Comparison of the mutagenicity of aristolochic acid I and aristolochic acid II in the gpt delta transgenic mouse kidney

Idiopathic pulmonary fibrosis (IPF) is the most common manifestation of telomere-mediated disorders. Germline mutations in the essential telomerase genes, hTERT and hTR, are the causal genetic defect in up to one-sixth of pulmonary fibrosis families. The presence of telomerase mutations in this subset is significant for clinical decisions as affected individuals can develop extra-pulmonary complications related to telomere shortening such as bone marrow failure and cryptogenic liver cirrhosis. There is also evidence that IPF is an ancestral manifestation of autosomal dominant telomere syndromes where, with successive generations, the disease evolves from pulmonary fibrosis into a bone marrow failure-predominant disorder, defining a unique form of genetic anticipation. Here I review the significance of telomere defects for understanding the genetics, disease patterns and pathophysiology of IPF. The importance of this diagnosis for patient care decisions will also be discussed.     

Effects of arachidonic acid on leptin secretion and expression in primary cultured rat adipocytes

Leptin, a hormone produced in adipocytes, is a key signal in the regulation of food intake and energy expenditure. Several studies have suggested that leptin can be regulated by macronutrients intake. Arachidonic acid is a dietary fatty acid known to affect cell metabolism. Controversial effects of this fatty acid on leptin have been reported. The aim of this experimental trial was to evaluate the effect of the arachidonic acid on basal and insulin-stimulated leptin secretion and expression in isolated rat adipocytes. Because insulin-stimulated glucose metabolism is an important regulator of leptin expression and secretion by the adipocytes, the effects of the arachidonic acid on indices of adipocyte metabolism were also examined. Isolated adipocytes were incubated with arachidonic acid (1-200 microM) in the absence and presence of insulin (1.6 nM). Leptin secretion and expression, glucose utilization and lactate production were determined at 96 h. The arachidonic acid (200 microM) inhibited both the basal and insulin stimulated leptin secretion and expression. Glucose utilization was not affected by the acid. Basal lactate production was increased by the fatty acid at the highest concentration used (200 microM), however lactate production in presence of insulin was not modified. Finally, the percentage of glucose carbon released as lactate was significantly increased (200 microM). These results suggest that the inhibitory effect of the arachidonic acid on leptin secretion and expression may be due, al least in part, to the increase in the anaerobic utilization of glucose.     

Plasma hyperosmolality stimulates leptin secretion acutely by a vasopressin-adrenal mechanism

Glucose administration to rodents acutely stimulates leptin secretion. To investigate the mechanism, rats were infused intravenously with various concentrations of glucose, and plasma leptin concentrations were measured with time. The osmolality of the infusates was equalized with various concentrations of carbohydrates that are not metabolized. Hyperosmolar glucose stimulates leptin secretion in a dose-dependent manner, with peak plasma leptin concentrations occurring approximately 3 h after the end of the glucose infusion. Hypertonic infusions of galactose, mannitol, and sodium chloride independently stimulate leptin secretion with approximately one-half the strength of equivalent osmolar concentrations of glucose. Peak plasma leptin concentrations occur approximately 4 h after the end of the hypertonic solution infusion. Hypertonic solutions of mannitol do not stimulate leptin secretion in vasopressin-deficient or in adrenalectomized animals. In conclusion, intravenous infusions of hypertonic glucose and hypertonic mannitol independently stimulate leptin secretion. Hyperosmolality stimulates leptin secretion by a vasopressin-adrenal mechanism.     

Zinc deficiency reduces leptin gene expression and leptin secretion in rat adipocytes

The present study was conducted to measure ob mRNA abundance in the zinc-deficient (ZD) rats and the secretion of leptin from adipose tissue obtained from ZD, zinc-adequate (ZA), and pair-fed (PF) rats. It was found that ob mRNA abundance was greatest (P < 0.05) in adipose tissue obtained from ZA and PF rats. Ob mRNA abundance was similar in PF and ZD rats. To study leptin secretion from adipose tissue in a cell culture model, a method was developed to use excised epididymal adipose tissue from ZD, ZA, and PF rats. Tissue was incubated in Opti-modified Eagle's medium (MEM) cell culture medium in which concentrations of zinc and insulin were manipulated. It was observed that leptin secretion was higher (P < 0.05) in adipose tissue obtained from ZA than ZD and PF rats. Secretion of leptin was higher in adipose tissue of PF than ZD rats (P < 0.05). Surprisingly, media zinc content in this ex vivo model tended to suppress secretion of leptin. This suppression seems to be zinc specific and might be caused by the sequestration of insulin in the culture medium. Our results indicate that the reduction in serum leptin observed in ZD rats is likely caused by not only a reduction in body fat, but also by a decrease in leptin synthesis and secretion per gram of adipose tissue. Taking these results into account along with a prior study (1), it is possible that even a marginal zinc deficiency could affect leptin secretion and serum leptin concentrations. Impaired leptin secretion caused by zinc deficiency might be one factor contributing to hypogonadism observed in zinc deficiency.     

Regulation of leptin gene expression and secretion by steroid hormones.

Previous work has shown that 17 beta-estradiol is the primary ovarian signal regulating body weight and adiposity, although its mechanisms of action remain unclear. We hypothesized that 17 beta-estradiol could enhance leptin levels as a mechanism of its anorectic effects. Administration of 5 microg 17 beta-estradiol subcutaneously (s.c.) for 2 days significantly elevated leptin mRNA levels in adipose tissue as compared to vehicle controls (P < 0.003). A time-course administration of estrogen showed increased mRNA levels in adipose tissue between 6 and 12 h after estrogen injection as compared to vehicle controls (P < 0.03). Corresponding to the increased leptin mRNA levels at 6 and 12 h, elevated plasma leptin levels were observed at 12 h after estrogen administration as compared to controls (P < 0.05). Administration of progesterone (1 mg/rat) after estradiol injection did not enhance the elevated leptin mRNA levels in adipose tissue. Serum leptin levels from cycling rats did not differ significantly between metestrous and proestrous animals. In conclusion, the present studies demonstrate that 17 beta-estradiol can regulate leptin gene expression and secretion in the female rat, thus providing a better understanding of the possible anorectic effect of estrogens.     

Para-aminohippuric acid secretion by the vertebrate kidney

Tubular secretory capacity has been investigated in the kidney of marine fishes Scorpaena porcus, Spicara smaris, frog Rana ridibunda and albino rat by determination of maximal transport of p-aminohippurate--TmPAH, Following values of Tmax expressed in mg/hour per 100 g of body weight were obtained: 0.73 +/- 0.17 for S. porcus, 0.92 +/- 0.09 for S. smaris, 0.93 +/- 0.10 for R. ridibunda and 15.6 +/- 1.6 for rats.     

Glutamate dehydrogenase requirement for apoptosis induced by aristolochic acid in renal tubular epithelial cells

Ingestion of aristolochic acids (AA) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adduct formation, is well-documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. Epithelial cell death is a critical characteristic of these pathological conditions. To elucidate the mechanisms of AA-induced cytotoxicity, we explored AA-interacting proteins in tubular epithelial cells (TEC). We found that AA interacts with a mitochondrial enzyme glutamate dehydrogenase (GDH) and moderately inhibits its activity. We report that AA induces cell death in GDH-knockdown TEC preferentially via non-apoptotic means, whereas in GDH-positive cells, death was executed by both the non-apoptotic and apoptotic mechanisms. Apoptosis is an energy-reliant process and demands higher adenosine 5′-triphosphate (ATP) consumption than does the non-apoptotic cell death. We found that, after AAI treatment, the ATP depletion is more pronounced in GDH-knockdown cells. When we reduced ATP in TEC cells by inhibition of glycolysis and mitochondrial respiration, cell death mode switched from apoptosis and necrosis to necrosis only. In addition, in cells incubated at low glucose and no glutamine conditions, oxaloacetate and pyruvate reduced AAI-induced apoptosis our data suggest that AAI-GDH interactions in TEC are critical for the induction of apoptosis by direct inhibition of GDH activity. AA binding may also induce changes in GDH conformation and promote interactions with other molecules or impair signaling by GDH metabolic products, leading to apoptosis.     

Regulation of adiponectin and leptin secretion and expression by insulin through a PI3K-PDE3B dependent mechanism in rat primary adipocytes

Adiponectin is intimately involved in the regulation of insulin sensitivity, carbohydrate and lipid metabolism, and cardiovascular functions. The circulating concentration of adiponectin is decreased in obesity and Type 2 diabetes. The present study attempts to elucidate the mechanisms underlying the regulation of adiponectin secretion and expression in rat primary adipocytes. The beta-agonist, isoprenaline, decreased adiponectin secretion and expression in a dose-dependent manner in primary adipocytes. Importantly, such an inhibitory effect could be blocked by insulin. The opposing effects of isoprenaline and insulin could be explained by differential regulation of intracellular cAMP levels, since cAMP analogues suppressed adiponectin secretion and expression in a fashion similar to isoprenaline, and insulin blocked the inhibitory effects of the cAMP analogue hydrolysable by PDE (phosphodiesterase). A specific PDE3 inhibitor, milrinone, and PI3K (phosphoinositide 3-kinase) inhibitors abolished the effects of insulin on adiponectin secretion and expression. In the same studies, leptin secretion and expression displayed a similar pattern of regulation to adiponectin. We conclude that insulin and beta-agonists act directly at the adipocytes in opposing fashions to regulate the production of adiponectin and leptin, and that a PI3K-PDE3B-cAMP pathway mediates the effects of insulin to restore beta-agonist/cAMP-suppressed secretion and expression of these two adipokines.     

Regulation of in vivo TSH secretion by leptin

Leptin, the product of the ob gene, is a hormone secreted by adipocytes that regulates food intake and energy expenditure. The hypothalamus-pituitary-thyroid axis is markedly influenced by the metabolic status, being suppressed during food deprivation.The aim of the present study was to assess whether leptin can act as a metabolic signal connecting the adipose tissue with the pituitary-thyroid axis. We studied the effect of leptin administration (10 microg, i.c.v.) on spontaneous TSH secretion and TSH responses to TRH in euthyroid and hypothyroid food-deprived rats. Spontaneous TSH secretion was assessed over 6 h with samples taken every 7 min. Administration of leptin to food-deprived euthyroid rats led to a reversal of the inhibitory effect exerted by fasting on spontaneous TSH secretion. This stimulatory effect of leptin on spontaneous TSH appears to be dependent on the thyroid status since it could not be observed in hypothyroid rats. This data suggests that blunted spontaneous TSH secretion in food-deprived rats is a functional and reversible state, and that the decreased leptin concentrations could be the primary event responsible for the suppression of the hypothalamic-pituitary-thyroid-axis in food-deprived rats.     

地塞米松对TRAIL双向诱导猪肾成纤维细胞增殖与凋亡作用的影响

采用MTT掺入法检测细胞活率变化,分别筛选出诱导细胞增殖与凋亡的药物浓度;半定量PCR法检测不同浓度下地塞米松对骨保护素(Osteoprotegerin,OPG)和核因子κB受体激活剂受体配体(Ligand of receptor activator of nuclear factor kappa B,RANKL)基因在mRNA水平上的调控作用;流式细胞术检测细胞周期分布和凋亡率变化,显微镜观察FRSs存活、增殖和凋亡变化.经MTT检测和浓度筛选,发现TRAIL在0.01-5 mg/L浓度范围内诱导FRSs增殖,浓度增加到10 mg/L出现凋亡趋势.地塞米松可协同TRAIL双向诱导FRSs增殖或凋亡,有效浓度约10-6-10-10 mol/L.细胞周期分析与凋亡率检测结果表明RAIL诱导FRSs增殖峰浓度为5 mg/L.TRAIL(5 mg/L)与地塞米松协同作用,细胞增殖指数(Prl.)较TRAIL5 mg/L组上升2.49%(P<0.05);TRAIL 10 mg/L组与空白对照组比较细胞增殖指数和凋亡率无显著变化,TRAIL 10 mg/L与地塞米松协同作用G0/G1期比例较TRAIL10 mg/L组增加2.36%(P<0.05),凋亡率较之上升6.79%(P<0.01).地塞米松作用下,OPG基因mRNA水平表现下调,RANKL基因则表现上调,二者均表现为剂量依赖型.综上所述,TRAIL对FRSs呈诱导增殖或凋亡的双向调控作用,并呈剂量依赖型.地塞米松可协同TRAIL对FRSs作用,该现象与TRAIL和地塞米松诱导FRSs细胞周期的改变以及地塞米松对OPG/RANK/RANKL信号通路的OPG和RANKL基因的表达调控相关.     

The interdependence of EGF-R and SGK-1 in fibronectin expression in primary kidney cortical fibroblast cells

BACKGROUND: Epidermal growth factor (EGF) has been shown to play a role in the nephromegaly and enhanced sodium reabsorption observed in diabetic nephropathy. This is recognized to be dependent on activation of serine threonine glucocorticoid kinase-1 (SGK-1). However, the roles of EGF and SGK-1 in renal fibrogenesis observed under high glucose conditions have not been established. METHODS: Primary cultures of human cortical fibroblasts (CFs) were used as the model in which to study the dependent and independent effects of high glucose, EGF and SGK-1 on the expression of the extracellular matrix protein (ECM) fibronectin. Wild type CFs expressing SGK-1, or cells in which SGK-1 was effectively silenced using siRNA methodology, were exposed to normal (5mM) or high (25mM) glucose, or EGF (10ng/ml) for 48hr and fibronectin assessed. The role of the EGF-receptor and its relationship to SGK-1 signaling was studied using concurrent treatment with PKI166, a specific inhibitor of EGF-receptor. RESULTS: Exposure of CF to high glucose and EGF increased phosphorylated EGF-R, SGK-1, and fibronectin expression in wild-type cells. Inhibition of the EGF-R reduced SGK-1 and fibronectin expression in control, and following exposure to EGF and high glucose conditions. In cells in which SGK-1 was silenced, fibronectin was reduced and there was no significant increase in pEGF-R, suggesting that SGK-1 is downstream of the EGF-R and negatively inhibits EGF-R activation. CONCLUSION: These results suggest that high glucose induced fibronectin expression is mediated through the EGF-R and downstream expression of SGK-1.     

The type II secretion system: biogenesis, molecular architecture and mechanism

Many gram-negative bacteria use the sophisticated type II secretion system (T2SS) to translocate a wide range of proteins from the periplasm across the outer membrane. The inner-membrane platform of the T2SS is the nexus of the system and orchestrates the secretion process through its interactions with the periplasmic filamentous pseudopilus, the dodecameric outer-membrane complex and a cytoplasmic secretion ATPase. Here, recent structural and biochemical information is reviewed to describe our current knowledge of the biogenesis and architecture of the T2SS and its mechanism of action.