Direct regulation of Na(+)-dependent myo-inositol transport by sugars in retinal pigment epithelium: role of phorbol ester and staurosporin

An Na(+)-dependent active process for myo-inositol (MI) uptake, sharing a common carrier system with glucose and sensitive to phlorizin, was previously established in primary cultures of bovine retinal pigment epithelial (RPE) cells (26, 32). The present report further examines the nature of glucose-induced inhibition of MI transport in primary cultures of RPE cells. RPE cells were grown in supplemented Dulbecco's modification of Eagle's medium (DMEM) containing 5 mM D-glucose (basic growth media) or 40 mM D-glucose or its nonmetabolizable analogue, alpha-methyl-D-glucoside (alpha MG); 1-5 mM nonradioactive MI, pyruvate, or lactate; or 0.2-20 microM phorbol 12-myristate 13-acetate (TPA) or straurosporin (modified growth media), for up to 4 weeks. The capacity of RPE cells to accumulate 3H-MI (ratios of intracellular transported radioactive MI, [MI]i, to external free MI concentration, [MI]i/[MI]o) decreased by up to 41% or 34% when cells were grown for 10 days or longer with 40 mM D-glucose or 40 mM alpha MG, respectively, compared to cells grown in basic growth media. The rate of uptake of 3H-MI also was reduced to 63 +/- 15% or 48 +/- 8% of the control values when cells were fed 1 or 5 mM nonradioactive MI, respectively. In addition, cellular capacity to bind to [3H]phlorizin was reduced to 52 +/- 7%, 61 +/- 5%, or 38 +/- 6% of the controls when RPE cells were fed 40 mM D-glucose, 40 mM alpha MG, or 5 mM nonradioactive MI, respectively. Growth media containing either pyruvate or lactate, the glucose metabolites, did not suppress the ability of RPE cells to accumulate MI. An 18 +/- 8% reduction in [3H]thymidine incorporation into DNA occurred when cells were grown in 40 mM glucose for 12-14 days, compared to cells grown with 5 mM glucose. Chronic treatment (12-14 days) of the cells with phorbol ester, an activator of protein kinase C, caused up to twofold increase in MI uptake, [3H]phlorizin binding, cell number, and DNA synthesis. However, when the rates of MI uptake into cells grown in basic growth media or TPA-treated media were normalized to cell number, no significant difference in MI uptake was found between the treated and untreated cells. Addition of staurosporin, a protein kinase C inhibitor, together with TPA, in the growth media reversed the phorbol-induced increase of MI uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Methylglyoxal and high glucose co-treatment induces apoptosis or necrosis in human umbilical vein endothelial cells

Hyperglycemia and elevation of methylglyoxal (MG) are symptoms of diabetes mellitus (DM). We previously showed that high glucose (HG; 30 mM) or MG (50-400 microM) could induce apoptosis in mammalian cells, but these doses are higher than the physiological concentrations of glucose and MG in the plasma of DM patients. The physiological concentration of MG and glucose in the normal blood circulation is about 1 microM and 5 mM, respectively. Here, we show that co-treatment with concentrations of MG and glucose comparable to those seen in the blood circulation of DM patients (5 microM and 15-30 mM, respectively) could cause cell apoptosis or necrosis in human umbilical vein endothelial cells (HUVECs) in vitro. HG/MG co-treatment directly increased the reactive oxygen species (ROS) content in HUVECs, leading to increases in intracellular ATP levels, which can control cell death through apoptosis or necrosis. Co-treatment of HUVECs with 5 microM MG and 20 mM glucose significantly increased cytoplasmic free calcium levels, activation of nitric oxide synthase (NOS), caspase-3 and -9, cytochrome c release, and apoptotic cell death. In contrast, these apoptotic biochemical changes were not detected in HUVECs treated with 5 microM MG and 30 mM glucose, which appeared to undergo necrosis. Pretreatment with nitric oxide (NO) scavengers could inhibit 5 microM MG/20 mM glucose-induced cytochrome c release, decrease activation of caspase-9 and caspase-3, and increase the gene expression and protein levels of p53 and p21, which are known to be involved in apoptotic signaling. Inhibition of p53 protein expression using small interfering RNA (siRNA) blocked the activation of p21 and the cell apoptosis induced by 5 microM MG/20 mM glucose. In contrast, inhibition of p21 protein expression by siRNA prevented apoptosis in HUVECs but had no effect on p53 expression. These results collectively suggest that the treatment dosage of MG and glucose could determine the mode of cell death (apoptosis vs. necrosis) in HUVECs, and both ROS and NO played important roles in MG/HG-induced apoptosis of these cells.     

Impact of nutritional supplements and monosaccharides on growth, oxalate accumulation, and culture pH by Sclerotinia sclerotiorum

Sclerotinia sclerotiorum D-E7 was studied to determine the impact of nutritional supplements and monosaccharides on growth, oxalate accumulation, and culture pH in broth media (initial pH c. 5). Cultures with 0.1% nutritional supplement (tryptone, yeast extract, or soytone) yielded minimal growth, 2-3 mM oxalate, and a final culture pH of 4.2-4.8. In contrast, cultures with 0.1% nutritional supplement and 25 mM glucose yielded significant growth, minimal oxalate (<1 mM), and a final culture pH of 2.8-3.7. Similar trends were observed when glucose in 0.1% soytone cultures was replaced with 25 mM d-mannose, l-arabinose, or d-xylose. With 1% soytone-25 mM glucose cultures, growth and oxalate accumulation ( approximately 21 mM) occurred with little change in initial pH. This was not the case with 1% soytone-250 mM glucose cultures; increased glucose levels resulted in a decrease in oxalate accumulation ( approximately 7 mM) and in final culture pH (3.4). Time-course studies with these cultures revealed that oxalate accumulation was suppressed during growth when the culture pH dropped to <4. Overall, these results indicate that (1) the decrease in external pH (i.e. acidification) was independent of oxalate accumulation and (2) acidification coupled to glucose-dependent growth regulated oxalate accumulation by Sclerotinia sclerotiorum.     

Culture of Plasmodium falciparum: the role of pH, glucose, and lactate

Yields of P. falciparum in intraerythrocytic in vitro cultures were maximized when extracellular pH was maintained between 7.2 and 7.45, and extracellular lactate was kept below 12 mM. Host erythrocytes metabolized 4.6 +/- 1.5 microM glucose/10(9) RBC/24 hr and produced 7.9 +/- 1.8 microM lactate/10(9) RBC/24 hr. Asynchronous parasite cultures used 122 +/- 34 microM glucose/10(9) parasitized RBC/24 hr and produced 143 +/- 47 microM lactate/10(9) parasitized RBC/24 hr. Synchronous cultures that were 80 to 100% ring forms after 24 hr in culture exhibited significantly lower glycolysis per 10(9) parasitized RBC than cultures that were 0 to 25% ring forms after 24 hr. The percent of glucose utilization accounted for by lactate production by parasites was significantly less than that of uninfected erythrocytes. These optimum ranges and metabolic rates can be used in the development of parasite culture techniques.     

Glucose toxicity in Prevotella ruminicola: methylglyoxal accumulation and its effect on membrane physiology.

When the ruminal bacterium prevotella ruminicola B(1)4-M was grown in a defined medium with an excess of glucose (3.6 mM ammonia and 50 mM glucose), the cells accumulated large amounts of cellular polysaccharide and the viable cell number decreased at least 1,000-fold. This decrease in viability was correlated with an accumulation of methylglyoxal in the supernatant (3 to 4 mM). Other genetically distinct strains of P. ruminicola produced methylglyoxal, but methylglyoxal production was not ubiquitous among the strains. When P. ruminicola B(1)4-M was grown in continuous culture (dilution rate, 0.1 h-1) with an excess of glucose, there was an oscillating pattern of growth and cell death which was correlated with the accumulation and washout of methylglyoxal from the culture vessel. Mutants which resisted an excess of glucose took up glucose at a slower rate and produced less methylglyoxal than the wild type. These mutants were, however, not stable. There was always a long lag time, and the mutants could only be maintained with a daily transfer schedule. When the mutants were transferred less frequently, methylglyoxal eventually accumulated and the cultures died. The mutants transported glucose at a threefold-slower rate than the wild type, and in each case the carrier had more than one binding site for glucose. Because glucose transport could not be driven by phosphoenolpyruvate or ATP, the glucose carrier of P. ruminicola is probably a proton symport system. When P. ruminicola B(1)4-M cultures were treated with 4 mM methylglyoxal, the delta psi decreased even though intracellular ATP concentrations were high.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of calcium ion uptake on Candida albicans morphology

In liquid culture using a synthetic medium, added magnesium but not calcium was required for exponential growth of Candida albicans yeast cells. However, medium without added divalent cations supported 2-3 generations of yeast growth or germ tube induction. The addition of calcium ions (1.0 mM) at any stage during the induction of germ tube formation caused reversion to a yeast mode of growth, in contrast to the effect of zinc and cobalt ions which were toxic to all growth. Inhibition of germ tube formation by calcium was not observed in the presence of either magnesium (10 microM) or manganese (100 microM). The presence of either of these ions caused inhibition of 45Ca uptake in yeast cultures. We conclude that unrestricted calcium uptake resulted in the specific inhibition of C. albicans mycelial growth, indicating a critical role for calcium in the regulation of C. albicans morphogenesis.     

Fructose-induced peroxynitrite production is mediated by methylglyoxal in vascular smooth muscle cells

Methylglyoxal (MG), a highly reactive molecule, has been implicated in the development of insulin resistance. We investigated whether fructose, a precursor of MG, induced ONOO(-) generation and whether this process was mediated via endogenously increased MG formation. Fructose significantly increased MG generation in vascular smooth muscle cells (VSMCs) in a concentration and time dependent manner. The intracellular production of MG was increased by 153+/-23% or 259+/-28% after cells were treated 6 h with fructose (15 mM or 30 mM), compared with production from untreated cells (p<0.01, n=4 for each group). A significant increase in the production of ONOO(-), NO, and O(2)(*-), was found in the cells treated with fructose (15 mM) or MG (10 microM). Fructose- or MG-induced ONOO(-) generation was significantly inhibited by MG scavengers, including reduced glutathione or N-acetyl-l-cysteine, and by O(2)(*-) or NO inhibitors, such as diphenylene iodonium, superoxide dismutase or N-nitro-l-arginine methyl ester. Moreover, an enhanced iNOS expression was also observed in the cells treated directly with MG which was significantly inhibited when co-application with N-acetyl-l-cysteine. Our results demonstrated that fructose is capable of inducing a significant increase in ONOO(-) production, which is mediated by an enhanced formation of endogenous MG in VSMCs.     

Advanced glycation endproduct induces ROS accumulation, apoptosis, MAP kinase activation and nuclear O-GlcNAcylation in human cardiac myocytes

Accumulation of advanced glycation endproduct (AGE) has been implicated in the pathogenesis of diabetic complications. However, the precise role and mechanism behind AGE-associated diabetic heart injury are not fully clear. This study was designed to evaluate the effect of AGE on accumulation of reactive oxygen species (ROS), apoptosis, mitogen-activated protein kinase (MAPK) activation and nuclear O-GlcNAcylation in fetal human cardiac myocytes. Myocytes were maintained for 24-72 h in a defined culture medium containing high glucose, the AGE carbon precursor methylglyoxal (MG), and MG-AGE derived from MG and bovine serum albumin (BSA). Generation of ROS was detected by 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by caspase-3 activity and quantitative DNA fragmentation. Both high glucose (25.5 mM) and MG (200 microM) significantly enhanced ROS and AGE formation with greater effects elicited by MG. Both high glucose and MG-AGE significantly facilitated apoptosis with a more predominant effect from MG-AGE. In addition, phosphorylation of MAPK cascade [extracellular signal-regulated kinase-1/2 (ERK1/2) and p38] and nuclear O-GlcNAcylation were enhanced in MG-AGE-treated myocytes, similar to those elicited by high glucose. MG-AGE-induced phosphorylation of ERK1/2 and p38 was nullified by neutralizing AGE with specific anti-AGE antibody but not nonspecific antiserum. Collectively, these results indicated that AGE or its precursor MG may trigger ROS generation, apoptosis, MAPK activation and nuclear O-GlcNAcylation in human cardiac myocytes, in a manner reminiscent of high extracellular glucose.     

Hexose transport in preimplantation rabbit blastocysts

Transtrophectodermal 3-0-methyl glucose (3-0MG) transport in the rabbit blastocyst at Days 6 and 7 post coitum was investigated to understand better how the trophectoderm can regulate inner cell mass growth by controlling substrate availability. 3-0MG rapidly traversed the trophectoderm and displayed saturation kinetics (Km = 4.3 +/- 0.5 mM, Vmax = 79 +/- 3.8 nmol.cm-2). The flux of 3-0MG was inhibited nearly 95% by 10(-4) M-phloretin, and only 15% by 10(-4) M-phlorizin. Furthermore, 3-0MG influx was inhibited by cytochalasin B (5 microM) and was unaffected by removal of sodium. The transport system had a high specificity for 2-deoxy-D-glucose and glucose, and a very low specificity for fructose and 4-alpha-methyl glucoside. Western blots probed with a polyclonal antibody to the human erythrocyte glucose transport protein and also with a polyclonal antibody to the C-terminus of the glucose transport protein of the rat brain revealed a broad band with a molecular weight of 55,000. Using immuno-gold labelling techniques, Na(+)-independent glucose transporters were localized to both the apical and basolateral borders of the trophectodermal cell. These results suggest that the mechanism in the trophectoderm responsible for transport of glucose is similar to other sodium-independent glucose transport systems. In addition, 3-0MG influx was unaffected by short-term incubation with progesterone, the progesterone antagonist mifepristone (RU-486), PGF-2 alpha, PGE-2, insulin, or cAMP. Day-7 p.c. embryos also transported hexoses by a similar system because the influx rate and the phlorizin/phloretin sensitivity were the same as in the Day-6 p.c. embryo.     

Mercaptopyridine-N-oxide, an NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts

The enzyme NADH-fumarate reductase is not found in mammalian cells but it is present in several parasitic protozoa including Trypanosoma cruzi, the parasite that causes Chagas' disease. This study shows that the drug 2-mercaptopyridine-N-oxide (MPNO) inhibits NADH-fumarate reductase purified from T. cruzi (ID50 = 35 microM). When added to intact cells, MPNO inhibited the growth of T. cruzi epimastigotes in culture (ID50 = 0.08 microM) as well as the infection of mammalian myoblasts by T. cruzi trypomastigotes (ID50 = 20 microM). At a concentration of 2.4 microM, MPNO also inhibited the growth of amastigotes (intracellular dividing forms) in cultured mammalian myoblasts. Supplementation of culture media with 5 mM succinate, the product of fumarate reductase, partially protected against the inhibition of the growth of epimastigotes by MPNO. Moreover, MPNO inhibited the accumulation of succinate in cultures of epimastigotes, as measured by high performance liquid chromatography. Although MPNO may have other intracellular targets in addition to fumarate reductase, these results support the hypothesis that compounds which inhibit the enzyme fumarate reductase may be potential chemotherapeutic agents against Chagas' disease.     

Inhibition of methicillin-resistant Staphylococcus aureus by an in vitro continuous-flow culture containing human stool microflora

We used an in vitro continuous-flow culture model of human stool microflora to examine the ability of human stool microflora to inhibit growth of two methicillin-resistant S. aureus (MRSA) strains. Continuous-flow cultures consistently eliminated MRSA inocula of 10(6) cfu/mL within 4 days, and addition of continuous-flow culture resulted in elimination of a pre-established MRSA culture ( approximately 10(8) cfu/mL) within 6-8 days. Anaerobic or "aerobic" (i.e., continuous bubbling of room air to eliminate obligate anaerobes) cultures eliminated MRSA at similar rates. The MRSA strains were unable to replicate under anaerobic conditions in sterile filtrates produced from the continuous-flow culture, but rapid growth occurred when glucose was added. These data demonstrate that indigenous stool microflora efficiently eliminate MRSA colonization and obligate anaerobes are not essential for inhibition. Our findings also suggest that inhibition of MRSA in continuous-flow cultures is due to depletion of nutrients rather than production of inhibitory conditions.     

Effects of amiloride on the induction of DNA synthesis and casein gene expression in rabbit mammary explants

Amiloride, an inhibitor of Na+/H+ exchange, was added at various concentrations to the culture medium of rabbit mammary explants. In the concentration range 100-250 microM, amiloride progessively inhibited 14C-thymidine incorporation induced by insulin, EGF or prolactin. Up to 250 microM, amiloride, which did not inhibit basal protein synthesis, was not cytotoxic, but it reduced basal DNA synthesis at the highest concentration. Addition of amiloride to the culture medium of mammary explants also strongly inhibited the induction of casein synthesis and casein mRNA accumulation by prolactin. The inhibition by amiloride is therefore not specific of the mitogenic action of prolactin since this drug also prevented its lactogenic action. The data reported here describe a new inhibitory action of amiloride on the transmission of the lactogenic signals.     

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)     

Kinetics of myo-inositol transport in corneal endothelial cells: diverse effects of sugars and implications in corneal deutergensence [corrected

Kinetics of myo-inositol (MI) uptake into primary cultures of bovine corneal endothelial cells (CEC) were studied. Confluent corneal endothelial cells accumulated 3H-MI in a time dependent and saturable process. At a narrow range of external concentrations of 3H-MI (4-50 microM), the Na(+)-dependent MI uptake followed saturation kinetics. The apparent Km value was 20 microM with a maximum velocity (Vmax) of 16 pmol/20 min/micrograms DNA. At low external 3H-MI concentrations the uptake was dependent on Na ions, but at higher levels the Na(+)-independent fraction of MI uptake significantly increased. The uptake was sensitive to removal of Ca ions and to the presence of inhibitors such as n-ethyl maleimide, phlorizin, ouabain, and amiloride (an inhibitor of Na+/H+ exchanger). The sensitivity of MI uptake toward inhibitors and ionic changes in the bathing media was reduced as external concentrations of 3H-MI increased. Citrate at 0.5 mM increased the uptake, suggesting involvement of mitochondrial oxidative metabolism in the MI uptake. Percent release of radioactivity by 2 min, after an initial 40-min incubation with 20 microM 3H-MI, was 6.6% +/- 0.8 or 35% +/- 4 when release media contained BSS alone or BSS containing 5 mM nonradioactive MI, respectively. Efflux of radioactivity from the cells also was enhanced when release media contained 40 mM glucose. Glucose and galactose as well as nonmetabolizable glucose analogues, such as 3O-methyl glucose or alpha-methyl glucose, at high concentrations (40 mM), acutely (in the incubation media) or chronically (in the growth media) inhibited MI uptake into CEC, and the extent of inhibition was inversely proportional to the external levels of 3H-MI. However, glucose at lower levels (less than or equal to 10 mM) slightly increased MI uptake. These studies indicated that the uptake of MI into corneal endothelial cells was an Na(+)-dependent active process at a narrow range of external radioactive MI concentrations. Higher levels of MI were taken up by the cells via a passive diffusion mechanism, independent of carrier protein(s). Glucose influenced the uptake of MI in a complex manner. The increased MI efflux by glucose or by MI was perhaps due to the limited capacity of CEC for accumulation or compartmentalization of this or other solutes/osmolytes, a phenomenon that may be related to the role of CEC in maintenance of corneal deutergence. High glucose-induced inhibition of Na(+)-dependent MI uptake may be in part due to glucose regulation of Na+ fluxes and cell volume.(ABSTRACT TRUNCATED AT 400 WORDS)     

Removal of inhibitors of bacterial growth by dialysis culture.

Dialysis culture was used to investigate the extent to which growth inhibition in bacterial cultures may be caused by accumulation of metabolites. Escherichia coli B was grown in a glucose/salts medium. A concentrated nutrient solution was pumped at a constant rate into the growing culture to ensure that growth was not limited by exhaustion of nutrients. In this way the only difference between growth conditions in dialysis and non-dialysis cultures was the transfer of dialysable metabolites from the culture vessel to the reservoir in the dialysis culture system. By adjusting the glucose concentration in the feed and maintaining a constant rate of feeding, glucose-limited growth could be achieved. Under these conditions, with oxygen in excess, bacterial yields of 140 to 150 g dry wt l-1 were obtained in dialysis culture compared with 30 to 40 g l-1 in non-dialysis culture. The high yields in dialysis culture depended on the removal of end-products of glucose metabolism. Growth inhibition was demonstrated to be the result of the combined influence of acetate, lactate, pyruvate, succinate, propionate and isobutyrate in concentrations found at the end of growth in non-dialysis cultures of Escherichia coli B.     

Regulation of uptake of inositol by glucose in cultured retinal pigment epithelial cells

Long term and acute effects of glucose on myo-inositol (MI) uptake were studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. RPE cells were grown under low (5 mM) or high (20, 40, or 50 mM) glucose levels in the growth medium for up to 18 days. The concentrative capacity of confluent RPE cells to accululate [3H]MI (10 microM) was reduced up to 41% as the glucose concentration in the growth medium increased. When the growth medium glucose was switched from 5 to 40 mM, or vice versa, the capacity of cells to accumulate MI was reversed. Treatment of cells grown in 40 or 50 mM glucose with 0.1 mM Sorbinil (an aldose reductase inhibitor) minimally reversed the ability of cells to accumulate MI. RPE cells, grown in 5 mM glucose, were incubated with 10-60 mM D-glucose or its nonmetabolizable analogues (acute effect). Kinetics of MI uptake inhibition by alpha-methyl glucose according to Dixon plots were characteristic of competitive inhibition (Ki = 28 mM). MI uptake was strongly inhibited by phlorizin. The ability of RPE cells to bind 5 microM [3H]phlorizin also was reduced by increased glucose levels in the growth medium. These studies indicated that MI and glucose shared the same transporter system. Glucose in the incubation medium directly interfered with MI binding to the transporter. High glucose feeding of the cells also down-regulated the transporter density. The uptake and function of solutes such as MI in tissues that operate on the glucose carrier system may be severely impaired in diabetes.     

Influence of auxins and sucrose in monoterpenoid oxindole alkaloid production by Uncaria tomentosa cell suspension cultures

Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.     

Estrogen Hormones Reduce Lipid Peroxidation in Cells and Tissues of the Central Nervous System

Effects of estrogen hormones on lipid peroxidation (LPO) were examined in rat brain homogenates (RBHs), hippocampal HT 22 cells, rat primary neocortical cultures, and human brain homogenates (HBHs). Dose-response curves indicated half-maximal effective concentrations (EC50) of 5.5 and 5.6 mM for iron-induced LPO in RBHs and HT 22 homogenates. Incubation of living rat primary neocortical cultures with iron resulted in an EC50 of 0.5 mM, whereas culture homogenates showed an EC50 of 1.2 mM. Estrogen hormones reduced LPO in all systems: In RBHs, estrone inhibited iron-induced LPO to 74.1 +/- 5.8% of control levels (17beta-estradiol: 71.3 +/- 0.1%) at a concentration of 10 microM. In hippocampal HT 22 cell homogenates, levels of LPO were reduced to 74.8 +/- 5.5% by estrone and to 47.8 +/- 6.2% by 17beta-estradiol. In living neocortical cultures, 17beta-estradiol decreased iron-induced LPO to 79.2 +/- 4.8% and increased the survival of cultured neuronal cells. Of the other steroid compounds tested (corticosterone, progesterone, testosterone), only progesterone decreased LPO in HT 22 cell homogenates. In HBHs, LPO was dose-dependently increased by iron concentrations from 2.7 to 6.0 mM. Incubation with estrogens resulted in a dose-dependent inhibition of LPO to 53.8 +/- 8.6% with 10 microM 17beta-estradiol, whereas estrone failed to affect iron-induced LPO to a significant extent. Nonestrogenic steroids, including hydrocortisol, did not show significant effects on LPO in HBHs.     

In vitro studies on the growth of Shigella sonnei by Lactobacillus casei and Lact. acidophilus.

The inhibitory effect of lactobacilli on growth of Shigella sonnei was studied. The effect was not due to pH alone, as addition of hydrochloric, lactic or acetic acids to culture media did not inhibit the normal growth of the shigellas. The degree of inhibition was measured by disc assay and showed that the inhibitory substance(s) can be extracellular and diffusible, varying the degrees of inhibition depending on the media tested. When broth was inoculated with mixed cultures of Lactobacillus and Shigella strains, the inhibition began at 6 h and the death phase at 9 h. The higher inhibition was produced by the mixture of lactobacilli (35.5 +/- 2.5% at 6 h culture, 57.4 +/- 1.9% at 9 h and 91.2 +/- 1.2% at 14 h). The degree of inhibition was higher when the relationship pathogen : lactobacilli was 1:10(3). The specific growth rate of lactobacilli and shigella was different in pure or mixed cultures. When the lactobacillus alone was grown for 12 h and the shigellas then added, the numbers of shigellas began to decrease immediately at 37 degrees C. This work shows that the Lactobacillus strains employed in fermented milk can be used to inhibit the growth of Sh. sonnei.     

Histamine Stimulation of Cyclic AMP Accumulation in Astrocyte-Enriched and Neuronal Primary Cultures from Rat Brain

Histamine stimulates cyclic AMP accumulation in astrocyte-enriched and neuronal primary cultures from rat brain in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. The response in the astrocyte cultures (Emax = 304 +/- 44% over basal, EC50 = 43 +/- 5 microM) was much higher than in neuronal cultures (Emax = 24 +/- 2%, EC50 = 14 +/- 7 microM). The histamine effect in astrocytes was competitively inhibited by the H2 antagonists cimetidine (Ki = 1.1 +/- 0.2 microM) and ranitidine (Ki = 46 +/- 10 nM) but was insensitive to the H1 antagonist mepyramine (1 microM). The two selective H2 agonists impromidine and dimaprit behaved as partial agonists and showed relative potencies (139 and 0.5, respectively) consistent with an interaction with H2 receptors. The more selective H1 agonist 2-thiazolylethylamine (0.01-1 mM) did not potentiate the response to impromidine (10 microM). Thus, in contrast to what is generally observed in intact cell preparations from brain, the histamine-induced cyclic AMP accumulation in astroglial cells is mediated solely by H2 receptors. The small effect shown in neuronal cultures also appears to be mediated by H2 receptors.