Regulation of MI transport in retinal pigment epithelium by sugars, amiloride, and pH gradients: potential impairment of pump-leak balance in diabetic maculopathy

Impairment of transport and metabolism of retinal pigment epithelium (RPE) has been recognized to play a role in the development of diabetic macular edema. To understand the mechanism(s) of action of high glucose levels in alteration of RPE metabolism, primary cultures of RPE cells were used as an in vitro model of diabetic retinopathy/maculopathy. RPE cells were grown with 5 mM (control) or 40 mM glucose (a monosaccharide that enters the cells), or 40 mM sucrose (a disaccharide that does not enter the cells), and the extent of Na(+)-dependent active transport of an osmolyte ([3H]-myo-inositol, MI, 10 microM) into cells was determined. While 40 mM glucose down-regulated 3H-MI transport, 40 mM sucrose stimulated it, compared to 5 mM glucose feeding. Addition of 1 mM amiloride, an inhibitor of Na+/H+ exchanger, in the incubation media, significantly inhibited MI transport. Cells treated with high sucrose or high glucose were more sensitive toward amiloride inhibition, compared to controls. Inhibition of either pump or leak pathway alone was not sufficient to completely inhibit MI transport, but simultaneous inhibition of both pathways, by amiloride and ouabain (1 mM each), strongly inhibited osmolyte accumulation. The strongest inhibition of uptake occurred when 150 mM NaCl in the incubation media was replaced by 150 mM choline-Cl, and the percent inhibition of uptake, with choline-Cl, was highest with sucrose-fed cells, compared to normal or high glucose-fed cells. Imposition of a pH gradient [pHi (6.1) less than pH0 (8.0)] across the cell membrane, a condition that stimulates Na+/H+ exchange activity, also reduced MI accumulation. Cellular water content, measured by the extent of [3H]-3-O-methyl glucose uptake, in the presence of balanced salt solution (BSS), BSS containing half the ionic strength (hypotonic solution), or BSS containing 20 mM K+, for induction of cell swelling, varied when cells were fed with various sugars. Cells fed with high glucose were less sensitive toward media tonicity compared to normal. These results suggested that in cultured RPE cells, changes in Na+/H+ exchanger activity (intracellularly or extracellularly), through its inhibition by amiloride, its activation via intracellular acidification, or perhaps by chronic feeding with high sucrose or high glucose, affected the Na(+)-dependent active accumulation of MI. A metabolic factor involved in the development of diabetic macular edema is perhaps associated with glucose-induced alterations in Na+ fluxes (e.g., changes in Na+/H+ exchanger activity), which can secondarily influence osmolyte accumulation, impairment of pump-leak balance, and/or intracellular pH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mannitol, a novel bacterial compatible solute in Pseudomonas putida S12.

The aim of this study was to identify the compatible solutes accumulated by Pseudomonas putida S12 subjected to osmotic stress. In response to reduced water activity, P. putida S12 accumulated Nalpha-acetylglutaminylglutamine amide (NAGGN) simultaneously with a novel compatible solute identified as mannitol (using 13C- and 1H-nuclear magnetic resonance, liquid chromatography-mass spectroscopy and high-performance liquid chromatography methods) to maximum concentrations of 74 and 258 micromol g (dry weight) of cells(-1), respectively. The intracellular amounts of each solute varied with both the type and amount of osmolyte applied to induce osmotic stress in the medium. Both solutes were synthesized de novo. Addition of betaine to the medium resulted in accumulation of this compound and depletion of both NAGGN and mannitol. Mannitol and NAGGN were accumulated when sucrose instead of salts was used to reduce the medium water activity. Furthermore, both compatible solutes were accumulated when glucose was substituted by other carbon sources. However, the intracellular quantities of mannitol decreased when fructose, succinate, or lactate were applied as a carbon source. Mannitol was also raised to high intracellular concentrations by other salt-stressed Pseudomonas putida strains. This is the first study demonstrating a principal role for the de novo-synthesized polyol mannitol in osmoadaptation of a heterotrophic eubacterium.     

Lactose metabolism in Streptococcus lactis: phosphorylation of galactose and glucose moieties in vivo.

Starved cells of Streptococcus lactis ML3 grown previously on lactose, galactose, or maltose were devoid of adenosine 5'-triphosphate contained only three glycolytic intermediates: 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate (PEP). The three metabolites (total concentration, ca 40 mM) served as the intracellular PEP potential for sugar transport via PEP-dependent phosphotransferase systems. When accumulation of [14C]lactose by iodoacetate-inhibited starved cells was abolished within 1 s of commencement of transport, a phosphorylated disaccharide was identified by autoradiography. The compound was isolated by ion-exchange (borate) chromatography, and enzymatic analysis showed that the derivative was 6-phosphoryl-O-beta-D-galactopyranosyl (1 leads to 4')-alpha-D-glucopyranose (lactose 6-phosphate). After maximum lactose uptake (ca. 15 mM in 15 s) the cells were collected by membrane filtration and extracted with trichloroacetic acid. Neither free nor phosphorylated lactose was detected in cell extracts, but enzymatic analysis revealed high levels of galactose 6-phosphate and glucose 6-phosphate. The starved organisms rapidly accumulated glucose, 2-deoxy-D-glucose, methyl-beta-D-thiogalactopyranoside, and o-nitrophenyl-beta-D-galactopyranoside in phosphorylated form to intracellular concentrations of 32, 32, 42, and 38.5 mM, respectively. In contrast, maximum accumulation of lactose (ca. 15 mM) was only 40 to 50% that of the monosaccharides. From the stoichiometry of PEP-dependent lactose transport and the results of enzymatic analysis, it was concluded that (i) ca. 60% of the PEP potential was utilized via the lactose phosphotransferase system for phosphorylation of the galactosyl moiety of the disaccharide, and (ii) the residual potential (ca. 40%) was consumed during phosphorylation of the glucose moiety.     

Transient Accumulation of Glycine Betaine and Dynamics of Endogenous Osmolytes in Salt-Stressed Cultures of Sinorhizobium meliloti

The fate of exogenously supplied glycine betaine and the dynamics of endogenous osmolytes were investigated throughout the growth cycle of salt-stressed cultures of strains of Sinorhizobium meliloti which differ in their ability to use glycine betaine as a growth substrate, but not as an osmoprotectant. We present (sup13)C nuclear magnetic resonance spectral and radiotracer evidence which demonstrates that glycine betaine is only transiently accumulated as a cytoplasmic osmolyte in young cultures of wild-type strains 102F34 and RCR2011. Specifically, these strains accumulate glycine betaine as a preferred osmolyte which virtually prevents the accumulation of endogenous osmolytes during the lag and early exponential phases of growth. Then, betaine levels in stressed cells decrease abruptly during the second half of the exponential phase. At this stage, the levels of glutamate and the dipeptide N-acetylglutaminylglutamine amide increase sharply so that the two endogenous solutes supplant glycine betaine in the ageing culture, in which it becomes a minor osmolyte because it is progressively catabolized. Ultimately, glycine betaine disappears when stressed cells reach the stationary phase. At this stage, wild-type strains of S. meliloti also accumulate the disaccharide trehalose as a third major endogenous osmolyte. By contrast, glycine betaine is always the dominant osmolyte and strongly suppresses the buildup of endogenous osmolytes at all stages of the growth cycle of a mutant strain, S. meliloti GMI766, which does not catabolize this exogenous osmoprotectant under any growth conditions.     

Osmoadaptative responses in the rhizobia nodulating Acacia isolated from south-eastern Moroccan Sahara

Four strains of rhizobia nodulating Acacia were isolated from the Moroccan desert soil by trapping with seedlings of Acacia gummifera and Acacia raddiana, and were studied for their ability to tolerate high salinity and dryness conditions. The strains MDSMC 2, MDSMC 18 and MDSMC 50 were halotolerant (they tolerated up to 1 M NaCl) and they accumulated glutamate and mannosucrose. The synthesis of the latter solute, which is the major endogenous osmolyte, is partially repressed in the presence of glycine betaine. The strain MDSMC 34 was less halotolerant (growth inhibited by a concentration greater than 0.5 M NaCl), and accumulated trehalose (as the main endogenous osmolyte) and glutamate. Rhizobia from the Moroccan desert soil were highly resistant to desiccation and their tolerance to dryness was stimulated by osmotic pretreatment. Thus, the accumulation of mannosucrose or trehalose by desert rhizobia represents both an osmoadaptative response and a part of a desiccation tolerance mechanism.     

Osmotic adaptation in Brevibacterium linens: differential effect of proline and glycine betaine on cytoplasmic osmolyte pool

In the coryneform Brevibacterium linens, ectoine constitutes the major intracellular solute accumulated under elevated medium osmolarity. Here we report that exogenously supplied proline, choline, glycine betaine, and even ectoine, protected bacterial cells against deleterious effects of a hyperosmotic constraint (i.e. 1.5 M NaCl). In all cases, a significant improvement of growth was observed; in parallel, intracellular osmolyte pools composed mainly of glutamate and ectoine substantially increased, either with added glycine betaine (under limiting supply) or with proline. However, these two osmoprotectants behaved differently: glycine betaine acted as a genuine osmoprotectant, whereas proline was accumulated only transiently and participated actively in the biosynthesis of glutamate, ectoine, and trehalose. The strategy developed by B. linens cells allows the proposal of a novel role for proline in the osmoprotection process through its conversion to the apparently preferred endogenous osmolyte ectoine.     

High specific rates of glucose utilisation under conditions of restricted growth are required for citric acid accumulation by Yarrowia lipolytica IMK 2

Yarrowia lipolytica strain IMK 2, a yeast capable of producing and excreting citric acid, only accumulated citric acid when cell growth was restricted by specific nutrient limitations. Effective citrate accumulation was linked with the ability of cells to maintain a high rate of glucose utilisation when growth was limited. Cells limited by nitrogen (N), sulphur (S), magnesium (Mg) or phosphorus (P) accumulated between 50 and 220 mm citric acid after 168 h, with N and S limitation resulting in the highest specific rates of production. In contrast, potassium (K)-limited cells accumulated 6 mm citric acid in the same time period. Cells limited by K or lower levels of Mg or P had rates of glucose utilisation that were less than 50% of those measured in cells limited by N or S. Although limitation of strain IMK 2 by Mg or P led to citrate production, significant accumulation occured only when the threshold concentration of the limiting nutrient was exceeded. There was no large accumulation of other tricarboxylic acid (TCA) cycle acids, acetate, pyruvate, lactate or ethanol, although K-limited cells produced quantities of intracellular glycogen. Mannitol was accumulated under N, S and P limitation (up to 40 mm), as were small quantities of 2-oxoglutarate, which preceded the accumulation of citrate and all of the other TCA cycle acids measured. A clear difference was evident between the rates of glucose utilisation under N limitation between two citrate-accumulating strains (IMK 2 and Candida guilliermondii IMK 1) and a citrate non-accumulating strain, Y. lipolytica YB 423. Although these strains had similar rates of glucose utilisation during exponential growth, both strains IMK 1 and IMK 2 had specific rates of glucose utilisation under N limitation more than double that of strain YB 423.Correspondence to: J. D. Brooks 2     

Role of osmolytes in adaptation of osmotically stressed and chill-stressed Listeria monocytogenes grown in liquid media and on processed meat surfaces.

Listeria monocytogenes is a food-borne pathogen that is widely distributed in nature and is found in many kinds of fresh and processed foods. The pervasiveness of this organism is due, in part, to its ability to tolerate environments with elevated osmolarity and reduced temperatures. Previously, we showed that L. monocytogenes adapts to osmotic and chill stress by transporting the osmolyte glycine betaine from the environment and accumulating it intracellularly (R. Ko, L. T. Smith, and G. M. Smith, J. Bacteriol. 176:426-431, 1994). In the present study, the influence of various environmental conditions on the accumulation of glycine betaine and another osmolyte, carnitine, was investigated. Carnitine was shown to confer both chill and osmotic tolerance to the pathogen but was less effective than glycine betaine. The absolute amount of each osmolyte accumulated by the cell was dependent on the temperature, the osmolarity of the medium, and the phase of growth of the culture. L. monocytogenes also accumulated high levels of osmolytes when grown on a variety of processed meats at reduced temperatures. However, the contribution of carnitine to the total intracellular osmolyte concentration was much greater in samples grown on meat than in those grown in liquid media. While the amount of each osmolyte in meat was less than 1 nmol/mg (fresh weight), the overall levels of osmolytes in L. monocytogenes grown on meat were about the same as those in liquid samples, from about 200 to 1,000 nmol/mg of cell protein for each osmolyte. This finding suggests that the accumulation of osmolytes is as important in the survival of L. monocytogenes in meat as it is in liquid media.     

Preferential Osmolyte Accumulation: a Mechanism of Osmotic Stress Adaptation in Diazotrophic Bacteria

A common cellular mechanism of osmotic-stress adaptation is the intracellular accumulation of organic solutes (osmolytes). We investigated the mechanism of osmotic adaptation in the diazotrophic bacteria Azotobacter chroococcum, Azospirillum brasilense, and Klebsiella pneumoniae, which are adversely affected by high osmotic strength (i.e., soil salinity and/or drought). We used natural-abundance ¹³C nuclear magnetic resonance spectroscopy to identify all the osmolytes accumulating in these strains during osmotic stress generated by 0.5 M NaCl. Evidence is presented for the accumulation of trehalose and glutamate in Azotobacter chroococcum ZSM4, proline and glutamate in Azospirillum brasilense SHS6, and trehalose and proline in K. pneumoniae. Glycine betaine was accumulated in all strains grown in culture media containing yeast extract as the sole nitrogen source. Alternative nitrogen sources (e.g., NH₄Cl or casamino acids) in the culture medium did not result in measurable glycine betaine accumulation. We suggest that the mechanism of osmotic adaptation in these organisms entails the accumulation of osmolytes in hyperosmotically stressed cells resulting from either enhanced uptake from the medium (of glycine betaine, proline, and glutamate) or increased net biosynthesis (of trehalose, proline, and glutamate) or both. The preferred osmolyte in Azotobacter chroococcum ZSM4 shifted from glutamate to trehalose as a consequence of a prolonged osmotic stress. Also, the dominant osmolyte in Azospirillum brasilense SHS6 shifted from glutamate to proline accumulation as the osmotic strength of the medium increased.     

Glutamine, Glutamate, and α-Glucosylglycerate Are the Major Osmotic Solutes Accumulated by Erwinia chrysanthemi Strain 3937

Erwinia chrysanthemi is a phytopathogenic soil enterobacterium closely related to Escherichia coli. Both species respond to hyperosmotic pressure and to external added osmoprotectants in a similar way. Unexpectedly, the pools of endogenous osmolytes show different compositions. Instead of the commonly accumulated glutamate and trehalose, E. chrysanthemi strain 3937 promotes the accumulation of glutamine and α-glucosylglycerate, which is a new osmolyte for enterobacteria, together with glutamine. The amounts of the three osmolytes increased with medium osmolarity and were reduced when betaine was provided in the growth medium. Both glutamine and glutamate showed a high rate of turnover, whereas glucosylglycerate stayed stable. In addition, the balance between the osmolytes depended on the osmolality of the medium. Glucosylglycerate and glutamate were the major intracellular compounds in low salt concentrations, whereas glutamine predominated at higher concentrations. Interestingly, the ammonium content of the medium also influenced the pool of osmolytes. During bacterial growth with 1 mM ammonium in stressing conditions, more glucosylglycerate accumulated by far than the other organic solutes. Glucosylglycerate synthesis has been described in some halophilic archaea and bacteria but not as a dominant osmolyte, and its role as an osmolyte in Erwinia chrysanthemi 3937 shows that nonhalophilic bacteria can also use ionic osmolytes.     

Glucose Metabolic Trapping in Mouse Arteries: Nonradioactive Assay of Atherosclerotic Plaque Inflammation Applicable to Drug Discovery

Background

¹⁸F-Fluorodeoxyglucose (FDG)-positron emission tomography (PET) imaging of atherosclerosis in the clinic is based on preferential accumulation of radioactive glucose analog in atherosclerotic plaques. FDG-PET is challenging in mouse models due to limited resolution and high cost. We aimed to quantify accumulation of nonradioactive glucose metabolite, FDG-6-phosphate, in the mouse atherosclerotic plaques as a simple alternative to PET imaging.

Methodology/Principal Findings

Nonradioactive FDG was injected 30 minutes before euthanasia. Arteries were dissected, and lipids were extracted. The arteries were re-extracted with 50% acetonitrile-50% methanol-0.1% formic acid. A daughter ion of FDG-6-phosphate was quantified using liquid chromatography and mass spectrometry (LC/MS/MS). Thus, both traditional (cholesterol) and novel (FDG-6-phosphate) markers were assayed in the same tissue. FDG-6-phosphate was accumulated in atherosclerotic lesions associated with carotid ligation of the Western diet fed ApoE knockout mice (5.9 times increase compare to unligated carotids, p<0.001). Treatment with the liver X receptor agonist T0901317 significantly (2.1 times, p<0.01) reduced FDG-6-phosphate accumulation 2 weeks after surgery. Anti-atherosclerotic effects were independently confirmed by reduction in lesion size, macrophage number, cholesterol ester accumulation, and macrophage proteolytic activity.

Conclusions/Significance

Mass spectrometry of FDG-6-phosphate in experimental atherosclerosis is consistent with plaque inflammation and provides potential translational link to the clinical studies utilizing FDG-PET imaging.     

The role of trehalose in the osmoadaptation of Escherichia coli NCIB 9484: interaction of trehalose, K+ and glutamate during osmoadaptation in continuous culture.

Natural abundance 13C nuclear magnetic resonance spectroscopy identified the disaccharide trehalose as the major organic osmolyte synthesized by Escherichia coli grown in continuous culture under nitrogen limitation in the presence of 0.5 M-NaCl. Trehalose accumulation was dependent on both the growth phase of the culture and the osmolality of the growth medium, but independent of the solute used to increase the osmolality as long as the solute was non-penetrant. The penetrant solute glycerol did not induce trehalose synthesis indicating that the loss of cell turgor rather than increasing medium osmolality per se was the mechanism stimulating trehalose synthesis. Under conditions of either carbon or nitrogen limitation osmoadaptation was distinctly biphasic. The initial response consisted of a rapid (within 30 min) accumulation of K+ and a concurrent synthesis of the amino acid glutamate; trehalose synthesis occurred during the second slower phase of osmoadaption. Chloramphenicol severely inhibited trehalose accumulation indicating that the enzyme(s) involved in trehalose synthesis were inducible.     

Regional Central Nervous System Oligosaccharide Storage in Caprine β-Mannosidosis

Goats affected with beta-mannosidosis, an autosomal recessive disease of glycoprotein metabolism, have deficient activity of the lysosomal enzyme beta-mannosidase along with tissue storage of oligosaccharides, including a trisaccharide [Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc] and a disaccharide [Man(beta 1-4)GlcNAc]. CNS myelin deficiency, with regional variation in severity, is a major pathological characteristic of affected goats. This study was designed to investigate regional CNS differences in oligosaccharide accumulation to assess the extent of correlation between oligosaccharide accumulation and severity of myelin deficits. The concentrations of accumulated disaccharide and trisaccharide and the activity of beta-mannosidase were determined in cerebral hemisphere gray and white matter and in spinal cord from three affected and two control neonatal goats. In affected goats, the content of trisaccharide and disaccharide in spinal cord (moderate myelin deficiency) was similar to or greater than that in cerebral hemispheres (severe myelin deficiency). Thus, greater oligosaccharide accumulation was not associated with more severe myelin deficiency. Regional beta-mannosidase activity levels in control goats were consistent with the affected goat oligosaccharide accumulation pattern. The similarity of trisaccharide and disaccharide content in cerebral hemisphere gray and white matter suggested that lysosomal storage vacuoles, more numerous in gray matter, may not be the only location of stored CNS oligosaccharides.     

Analysis of Strains Lacking Known Osmolyte Accumulation Mechanisms Reveals Contributions of Osmolytes and Transporters to Protection against Abiotic Stress

Osmolyte accumulation and release can protect cells from abiotic stresses. In Escherichia coli, known mechanisms mediate osmotic stress-induced accumulation of K⁺ glutamate, trehalose, or zwitterions like glycine betaine. Previous observations suggested that additional osmolyte accumulation mechanisms (OAMs) exist and their impacts may be abiotic stress specific. Derivatives of the uropathogenic strain CFT073 and the laboratory strain MG1655 lacking known OAMs were created. CFT073 grew without osmoprotectants in minimal medium with up to 0.9 M NaCl. CFT073 and its OAM-deficient derivative grew equally well in high- and low-osmolality urine pools. Urine-grown bacteria did not accumulate large amounts of known or novel osmolytes. Thus, CFT073 showed unusual osmotolerance and did not require osmolyte accumulation to grow in urine. Yeast extract and brain heart infusion stimulated growth of the OAM-deficient MG1655 derivative at high salinity. Neither known nor putative osmoprotectants did so. Glutamate and glutamine accumulated after growth with either organic mixture, and no novel osmolytes were detected. MG1655 derivatives retaining individual OAMs were created. Their abilities to mediate osmoprotection were compared at 15°C, 37°C without or with urea, and 42°C. Stress protection was not OAM specific, and variations in osmoprotectant effectiveness were similar under all conditions. Glycine betaine and dimethylsulfoniopropionate (DMSP) were the most effective. Trimethylamine-N-oxide (TMAO) was a weak osmoprotectant and a particularly effective urea protectant. The effectiveness of glycine betaine, TMAO, and proline as osmoprotectants correlated with their preferential exclusion from protein surfaces, not with their propensity to prevent protein denaturation. Thus, their effectiveness as stress protectants correlated with their ability to rehydrate the cytoplasm.     

Effects of intracellular trehalose content on Streptomyces griseus spores.

The disaccharide trehalose is accumulated as a storage product by spores of Streptomyces griseus. Growth on media containing excess glucose yielded spores containing up to 25% of their dry weight as trehalose. Spores containing as little as 1% of their dry weight as trehalose were obtained during growth on media containing a limiting amount of glucose. Spores containing low levels of trehalose accumulated this sugar when incubated with glucose. The increase in trehalose content coincided with increases in spore refractility, heat resistance, desiccation resistance, and the time required for spore germination in complex media. Trehalose is accumulated by a wide variety of actinomycetes and related bacteria and may be partially responsible for their resistance properties.     

Role of Glycine Betaine and Related Osmolytes in Osmotic Stress Adaptation in Yersinia enterocolitica ATCC 9610

Yersinia enterocolitica is a gram-negative, food-borne pathogen that can grow in 5% NaCl and at refrigerator temperatures. In this report, the compatible solutes (osmolytes) which accumulate intracellularly and confer the observed osmotic tolerance to this pathogen were identified. In minimal medium, glutamate was the only detectable osmolyte that accumulated in osmotically stressed cells. However, when the growth medium was supplemented with glycine betaine, dimethylglycine, or carnitine, the respective osmolyte accumulated intracellularly to high levels and the growth rates of the osmotically stressed cultures improved from 2.4- to 3.5-fold. Chill stress also stimulated the intracellular accumulation of glycine betaine, but the growth rate was only slightly improved by this osmolyte. Both osmotic upshock and temperature downshock stimulated the rate of uptake of [(sup14)C]glycine betaine by more than 30-fold, consistent with other data indicating that the osmolytes are accumulated from the growth medium via transport.     

Role of a nonmitochondrial Ca2+ pool in the synergistic stimulation by cyclic AMP and vasopressin of Ca2+ uptake in isolated rat hepatocytes.

The subcellular distribution of 45Ca2+ accumulated by isolated rat hepatocytes exposed to dibutyryl cyclic AMP (dbcAMP) followed by vasopressin (Vp) was studied by means of a nondisruptive technique. When treated with dbcAMP followed by vasopressin, hepatocytes obtained from fed rats accumulated an amount of Ca2+ approximately fivefold higher than that attained under control conditions. Ca2+ released from the mitochondrial compartment by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) accounted for only a minor portion of the accumulated Ca2+. The largest portion was released by the Ca2+ ionophore A23187 and was attributable to a nonmitochondrial compartment. DbcAMP + Vp-treatment also caused a maximal stimulation of glucose production and a twofold increase in cellular glucose 6-phosphate levels. In hepatocytes obtained from fasted rats, dbcAMP + Vp-stimulated Ca2+ accumulation was lower, although with the same subcellular distribution, and was associated with a minimal glucose production. In the presence of gluconeogenetic substrates (lactate plus pyruvate) hepatocytes from fasted rats were comparable to cells isolated from fed animals. However, Ca2+ accumulation and glucose 6-phosphate production could be dissociated in the absence of dbcAMP, in the presence of lactate/pyruvate alone. Under this condition in fact Vp induced only a minimal accumulation of Ca2+ in hepatocytes isolated from fasted rats, although glucose production was markedly increased. Moreover, treatment of fed rat hepatocytes with 1 mM ATP caused a maximal activation of glycogenolysis, but only a moderate stimulation of cellular Ca2+ accumulation. In this case, sequestration of Ca2+ occurred mainly in the mitochondrial compartment. By contrast, the addition of ATP to dbcAMP-pretreated hepatocytes induced a large accumulation of Ca2+ in a nonmitochondrial pool. Additional experiments using the fluorescent Ca2+ indicator Fura-2 showed that dbcAMP pretreatment can enlarge and prolong the elevation of cytosolic free Ca2+ caused by Vp. A nonmitochondrial Ca2+ pool thus appears mainly responsible for the Ca2+ accumulation stimulated by dbcAMP and Vp in isolated hepatocytes, and cyclic AMP seems able to activate Ca2+ uptake in such a nonmitochondrial pool.     

Uptake and metabolism of sucrose by Streptococcus lactis

Transport and metabolism of sucrose in Streptococcus lactis K1 have been examined. Starved cells of S. lactis K1 grown previously on sucrose accumulated [14C]sucrose by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) (sucrose-PTS; Km, 22 microM; Vmax, 191 mumol transported min-1 g of dry weight of cells-1). The product of group translocation was sucrose 6-phosphate (6-O-phosphoryl-D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside). A specific sucrose 6-phosphate hydrolase was identified which cleaved the disaccharide phosphate (Km, 0.10 mM) to glucose 6-phosphate and fructose. The enzyme did not cleave sucrose 6'-phosphate(D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside-6'-phosphate). Extracts prepared from sucrose-grown cells also contained an ATP-dependent mannofructokinase which catalyzed the conversion of fructose to fructose 6-phosphate (Km, 0.33 mM). The sucrose-PTS and sucrose 6-phosphate hydrolase activities were coordinately induced during growth on sucrose. Mannofructokinase appeared to be regulated independently of the sucrose-PTS and sucrose 6-phosphate hydrolase, since expression also occurred when S. lactis K1 was grown on non-PTS sugars. Expression of the mannofructokinase may be negatively regulated by a component (or a derivative) of the PTS.     

Trehalose and glycogen accumulation is related to the duration of the G1 phase of Saccharomyces cerevisiae

Several factors may control trehalose and glycogen synthesis, like the glucose flux, the growth rate, the intracellular glucose-6-phosphate level and the glucose concentration in the medium. Here, the possible relation of these putative inducers to reserve carbohydrate accumulation was studied under well-defined growth conditions in nitrogen-limited continuous cultures. We showed that the amounts of accumulated trehalose and glycogen were regulated by the growth rate imposed on the culture, whereas other implicated inducers did not exhibit a correlation with reserve carbohydrate accumulation. Trehalose accumulation was induced at a dilution rate (D)相似文献   

Acclimation of potato plants to polyethylene glycol-induced water deficit II. Contents and subcellular distribution of organic solutes

Potato plants (Solanum tuberosum cv. Désirée) were grown hydroponically and subjected to water deficit induced by addition of 10% (w/v) PEG 6000. The potato plants were able to grow under water deficit by accumulating organic solutes (osmoregulation). Osmoregulation occurred in two phases. During the initial 2d hexoses were accumulated, and after 7 d of PEG treatment osmotic adjustment was mostly due to the accumulation of amino acids, especially proline, which accumulated up to 150 times the control content. Sucrose contents remained unchanged in leaves of PEG-treated plants compared with controls, whereas the starch content decreased during PEG treatment.In control leaves, the hexoses and malate were compartmented in the vacuole and sucrose was found in the cytosol and vacuole. Amino acids were distributed between the cytosol and stroma, but only minor amounts of amino acids could be detected in the vacuole. Under water deficit the subcellular distribution of hexoses, malate and sucrose remained unchanged. Most amino acids showed a slight to moderate higher concentration in the vacuole under water deficit. Proline, the metabolite contributing mainly to osmoregulation, was concentrated mostly in the chloroplast and the cytosol. This underlines the important role of proline as the osmolyte under water deficit.