Effects of lipid-lowering drugs on reverse cholesterol transport gene expressions in peripheral blood mononuclear and HepG2 cells

Aims: The ATP-binding cassette transporters, ABCA1 and ABCG1, are LXR-target genes that play an important role in reverse cholesterol transport. We examined the effects of inhibitors of the cholesterol absorption (ezetimibe) and synthesis (statins) on expression of these transporters in HepG2 cells and peripheral blood mononuclear cells (PBMCs) of individuals with primary (and nonfamilial) hypercholesterolemia (HC). Materials & methods: A total of 48 HC individuals were treated with atorvastatin (10 mg/day/4 weeks) and 23 were treated with ezetimibe (10 mg/day/4 weeks), followed by simvastatin (10 mg/day/8 weeks) and simvastatin plus ezetimibe (10 mg of each/day/4 weeks). Gene expression was examined in statin- or ezetimibe-treated and control HepG2 cells as well as PBMCs using real-time PCR. Results: In PBMCs, statins and ezetimibe downregulated ABCA1 and ABCG1 mRNA expression but did not modulate NR1H2 (LxR-beta) and NR1H3 (LXR-alpha) levels. Positive correlations of ABCA1 with ABCG1 and of NR1H2 with NR1H3 expressions were found in all phases of the treatments. In HepG2 cells, ABCA1 mRNA levels remained unaltered while ABCG1 expression was increased by statin (1.0-10.0 mu M) or ezetimibe (5.0 mu M) treatments. Atorvastatin upregulated NR1H2 and NR1H3 only at 10.0 mu M, meanwhile ezetimibe (1.0-5.0 mu M) downregulated NR1H2 but did not change NR1H3 expression. Conclusion: Our findings reveal that lipid-lowering drugs downregulate ABCA1 and ABCG1 mRNA expression in PBMCs of HC individuals and exhibit differential effects on HepG2 cells. Moreover, they indicate that the ABCA1 and ABCG1 transcript levels were not correlated directly to LXR mRNA expression in both cell models treated with lipid-lowering drugs.

Characterization of Caulobacter crescentus response to low temperature and identification of genes involved in freezing resistance

Free-living bacteria must respond to a wide range of temperature changes, and have developed specific mechanisms to survive in extreme environments. In this work we describe a remarkable resistance of mesophilic bacterium Caulobacter crescentus to several cycles of freezing at -80 degrees C, which was able to grow at low temperatures. Exponentially growing cells and late stationary-phase cells presented higher freezing resistance at both -20 and -80 degrees C than early stationary-phase cells. Cryotolerance was observed when log-phase cultures grown at 30 degrees C were preincubated at 5, 15 or 20 degrees C before freezing at -20 degrees C. A transposon library was screened to identify mutants sensitive to freezing at -80 degrees C and three strains presenting < 10% survival were isolated. Identification of genes disrupted in each mutant showed that they encoded an AddA family DNA helicase, a DEAD/DEAH box RNA helicase and a putative RND (resistance, nodulation, cell division) efflux system component. These strains showed longer generation times than wild-type cells when growing at 15 degrees C, with the RNA helicase mutant presenting a severe growth defect. These analyses suggest that the singular intrinsic resistance to freezing of C. crescentus is in fact a consequence of several independent traits, especially the maintenance of a proper degree of supercoiling of nucleic acids.

Variabilidade espacial da emissão de CO2, da temperatura e umidade de um latossolo desprovido de vegetação sob diferentes lâminas de molhamento

The irrigation application is one of the most useful techniques in tropical environments, especially during dry seasons. In this study, CO2 efflux, temperature and soil moisture were studied in a field sampled with a grid having 48 points distributed in 35 x 25 m, under irrigation promoted by a sprinkler located at the center of the area, provoking different levels of water deposition, with maximum irrigation levels of 44.4 and 62.2 mm in points closer to the sprinkler. The results show that the emissions, temperature and moisture were strongly affected by the two irrigations events, having a total water level added of 106,6 mm for the points next to the sprinkler and zero for the most distant points from it. The maps of space variation of the variables, as well as the linear correlation between them, indicate that the emissions were positively related to the soil moisture and negative correlated to the soil temperature only after the irrigations events. The special variability models of soil CO2 emission changed from exponential to spherical after the irrigations events. Such results indicate that soil moisture is among possible controlling factors of the soil CO2 emission, because even with reductions in soil temperature provoked by the wetness, emissions increased strongly.

Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells

The effect of tetracaine on Ca-45 efflux, cytoplasmic Ca2+ concentration [Ca2+](i), and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the Ca-45 efflux from isolated islets in a dose-dependant manner. Tetracaine did not affect the increase in Ca-45 efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+](i) in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 mu M D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 mu M thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in Ca-45 efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.

O efeito da complexidade estrutural da fonte de nitrogênio no transporte de amônio em Saccharomyces cerevisiae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

LOW-PROTEIN DIET IMPAIRS GLUCOSE-INDUCED INSULIN-SECRETION FROM AND CA-45 UPTAKE BY PANCREATIC RAT ISLETS

Glucose-induced insulin secretion rom and Ca-45 uptake by isolated pancreatic islets, derived from rats fed with normal (NPD) or low protein diet (LPD), were studied. Insulin secretion from both types of islets in response to increasing concentrations of glucose followed an S-shaped pattern. However, basal secretion observed at substimulatory concentrations of glucose (0-5.6 mM), as well as maximal release, obtained at 16.7 mM or higher glucose concentrations were significantly reduced in islets from LPD. Furthermore, in LPD rat islets, the dose-response curve to glucose was clearly shifted to the right compared with NPD islets, with the half-maximal response occurring at 8.5 and 14.4 mM glucose for NPD and LPD islets, respectively. In islets from NPD rats, the Ca-45 content, after 5 or 90 min in the presence of 8.3 mM glucose, was higher than that observed for islets kept at 2.8 mM glucose and increased further at 16.7 mM glucose. After 5 min of incubation, the Ca-45 uptake by LPD islets in the presence of 8.3 mM glucose was slightly higher than basal values (2.8 mM glucose); however, no further increase in the Ca-45 uptake was noticed at 16.7 mM glucose. In LPD islets a significant increase in Ca-45 uptake over basal values was registered only at 16.7 mM glucose, after 90 min of incubation. These data indicate that the poor secretary response to glucose observed in islets from LPD rats may be related to a defect in the ability of glucose to increase Ca2+ uptake and/or to reduce Ca2+ efflux from beta-cells.

Short-term soil CO2 emission after conventional and reduced tillage of a no-till sugar cane area in southern Brazil

The impact of tillage systems on soil CO2 emission is a complex issue as different soil types are managed in various ways, from no-till to intensive land preparation. In southern Brazil, the adoption of a new management option has arisen most recently, with no-tillage as well as no burning of crops residues left on soil surface after harvesting, especially in sugar cane areas. Although such practice has helped to restore soil carbon, the tillage impact on soil carbon loss in such areas has not been widely investigated. This study evaluated the effect of moldboard plowing followed by offset disk harrow and chisel plowing on clay oxisolCO(2) emission in a sugar cane field treated with no-tillage and high crop residues input in the last 6 years. Emissions after tillage were compared to undisturbed soil CO2 emissions during a 4-week period by using an LI-6400 system coupled to a portable soil chamber. Conventional tillage caused the highest emission during almost the whole period studied, except for the efflux immediately following tillage, when the reduced plot produced the highest peak. The lowest emissions were recorded 7 days after tillage, at the end of a dry period, when soil moisture reached its lowest rate. A linear regression between Soil CO2 effluxes and soil moisture in the no-till and conventional plots corroborate the fact that moisture, and not soil temperature, was a controlling factor. Total soil CO2 loss was huge and indicates that the adoption of reduced tillage would considerably decrease soil carbon dioxide emission in our region, particularly during the summer season and when growers leave large amounts of crop residues on the soil surface. Although it is known that crop residues are important for restoring soil carbon, our result indicates that an amount equivalent to approximately 30% of annual crop carbon residues could be transferred to the atmosphere, in a period of 4 weeks only, when conventional tillage is applied on no-tilled soils. (c) 2005 Elsevier B.V. All rights reserved.

PROLACTIN INDUCES MATURATION OF GLUCOSE SENSING MECHANISMS IN CULTURED NEONATAL RAT ISLETS

The effects of PRL treatment on insulin content and secretion, and Rb-86 and Ca-45 fluxes from neonatal rat islets maintained in culture for 7-9 days were studied. PRL treatment enhanced islet insulin content by 40% and enhanced early insulin secretion evoked by 16.7 mm glucose. Insulin release stimulated by oxotremorine-M, a muscarinic agonist, in the presence of glucose (8.3 or 16.7 mm) was unchanged by PRL treatment. However, PRL treatment potentiated phorbol 12,13-dibutyrate-stimulated insulin secretion in the presence of the above glucose concentrations. PRL treatment potentiated the reduction in Rb-86 efflux induced by glucose or tolbutamide and enhanced the increase in Rb-86 efflux evoked by diazoxide. PRL treatment slightly potentiated the increment in Ca-45 uptake induced by high concentrations of K+, but failed to affect the increment evoked by 16.7 mm glucose. Since glucose-induced Ca-45 uptake was not affected by PRL, we suggest that the enhancement in first phase insulin secretion evoked by glucose in the PRL-treated islets occurs at a step in the secretory process that may involve protein kinase-C. These data further support observations that PRL treatment increases islet sensitivity to glucose.

Dehydromonocrotaline induces cyclosporine A-insensitive mitochondrial permeability transition/cytochrome c release

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Using Efinol (R) L during transportation of marbled hatchetfish, Carnegiella strigata (Gunther)

The objective of this experiment was to test the efficacy of a probiotic (Efinol (R) L) during transportation of marbled hatchetfish, Carnegiella strigata. Wild specimens were captured from a small stream and transported for 24 h in plastic fish boxes with a probiotic (10 mg L-1) and probiotic-free water. The boxes were sampled at 3. 12 and 24 h of transport. At the end of the experiment, the survival rate was close to.100%) in both treatments. Dissolved oxygen diminished with time in both treatments, but the probiotic group had significantly higher levels. Conductivity. pH and ammonia increased significantly during the transport. demonstrating higher levels in the probiotic-free group. Fish from both treatments presented very high net Na+ and K+ effluxes after 3 h of transport. At 24 h, net K+ effluxes in fish of the probiotic treatment reached values close to zero and a significantly lower Na+ efflux was observed. Cortisol levels in both treatments at 3 and 12 h were significantly higher than that in control samples. Higher body cortisol levels were observed in the probiotic-frec group than that in the probiotic group at 3 and 12 h. The results demonstrate that addition of a probiotic during fish transport improves water quality and leads to fish presenting a lower stress response intensity.

INGAP-PP up-regulates the expression of genes and proteins related to K-ATP(+) channels and ameliorates Ca2+ handling in cultured adult rat islets

Islet Neogenesis Associated Protein (INGAP) increases pancreatic beta-cell mass and potentiates glucose-induced insulin secretion. Here, we investigated the effects of the pentadecapeptide INGAP-PP in adult cultured rat islets upon the expression of proteins constitutive of the K-ATP(+) channel, Ca2+ handling, and insulin secretion. The islets were cultured in RPMI medium with or without INGAP-PP for four days. Thereafter, gene (RT-PCR) and protein expression (Western blotting) of Foxa2, SUR1 and Kir6.2, cytoplasmic Ca2+ ([Ca2+](i)), static and dynamic insulin secretion, and Rb-86 efflux were measured. INGAP-PP increased the expression levels of Kir6.2, SUR1 and Foxa2 genes, and SUR1 and Foxa2 proteins. INGAP-PP cultured islets released significantly more insulin in response to 40 mM KCl and 100 mu M tolbutamide. INGAP-PP shifted to the left the dose-response curve of insulin secretion to increasing concentrations of glucose (EC50 of 10.0 +/- 0.4 vs. 13.7 +/- 1.5 mM glucose of the controls). It also increased the first phase of insulin secretion elicited by either 22.2 mM glucose or 100 mu M tolbutamide and accelerated the velocity of glucose-induced reduction of Rb-86 efflux in perifused islets. These effects were accompanied by a significant increase in [Ca2+](i) and the maintenance of a considerable degree of [Ca2+](i) oscillations. These results confirm that the enhancing effect of INGAP-PP upon insulin release, elicited by different secretagogues, is due to an improvement of the secretory function in cultured islets. Such improvement is due, at least partly, to an increased K-ATP(+) channel protein expression and/or changing in the kinetic properties of these channels and augmented [Ca2+](i) response. Accordingly, INGAP-PP could potentially be used to maintain the functional integrity of cultured islets and eventually, for the prevention and treatment of diabetes. (C) 2008 Elsevier B.V. All rights reserved.

Detection of P. aeruginosa harboring bla(CTX-M-2), bla(GES-1) and bla(GES-5), bla(IMP-1) and bla(SPM-1) causing infections in Brazilian tertiary-care hospital

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Long-term effect of prolactin treatment on glucose-induced insulin secretion in cultured neonatal rat islets

Insulin secretion and 45SCa2+ uptake and efflux were studied in neonatal rat islets maintained in culture for 7 or 19 days in the absence or presence of prolactin (PRL). Insulin secretion in response to glucose (G), leucine (Leu), arginine (Arg) and carbachol (Cch) was augmented after 7 and 19 days in culture, compared to basal secretion (G 2.8 mM), in both PRL- treated and control islets. However, the increase in insulin secretion induced by the above secretagogues was higher in islets cultured in the presence of PRL for 19 days. In PRL-treated islets, the 45Ca2+ content after a 5 min incubation in the presence of G, Leu, Arg and Cch was significantly higher than the control only in islets cultured for 19 days. Except with Arg, the 45Ca2+ uptake in PRL-treated islets after a 90 min incubation was also significantly higher than the control only in islets cultured for 19 days. Finally, Leu-induced alterations in the 45Ca2+ efflux were higher in PRL-treated than in control islets cultured for 7 or 19 days. In the absence of external Ca2+, the reduction in 45Ca2+ efflux induced by glucose was also significantly higher in PRL-treated than in control islets. This effect was slightly potentiated after 19 days in culture. These data further support the hypothesis that PRL treatment enhances maturation of the secretory mechanism in neonatal islets. This effect can be potentiated even more if the treatment is prolonged.

Efeito de um novo produto fermentado de soja sobre os lípides séricos de homens adultos normocolesterolêmicos

This study was undertaken to verify the effect of a daily intake of a new fermented soy milk produced with Enterococcus faecium and Lactobacillus jugurti on the serum lipid levels in normocholesterolemic middle-aged men. The study was randomized, double-blind and placebo-controlled and was performed for a period of 6 weeks. Forty-four normocholesterolemic healthy, male volunteers, aged 40-55 years old were randomly separated in two groups: The F-group received 200 ml of the fermented product daily and the P-group received 200 ml of placebo (chemically fermented). The blood samples were drawn initially and after 3 and 6 weeks and serum values for total cholesterol, HDL-cholesterol and triglyceride were determined. The LDL-cholesterol value was estimated. No significant changes in the fermented group (F) were observed for total cholesterol, LDL-cholesterol or triglyceride levels, while the HDL-cholesterol level was significantly higher (p≤ 0,05) after 6 weeks. The total cholesterol and LDL-cholesterol levels were significantly higer (p≤ 0,05) in the placebo group (P), but no changes were found for the HDL-cholestrol and triglyceride levels during the experimental period. In conclusion, the intake of 200 ml/day of the fermented soy milk, produced with E.faecium and L. jugurti, for 6 weeks, did not affect the serum total cholesterol and LDL-cholesterol, and led an increase of 10% in the HDL-cholesterol level.

ZmPUMP encodes a fully functional monocot plant uncoupling mitochondrial protein whose affinity to fatty acid is increased with the introduction of a His pair at the second matrix loop

Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins that uncouple respiration from ATP synthesis. In this study, cDNA encoding maize uncoupling protein (ZmPUMP) was expressed in Escherichia coli and recombinant ZmPUMP reconstituted in liposomes. ZmPUMP activity was associated with a linoleic acid (LA)-mediated H+ efflux with Km of 56.36 ± 0.27 μM and Vmax of 66.9 μmol H+ min-1 (mg prot)-1. LA-mediated H+ fluxes were sensitive to ATP inhibition with Ki of 2.61 ± 0.36 mM (at pH 7.2), a value similar to those for dicot UCPs. ZmPUMP was also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in plant UCPs. ZmPUMP with introduced His pair (Lys155His and Ala157His) displayed a 1.55-fold increase in LA-affinity while its activity remained unchanged. Our data indicate conserved properties of plant UCPs and suggest an enhancing but not essential role of the histidine pair in proton transport mechanism. © 2006 Elsevier Inc. All rights reserved.