shown to be significantly affected by enzyme reduction (KD of 30 and 500 microM for oxidized and the succinate-reduced enzyme, respectively). Binding of NADH-OH is shown to abolish the succinate-supported superoxide generation by complex I. Gradual inhibition of the rotenone-sensitive uncoupled NADH oxidase and the reverse electron transfer activities by NADH-OH yield the same final titration point (approximately 0.1 nmol/mg of protein). The titration of NADH oxidase appears as a straight line, whereas the titration of the reverse reaction appears as a convex curve. Possible models to explain the different titration patterns for the forward and reverse reactions are briefly discussed.

An open-label study to evaluate dose and cycle dependence of the pharmacokinetics of pegylated liposomal doxorubicin

There are no definitive data in humans on the dose dependence and/or cycle dependence of the pharmacokinetics (PK) of pegylated liposomal doxorubicin (PLD). This study examined the PK of PLD across a twofold dose variation and along 3 cycles. Fifteen patients received PLD in successive doses of 60, 30, and 45 mg/m(2) (Arm A) and 30, 60, and 45 mg/m(2) (Arm B), every 4 weeks. Twelve patients, six on each arm, completed all three cycles and were fully evaluable. Plasma levels of doxorubicin were analyzed by HPLC and fluorimetry. PK analysis was done by non-compartmental method. Repeated measures ANOVA and paired tests were used for statistical analysis. There was no significant difference in the PK parameters examined when the dose was increased from 30 to 60 mg/m(2). However, when we analyzed the effect of cycle number on the PK, we found a gradual and significant inhibition of clearance (P < 0.0001) from the 1st through the 3rd cycle of PLD, with a geometric mean increase of 43% in dose-normalized AUC (P = 0.0003). Dose-normalized C(max) and T(1/2) mean values increased by 17 and 18%, respectively between the 1st and 3rd cycles, but only the increase in T(1/2) was statistically significant (P = 0.0017). While the PK of PLD is not dose-dependent within the dose range of 30-60 mg/m(2), there is evidence of a cycle-dependent effect that results in inhibition of clearance when patients receive successive cycles of PLD. These results suggest the need for dose adjustments of PLD upon retreatment to minimize the risk of toxicity.

Effects of Cadmium Chloride on the Activity ofFour Enzymes in Gills of Mosquito FishGambusia affinis

: Gambusia affinis fishes were exposed to 2,4 and 8 mg Cd/L of water for 8 days and for 2,4, and 8 days to 10 mg Cd/L of water. Gills were used to investigate the effect of cadmium on the activity of four enzymes measured by calorimetric methods. There were no clear changes in the activity of Alkaline phosphatase at different concentrations but the activity increased at different periods of exposure to cadmium. Acid phosphatase activity showed a gradual inhibition after the first concentration, while the activity increased at the different periods. There was no change in the activity of aspartate aminotransferase (AST) at different concentrations and periods of exposure. Alanine aminotransferase (ALT) activity increased at the concentrations 2 and 8 mg Cd/L, but the activity increased after the first period and decreased gradually at other periods of exposure to the metal.

Rapid chlorophyll a fluorescence transient of Lemna gibba leaf as an indication of light and hydroxylamine effect on photosystem II activity

Rapid chlorophyll fluorescence transient induced by saturating flash (3000 micromol of photons m-2 s-1) was investigated when Lemna gibba had been exposed to light (100 micromol of photons m-2 s-1) causing the Kautsky effect or in low light intensity unable to trigger PSII photochemistry. Measurements were made by using, simultaneously, a pulse amplitude modulated fluorometer and plant efficiency analyzer system, either on non-treated L. gibba leaf or those treated with different concentrations of hydroxylamine (1-50 mM) causing gradual inhibition of the water splitting system. When any leaf was exposed to continuous light during the Kautsky effect, a rapid fluorescence transient may reflect current activity of photosystem II within the photosystem II complex. Under those conditions, a variation of transition steps appearing over time was related to a drastic change to the photosystem II functional properties. This value indicated that the energy dissipation through non-photochemical pathways was undergoing extreme change. The change of rapid fluorescence transient, induced under continuous light, when compared to those obtained under very low light intensity, confirmed the ability of photosystem II to be capable to undergo rapid adaptation lasting about two minutes. When the water splitting system was inhibited and electron donation partially substituted by hydroxylamine, the adaptation ability of photosystem II to different light conditions was lost. In this study, the change of rapid fluorescence kinetic and transient appearing over time was shown to be a good indication for the change of the functional properties of photosystem II induced either by light or by hydroxylamine.

Nickel toxicity inhibits ribonuclease and protease activities in rice seedlings: protective effects of proline

Seedlings of two rice cvs Malviya-36 and Pant-12, when grown under increasing levels of nickel (Ni2+: 200 and 400 μM) in the nutrient medium, showed increased levels of RNA, soluble proteins and free amino acids, especially proline over a total growth period of 5–20 days. Ribonuclease (RNase) and protease activities decreased in both roots and shoots due to Ni treatment in situ. Under in vitro conditions, a gradual inhibition of RNase activity was observed with increasing concentrations of Ni2+ (0–2500 μM) in the assay medium. Artificial desiccation simulated by 40% PEG or the presence of 2.5 mM Ni2+ in the reaction medium resulted in about 52–53% loss of RNase activity. Such a loss could be partially restored by 1 M proline in the assay medium. The activity staining of RNase revealed seven and four isoforms of RNase in roots and shoots, respectively. The intensity of most of the bands decreased with increasing levels of Ni2+ treatment in situ. The results suggest that Ni toxicity in rice seedlings suppresses the hydrolysis of RNA and proteins by inhibiting the activity of RNase and protease, respectively. Proline appears to act as a protectant of the enzyme RNase against metal- and PEG-induced damages.

Shear-induced reactive nitrogen species inhibit mitochondrial respiratory complex activities in cultured vascular endothelial cells

There is evidence that nitric oxide (NO), superoxide (O(2)(*-)), and their associated reactive nitrogen species (RNS) produced by vascular endothelial cells (ECs) in response to hemodynamic forces play a role in cell signaling. NO is known to impair mitochondrial respiration. We sought to determine whether exposure of human umbilical vein ECs (HUVECs) to steady laminar shear stress and the resultant NO production modulate electron transport chain (ETC) enzymatic activities. The activities of respiratory complexes I, II/III, and IV were dependent on the presence of serum and growth factor supplement in the medium. EC exposure to steady laminar shear stress (10 dyn/cm(2)) resulted in a gradual inhibition of each of the complexes starting as early as 5 min from the flow onset and lasting up to 16 h. Ramp flow resulted in inhibition of the complexes similar to that of step flow. When ECs were sheared in the presence of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 100 microM), the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; 100 microM), or the peroxynitrite (ONOO(-)) scavenger uric acid (UA; 50 microM), the flow-inhibitory effect on mitochondrial complexes was attenuated. In particular, L-NAME and UA abolished the flow effect on complex IV. Increased tyrosine nitration was observed in the mitochondria of sheared ECs, and UA blocked the shear-induced nitrotyrosine staining. In summary, shear stress induces mitochondrial RNS formation that inhibits the electron flux of the ETC at multiple sites. This may be a critical mechanism by which shear stress modulates EC signaling and function.

An open-label study to evaluate dose and cycle dependence of the pharmacokinetics (PK) of pegylated liposomal doxorubicin (PLD)

2012 Background: There are no definitive data in humans on the dose dependency and/or cycle dependency of the pharmacokinetics of PLD. The skin toxicity of PLD tends to worsen after several treatment cycles. Therefore, it is important to characterize the PK of PLD after 2 or more cycles. Methods: Fifteen patients with various solid tumors were randomized to two arms of treatment in an open-label study. Arm A received PLD at doses of 60, 30, and 45 mg/m2 in 3 successive cycles every 4 weeks (q4w). Arm B received PLD at doses of 30, 60, and 45 mg/m2 in 3 successive cycles q4w. Twelve patients, 6 on each arm, completed all three cycles and were fully evaluable. Plasma levels of doxorubicin were analyzed by HPLC and fluorimetry following a previously published method (J Chromatogr B, 779:259–69, 2002) with minor modifications. PK analysis was done by non-compartmental method. The following parameters were obtained: Cmax, AUC∞, terminal half-life (T1/2), and Clearance (CL). The paired t test was used for statistical analysis. Results: There was no significant difference in the parameters examined when the dose was increased from 30 to 60 mg/m2. However, when we analyzed the effect of cycle number on the PK, we found a gradual and significant inhibition of CL when patients advance from the 1st through the 3rd cycle of PLD (p=0.0003), with a mean increase of 44% in AUC/mg dose (p=0.0011). Cmax and T1/2 mean values increased by 17% and 18% respectively between the 1st and 3rd cycles, but only the increase in T1/2 was statistically significant (p=0.0127). Conclusion: While the PK of PLD is not dose-dependent within the dose range of 30 to 60mg/m2, there is evidence of a cycle-dependent effect that results in inhibition of CL when patients receive successive cycles of PLD. This effect may account for the delayed skin toxicity of PLD. These results suggest the need for dose adjustments of PLD to minimize the risk of toxicity, such as an initial high loading dose followed by reduced maintenance doses from the 2nd or 3rd cycle onwards. [Table: see text] [Table: see text]

Wakefulness-sleep cycle and hypothalamo-pituitary system of young rat pups on the background of photostimulation

Effects of multiple photostimulations (PS) on behavioral reactions of the 30-day old rats were studied in the “open field” test, on the dynamics of the time EEG parameters of the wakefulness-sleep cycle, and on morpho-functional characteristics of the hypothalamo-pituitary neuroendocrine system. It has been revealed that PS promotes the gradual inhibition of the locomotor activity and exploratory behavior (p < 0.05) and an enhancement of autonomic reactions (p < 0.05) as well as an initial increase of intensity of orientation reactions with a subsequent decrease of their number (in connection with adaptation to a light stimulus), A comparative analysis of time parameters of the wakefulness-sleep cycle (WSC) shows a decrease of the part of wakefulness, the slow-wave and rapid-wave sleep phases with a simultaneous increase of the cataleptic stage and the sleep stage transient to the rapid-wave stage, as well as of the number of microactivations appearing on the sleep background at once after the several first PS. By the 4–5th photostimulations the CWS parameters on the whole are restored, except for the cataleptic stage, whose value remains statistically significantly high. The obtained data indicate the initial total CNS excitation and a decrease of the CWS stability. This conclusion is confirmed by the data of analysis of spectral characteristics of electrograms of cortical, hippocampal, hypothalamic brain parts and of caudate nucleus, which revealed only a brief small increase of δ-waves in the slow-wave sleep phase, but a statistically significant long decrease of power of the EEG δ-and ϑ-oscillations with a simultaneous power increase of α-components in the rapid-wave sleep. The quantitative parameters of changes of the morpho-functional state of neurosecretory cells of the hypothalamus supraoptic and paraventricular nuclei indicate the stress effect of this performed stimulation. The aspects of interaction of the hypothalamic-cortical and thalamo-cortical levels of the CWS integration on the background of chronic stress are discussed.

Calcium Signals Are Affected by Ciprofloxacin as a Consequence of Reduction of Mitochondrial DNA Content in Jurkat Cells

The effects of ciprofloxacin on mitochondrial DNA (mtDNA) content, oxygen consumption, mitochondrial membrane potential, cellular ATP formation, and capacitative Ca(2+) entry into Jurkat cells were investigated. In cells incubated for several days with 25 mug/ml ciprofloxacin, a 60% reduction of mtDNA content, inhibition of the respiratory chain, and a significant decrease in mitochondrial membrane potential were observed. These changes led to a decrease in the calcium buffering capacity of mitochondria which, in turn, resulted in a gradual inhibition of the capacitative Ca(2+) entry. On days 4, 7, and 11 of incubation with ciprofloxacin, the initial rates of Ca(2+) entry were reduced by 33%, 50%, and 50%, respectively. Ciprofloxacin caused a transient decrease in the cellular capability for ATP formation. In cells incubated for 15 min with glucose, pyruvate, and glutamine as exogenous fuel, ciprofloxacin reduced ATP content by 16% and 35% on days 4 and 7, respectively, of incubation with the drug. However, on day 11 of incubation with ciprofloxacin, a recovery of cellular ATP formation was observed. In conclusion, long-term exposure of Jurkat cells to ciprofloxacin at a concentration of 25 mug/ml seriously affects cellular energy metabolism and calcium homeostasis.

Inhibition of ribonuclease and protease activities in arsenic exposed rice seedlings: Role of proline as enzyme protectant

When seedlings of two rice (Oryza sativa L.) cvs. Malviya-36 and Pant-12 were raised under 25 and 50 microM As2O3 in the medium an increase in the level of RNA, proteins and proline accompanied with a decline in the level of free amino acid pool was observed under arsenic supplementation compared to controls. In situ As3+ treatment caused a marked inhibition in activities of ribonuclease (RNase, EC 3.1.27.1), protease and leucine aminopeptidase (LAP, EC 3.4.11.1) whereas the activity level of carboxypeptidase (EC 3.4.16.5) was enhanced. In vitro supply of As2O3 in the enzyme assay medium beyond 400 microM resulted in gradual inhibition of RNase and beyond 5 microM inhibition of LAP activities. Addition of 1M proline in the assay medium significantly restored the loss in RNase activity due to in vitro arsenic treatment or due to osmotic stress created by incorporation of polyethylene glycol (PEG). Isoform pattern of RNase extracted from As3+ -exposed seedlings showed a significant alteration compared to its pattern in unexposed seedlings. Results suggest that arsenic exposure impairs hydrolysis of RNA and proteins in rice seedlings due to inhibition of RNase and proteases activities and that proline accumulating under As3+ toxicity appears to serve as enzyme protectant.

Physiological regulation of the properties of alcohol dehydrogenase II (ADH II) of Zymomonas mobilis: NADH renders ADH II resistant to cyanide and aeration

The variable cyanide-sensitivity of the iron-containing alcohol dehydrogenase isoenzyme (ADH II) of the ethanol-producing bacterium Zymomonas mobilis was studied. In aerobically grown permeabilized cells, cyanide caused gradual inhibition of ADH II, which was largely prevented by externally added NADH. Cyanide-sensitivity of ADH II was highest in cells grown under conditions of vigorous aeration, in which intracellular NADH concentration was low. Anaerobically grown bacteria, as well as those cultivated aerobically in the presence of cyanide, maintained higher intracellular NADH levels along with a more cyanide-resistant ADH II. It was demonstrated that cyanide acted as a competitive inhibitor of ADH II, competing with nicotinamide nucleotides. NADH increased both cyanide-resistance and oxygen-resistance of ADH II.