In vitro modulation of proliferation and melanization of melanoma cells by citrate

B16/F10 murine melanoma cells were grown for 24 and 36 h in Dulbecco's modified Eagle medium in presence of 10-20 mM trisodium citrate. The intracellular melanin concentration and the melanin secreted in the extracellular medium was estimated. It is observed that 20 mM citrate stimulates extracellular melanin secretion in B16/F10 melanoma cells by 200% at 36 h treatment. The intracellular melanin content increased by 90%. This stimulatory effect of citrate was totally abolished when these cells were grown in presence of 1 mM phenyl thiourea, a specific inhibitor of tyrosinase activity. Citrate (0.1-5 mM) had no effect on dopa oxidase activity either at pH 5.0 or at pH 6.8. There was no increase in the tyrosinase specific activity in presence of citrate. The increased melanin synthesis was shown to be due to stimulation of cellular tyrosine hydroxylase activity by citrate. It has been suggested that enhanced melanin synthesis results in an increased production of metabolites that are toxic to the growth of melanoma cells. We have studied the effect of citrate on cellular proliferation. Following 24 and 36 h treatment with citrate, the cells exhibited a dose-dependent decrease in proliferation. In presence of 20 mM citrate the cell number was only up to 50% of the control cultures after 36 h of incubation. The growth retardation was not due to cytotoxicity. Citrate, a natural metabolite, is a unique molecule which may be involved in the regulation of melanin biosynthetic pathway, since it enhances melanogenesis by increasing the hydroxylase activity of tyrosinase which is the regulatory enzyme of this pathway. These observations add further support to the critical role of intramelanosomal pH in regulation of melanogenesis.     

Insight into the role of sugars in bud burst under light in the rose

Bud burst is a decisive process in plant architecture that requires light in Rosa sp. This light effect was correlated with stimulation of sugar transport and metabolism in favor of bud outgrowth. We investigated whether sugars could act as signaling entities in the light-mediated regulation of vacuolar invertases and bud burst. Full-length cDNAs encoding two vacuolar invertases (RhVI1 and RhVI2) were isolated from buds. Unlike RhVI2, RhVI1 was preferentially expressed in bursting buds, and was up-regulated in buds of beheaded plants exposed to light. To assess the importance of sugars in this process, the expression of RhVI1 and RhVI2 and the total vacuolar invertase activity were further characterized in buds cultured in vitro on 100 mM sucrose or mannitol under light or in darkness for 48 h. Unlike mannitol, sucrose promoted the stimulatory effect of light on both RhVI1 expression and vacuolar invertase activity. This up-regulation of RhVI1 was rapid (after 6 h incubation) and was induced by as little as 10 mM sucrose or fructose. No effect of glucose was found. Interestingly, both 30 mM palatinose (a non-metabolizable sucrose analog) and 5 mM psicose (a non-metabolizable fructose analog) promoted the light-induced expression of RhVI1 and total vacuolar invertase activity. Sucrose, fructose, palatinose and psicose all promoted bursting of in vitro cultured buds under light. These findings indicate that soluble sugars contribute to the light effect on bud burst and vacuolar invertases, and can function as signaling entities.     

The alternative oxidase in roots of poa annua after transfer from high-light to low-light conditions

The activity of the alternative pathway can be affected by a number of factors, including the amount and reduction state of the alternative oxidase protein, and the reduction state of the ubiquinone pool. To investigate the importance of these factors in vivo, we manipulated the rate of root respiration by transferring the annual grass Poa annua L. from high-light to low-light conditions, and at the same time from long-day to short-day conditions for four days. As a result of the low-light treatment, the total respiration rate of the roots decreased by 45%, in vitro cytochrome c oxidase capacity decreased by 49%, sugar concentration decreased by 90% and the ubiquinone concentration increased by 31%, relative to control values. The absolute rate of oxygen uptake via the alternative pathway, as determined using the 18O-isotope fractionation technique, did not change. Conversely, the cytochrome pathway activity decreased during the low-light treatment; its activity increased upon addition of exogenous sugars to the roots. Interestingly, no change was observed in the concentration of the alternative oxidase protein or in the reduction state of the protein. Also, there was no change in the reduction state of the ubiquinone pool. In conclusion, the concentration and activity of the alternative oxidase were not changed, even under severe light deprivation.     

Sucrose uptake by the phloem parenchyma of carrot storage root

The characteristics of sucrose uptake into the symplast of phloemtissue discs harvested from fresh, actively-growing carrot storageroots are described. Sucrose uptake exhibited a curvilinearresponse with increasing sucrose concentration. The inhibitorsp-chloromercuribenzenesulphonic acid (PCMBS) and carbonyl cyanidem-chlorophenylhydrazone (CCCP) decreased uptake and resultedin solely linear relationships between uptake and sucrose concentration.These results suggest that active carrier-mediated transportoccurs at the plasmalemma in addition to a diffusive mechanism.The former saturates at a lower concentration (approximately20 mM) than the latter which does not saturate below 100 mM.Though similar in their effect on the ethanol-soluble fraction,CCCP and PCMBS had different effects on the conversion of sucroseto ethanol-insoluble material. Varying the osmotic environment with different mannitol concentrationsdid not affect uptake between 0 and 400 mM mannitol, but didcause an increase at 600 mM mannitol: an effect which may havebeen an artefact of plasmolysis. Metabolic conversion to ethanol-insolubleforms remained unchanged from 0 to 250 mM mannitol and declinedabove this. Thus metabolism, but not uptake may be responsiveto changes in turgor. Key words: carrot, sucrose, uptake, transport, turgor     

Solubilization of the alternative oxidase of cuckoo-pint (Arum maculatum) mitochondria. Stimulation by high concentrations of ions and effects of specific inhibitors.

Selective solubilization of cyanide- and antimycin-insensitive duroquinol oxidase activity from cuckoo-pint (Arum maculatum) mitochondria was achieved using taurocholate. Inhibitor-sensitivities and water-forming DQH2 (tetramethyl-p-hydroquinone, reduced form): O2 stoichiometry were the same for the alternative oxidase of intact Arum mitochondria. Cyanide-insensitive oxidation of DQH2 by intact and solubilized mitochondria was stimulated by up to four-fold by high concentrations of anions high in the Hofmeister series, such as phosphate, sulphate or citrate. Optimal (0.7 M) sodium citrate increased Vmax. for DQH2 oxidation by the solubilized preparation from 450 to 2400 nmol of O2 X min-1 X mg of protein-1 and decreased the apparent Km for DQH2 from 0.53 to 0.38 mM. Inhibition of solubilized DQH2 oxidase activity by CLAM (m-chlorobenzhydroxamic acid) and SHAM (salicylhydroxamic acid) was mixed competitive/non-competitive, with apparent inhibition constants for CLAM of 25 microM (Ki) and 81 microM (KI) and for SHAM of 53 microM (Ki) and 490 microM (KI). Propyl gallate and UHDBT were non-competitive inhibitors with respect to DQH2 (apparent Ki = 0.3 microM and 12 nM respectively). Low concentrations of C18 fatty acids selectively inhibited cyanide-insensitive oxidation by intact and solubilized mitochondria, and inhibition was reversed by 1% (w/v) bovine serum albumin. Inhibition was competitive with DQH2, suggesting that fatty acids interfere reversably with the binding of DQH2 to the oxidase. These results tend to support the view that quinol oxidation by the alternative pathway of Arum maculatum mitochondria is catalysed by a quinol oxidase protein, rather than by a non-enzymic mechanism involving fatty acid peroxidative reaction. [Rustin, Dupont & Lance (1983) Trends Biochem. Sci. 8, 155-157; (1983) Arch. Biochem. Biophys. 225, 630-639].     

Possible involvement of the mitochondrial alternative pathway in ethylene-induced apoptosis in tomato protoplasts

In this study, a monoclonal antibody to the terminal oxidase of the alternative pathway from Sauromatum guttatum was used to detect the expression of alternative oxidase (AOX) protein in tomato mitochondria. The results show that there was an obvious correlation between the ethylene-induced apoptosis and the levels of AOX protein in tomato cells undergoing ethylene-induced apoptosis. In addition, when tomato protoplasts were preincubated with 2 mM salicylhydroxamic acid (SHAM), an inhibitor of the alternative pathway, before their exposure to ethylene, the TUNEL positive reaction and DNA fragmentation were obviously accelerated. We suggest that AOX may play an important role in protecting tomato protoplasts against ethylene-induced apoptosis.     

Effects of Enhanced UV-B Radiation on the Activity and Expression of Alternative Oxidase in Red Kidney Bean Leaves

An increase in ultraviolet （UV） B radiation on the earth＇s surface is a feature of current global climate changes. It has been reported that alternative oxidase （AOX） may have a protective role against oxidative stress induced by environmental stresses, such as UV-B. To better understand the characteristic tolerance of plants to UV-B radiation, the effects of enhanced UV-B radiation on the activity and expression of AOX in red kidney bean （Phaseolus vulgaris） leaves were investigated in the present study. The results show that the total respiration rate and AOX activity in red kidney bean leaves increased significantly during treatment with enhanced UV-B. However, cytochrome oxidase （COX） activity did not change significantly. The H2O2 content was also markedly increased and reached a maximum of 4.45 mmol·L^-1·g^-1 DW （dry weight） at 24 h of UV-B treatment, before dropping rapidly. Both alternative pathway content and alternative pathway activity were increased in the presence of exogenous H2O2. Immunoblotting analysis with anti-AOX monoclonal antibody revealed that expression of the AOX protein increased in red kidney bean leaves under enhanced UV-B radiation, reaching a peak at 72 h. In addition, AOX expression in red kidney bean leaves was induced by exogenous H2O2. These data indicate that the increase in AOX activity in red kidney bean leaves under enhanced UV-B radiation was mainly due to H2O2-induced AOX expression.     

Regulation of the activity of the alternative oxidase in callus forming discs from potato tubers

Callus-forming discs from potato tubers lose 80% of their starch during one month of incubation on nutrient medium containing either 0, 3 or 6% (w/v) sucrose. The content of soluble sugar in the discs varies from 5 mg (incubated without sucrose) to 22 mg (on 3% sucrose) and 40 mg (on 6% sucrose) per g fresh weight. The activity of the cytochrome pathway (V_cyt) increases during the first week of incubation on all media. Thereafter V_cyt decreases again on 0% sucrose medium, while it remains constant on 3 and 6% sucrose media. Alternative pathway capacity (V_alt), absent in freshly sliced tissue, shows a sharp increase during the first days of incubation, independent of the sucrose concentration in the medium. This capacity further increases during prolonged incubation on 3 and 6% sucrose but decreases on 0% sucrose. The in vivo activity of the alternative pathway (the participation in uninhibited respiration, ?V_alt) varies with the sucrose concentration and with the culture time. In tissue incubated for 2-3 weeks on 6% sucrose as much as 45% of the electrons are transfered to oxygen via the alternative pathway. In this tissue the factor Q (the part of the alternative pathway capacity that is operative) is about 0.8, while in tissue incubated on 0 and 3% sucrose media p generally does not exceed 0.5. When chloramphenicol, an inhibitor of mitochondrial protein synthesis, is added to the medium together with 3% sucrose, the increase in V_yet does not occur, while the induction of V_alt during the first week of incubation is the same as without chloramphenicol. A greater part of the alternative pathway capacity becomes operative in this tissue, leading to values of Q of almost 1 after prolonged incubation. Apparently, incubation on high sugar medium leads to extra participation in respiration of the energetically inefficient alternative oxidase pathway Excess sugar leads to wasteful respiration suggesting that the alternative oxidase functions as an ‘energy overflow’.     

Influence of cell turgor on sucrose partitioning in potato tuber storage tissues

Sucrose uptake and partitioning in potato (Solanum tuberosum L.) tuber discs were examined under a range of mannitol and ethylene-glycol concentrations. Mannitol caused the same changes in turgor over a wide range of incubation periods (90 min-6 h), indicating that it did not penetrate the tissue. In comparison, ethylene glycol reduced turgor losses but did not eliminate them, even after 6 h. Between 100 mM and 300 mM mannitol, turgor fell by 350 kPa, compared with 35 kPa in ethylene glycol. Uptake experiments in mannitol alone showed that total sucrose uptake was strongly correlated with both osmotic potential and with turgor potential. In subsequent experiments sucrose uptake and partitioning were examined after 3 h equilibration in 100 mM and 300 mM concentrations of mannitol and ethylene glycol. Total sucrose uptake and the conversion of sucrose to starch were enhanced greatly only at 300 mM mannitol, indicating an effect of turgor, rather than osmotic potential on sucrose partitioning. The inhibitors p-chloromercuribenzenesulfonic acid and carbonylcyanide m-chlorophenylhydrazone (CCCP) both reduced sucrose uptake, but in quite different ways. p-Chloromercuribenzenesulfonic acid reduced total sucrose uptake but did not affect the partitioning of sucrose to starch. By contrast, CCCP inhibited total uptake and virtually eliminated the conversion of sucrose to starch. Despite this, sucrose uptake in the presence of CCCP continued to increase as the mannitol concentration increased, indicating an increase in passive transport at higher mannitol concentrations. Increased sucrose uptake above 400 mM mannitol was shown to be the result of uptake into the free space. The data show that starch synthesis is optimised at low but positive turgors and the relation between sucrose partitioning and the changing diurnal water relations of the tuber are discussed.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - PCMBS p-chloromercuribenzenesulfonic acid     

Mg2+-ATPase activity in wheat root plasma membrane vesicles: Time-dependence and effect of sucrose and detergents

Purified plasma membrane vesicles were isolated in the presence of 250 mM sucrose from 7-day-old roots of Triticum aestivum L. cv. Drabant by aqueous polymer two-phase partitioning. When added to a low-salt medium containing 9-aminoacridine (9-AA), the vesicles caused a much larger total decrease in 9-AA fluorescence when sucrose was absent than when sucrose was present. A slow component of the decrease was also larger in the absence of sucrose. Triton X-100 reduced the decrease in 9-AA fluorescence upon vesicle addition and abolished completely the slow component of the decrease. There was no correlation between the time-dependence of 9-AA fluorescence and that of the Mg²⁺-ATPase described below. The time course of Mg²⁺-ATPase activity was followed by sampling at short intervals (down to 10 s) and analyzing for P, released. In the absence of detergent, the rates of P, release were linear from zero minutes, whether 250 mM sucrose was present or not, but the rate was 10^?50% higher in the absence of sucrose than in its presence. Sucrose (250 mM) added during a minus-sucrose assay lowered Mg²⁺-ATPase activity within 2 min to the level observed with 250 mM sucrose present from the start. The effect of 25-1 100 mM sucrose was tested and there was little or no effect below KM) mM. Above 100 mM sucrose the rate of P, release decreased drastically; at 1 100 mM sucrose the rate was ca 20% the rate at 25 mM sucrose. The inhibitory effect of sucrose was not alleviated by increased concentrations of Mg²⁺ and/or ATP. nor was it affected by the presence or absence of Triton X-100. We conclude that sucrose somehow inhibits the Mg²⁺-ATPase directly or affects the conformation of the plasma membrane in such a way as to inhibit the enzyme. The presence of detergents increased Mg²⁺-ATPase activity in the order Triton X-100 (4–5-fold) > Zwittergent 3–14 = Na-cholate = octylglucoside > digitonin (2-fold). In all cases optimal activity was observed at detergent concentrations at or below the critical micellar concentration. The detergent concentration curves could be simulated by the sum of a stimulatory and an inhibitory reaction. At the optimal concentration, digitonin gave a linear time-course of P, release, whereas all the other detergents showed a distinct lag of 1–3 min before maximal rates were attained. The problems of using detergents in polarity assays are discussed.     

Retinol-induced elevation of ornithine decarboxylase activity in cultured rat Sertoli cells is attenuated by free radical scavenger and by iron chelator

We investigated retinol effects in ornithine decarboxylase activity in Sertoli cells. We also tested the hypothesis that free radical scavengers and iron chelators may attenuate the effect of retinol. Sertoli cells isolated from 15-day-old Wistar rats were previously cultured for 48 h and then treated with retinol by 24 h with or without mannitol (1 mM) or 1,10 phenanthroline (100 M). We measured ornithine decarboxylase and catalase activities and malondialdehyde concentrations in response to retinol treatment. In response to 7 M retinol treatment ornithine decarboxylase activity increased 30%. Retinol-induced ornithine decarboxylase activity was significantly decreased by addition of free radical scavenger (mannitol) or iron chelator (1,10 phenanthroline). In addition the same effect was observed in catalase increased activity and in malondialdehyde concentrations. These results suggest that retinol treatment induced ornithine decarboxylase and catalase activity and increased malondialdehyde concentration. These effects appear to be mediate by ROS.     

Nuclear Magnetic Resonance Studies on the Effects of Decreased External Sodium on Guinea Pig Cerebral Cortex Slices and the Permeabilities of Various Sodium Substitutes

Decreasing the external sodium concentration ([Na+]e) to 10 mM in the presence of 280 mM sucrose had no significant effect on phosphocreatine (PCr) or on intracellular pH (pHi) as assessed using 31P nuclear magnetic resonance spectroscopy. Zero [Na+]e in the presence of 300 mM sucrose caused a fall in PCr levels to 50% of control values, and the pHi fell to 6.85 from a control value of 7.30. 1H nuclear magnetic resonance spectroscopy confirmed that the sucrose had not entered the tissue. The decreases in PCr content and in pHi, known to occur on depolarization using 40 mM external potassium concentration ([K+]e), were further decreased in the presence of 10 mM [Na+]e), to 51.4 +/- 4.0 and 6.80 +/- 0.10% of control values, respectively. The free intracellular magnesium concentration was significantly increased from a control value of 0.37 +/- 0.10 mM to 0.66 +/- 0.13 mM (p less than 0.001), when [Na+]e was decreased to 10 mM, but was not further affected by high [K+]e or zero Na+. Membrane permeabilities of the sodium substitutes N-methyl-D-glucamine (NMG), tris(hydroxymethyl)aminomethane (Tris), tetramethylammonium (TMA), and choline were assessed using 1H nuclear magnetic resonance spectroscopy. In the presence of 10 mM [Na+]e, NMG, TMA, and choline (all at 140 mM) were taken up and remained within the tissue for at least 2 h, but no uptake of Tris (140 mM) or sucrose (above) could be detected. Tissue lactate levels (from the lactate/N-acetyl aspartate ratio) increased in the presence of the substitutes that were taken up, although no change in pH was detected.     

Mannitol enhances intracellular calcium diffusion in the rat ileum--a hypothesis

The addition of 92 or 136 mM mannitol to a modified saline solution that contained 1.25 mM Ca2+ led to a mannitol concentration-dependent increase in the amount of calcium absorbed in 1 h from 8 cm long ileal loops prepared from fasted male Sprague-Dawley rats, with body weights of 190 +/- 10 g. It is argued that this mannitol-enhanced movement of calcium out of the loop cannot have utilized the paracellular pathway, inasmuch as the luminal calcium concentration of the mannitol instillate decreased during the experiment, with a negative calcium gradient between luminal and body fluids. Instead it is proposed that uncomplexed mannitol and the uncharged calcium complex of mannitol entered the ileal cells. The uncomplexed intracellular mannitol would bind additional calcium that had crossed the brush border down its gradient. The increase in total intracellular calcium will raise the effective intracellular gradient and thereby amplify intracellular calcium diffusion. This in turn increases calcium absorption.     

Lactobacillus reuteri CRL 1101 highly produces mannitol from sugarcane molasses as carbon source

Mannitol is a natural polyol extensively used in the food industry as low-calorie sugar being applicable for diabetic food products. We aimed to evaluate mannitol production by Lactobacillus reuteri CRL 1101 using sugarcane molasses as low-cost energy source. Mannitol formation was studied in free-pH batch cultures using 3-10% (w/v) molasses concentrations at 37?°C and 30?°C under static and agitated conditions during 48?h. L. reuteri CRL 1101 grew well in all assayed media and heterofermentatively converted glucose into lactic and acetic acids and ethanol. Fructose was used as an alternative electron acceptor and reduced it to mannitol in all media assayed. Maximum mannitol concentrations of 177.7?±?26.6 and 184.5?±?22.5?mM were found using 7.5% and 10% molasses, respectively, at 37?°C after 24-h incubation. Increasing the molasses concentration from 7.5% up to 10% (w/v) and the fermentation period up to 48?h did not significantly improve mannitol production. In agitated cultures, high mannitol values (144.8?±?39.7?mM) were attained at 8?h of fermentation as compared to static ones (5.6?±?2.9?mM), the highest mannitol concentration value (211.3?±?15.5?mM) being found after 24?h. Mannitol 2-dehydrogenase (MDH) activity was measured during growth in all fermentations assayed; the highest MDH values were obtained during the log growth phase, and no correlation between MDH activities and mannitol production was observed in the fermentations performed. L. reuteri CRL 1101 successfully produced mannitol from sugarcane molasses being a promising candidate for microbial mannitol synthesis using low-cost substrate.     

The Effects of Salicylic Acid and Tobacco Mosaic Virus Infection on the Alternative Oxidase of Tobacco

Salicylic acid (SA) is a signal in systemic acquired resistance and an inducer of the alternative oxidase protein in tobacco (Nicotiana tabacum cv Xanthi nc) cell suspensions and during thermogenesis in aroid spadices. The effects of SA on the levels of alternative oxidase protein and the pathogenesis-related 1a mRNA (a marker for systemic acquired resistance), and on the partitioning of electrons between the Cyt and alternative pathways were investigated in tobacco. Leaves were treated with 1.0 mM SA and mitochondria isolated at times between 1 h and 3 d after treatment. Alternative oxidase protein increased 2.5-fold within 5 h, reached a maximum (9-fold) after 12 h, and remained at twice the level of control plants after 3 d. Measurements of isotope fractionation of 18O by intact leaf tissue gave a value of 23% at all times, identical to that of control plants, indicating a constant 27 to 30% of electron-flow partitioning to the alternative oxidase independent of treatment with SA. Transgenic NahG tobacco plants that express bacterial salicylate hydroxylase and possess very low levels of SA gave a fractionation of 23% and showed control levels of alternative oxidase protein, suggesting that steady-state alternative oxidase accumulates in an SA-independent manner. Infection of plants with tobacco mosaic virus resulted in an increase in alternative oxidase protein in both infected and systemic leaves, but no increase was observed in comparably infected NahG plants. Total respiration rate and partitioning of electrons to the alternative pathway in virus-infected plants was comparable to that in uninfected controls.