miR-143 decreases prostate cancer cells proliferation and migration and enhances their sensitivity to docetaxel through suppression of KRAS

As there is increasing evidence that Rho-Rho kinase (ROCK) pathway plays an important role in the proliferation and contraction in many tissues, we investigated the contractile role of a ROCK inhibitor, fasudil, and the distribution of RhoA, RhoB, RhoC, ROCK1, and ROCK2 in the rat prostate. Twelve-week-old Sprague-Dawley rat prostate was used in this study. Rat prostatic contractile responses induced by carbachol and norepinephrine were investigated in organ bath studies without or with 10(-7), 10(-6), and 10(-5) M of a non-selective ROCK inhibitor, fasudil. Immunoblot analysis and immunohistochemical staining were performed to investigate the participation levels of RhoA, RhoB, RhoC, ROCK1, and ROCK2. The E(max) values induced by carbachol and norepinephrine were similar in the rat prostate. Fasudil significantly inhibited carbachol- or norepinephrine-induced prostatic contractions in a dose-dependent manner. Fasudil 10(-5) M reduced the initial prostatic contraction (without fasudil) to 56.7 ± 5.9% for carbachol and to 45.7 ± 12.3% for norepinephrine. Amounts of RhoA, RhoB, RhoC, ROCK1, and ROCK2 were detected by immunoblot analysis in the prostate. Immunohistochemical study revealed that RhoA, RhoB, RhoC, ROCK1, and ROCK2 were all positive in the prostatic smooth muscle, while there were some differences of distributions of Immunoreactivities between these enzymes in the prostatic glandula. Our data indicated that rat prostate contains RhoA, RhoB, RhoC, ROCK1, and ROCK2, which play an important role in the autonomic nerve-mediated contractile responses in the prostate.     

Leptin enhances feeding suppression and neural activation produced by systemically administered bombesin

Leptin amplifies feeding inhibition and neural activation produced by either cholecystokinin or intragastric preloads, suggesting that leptin may increase the efficacy of gastrointestinal meal-related signals. To determine whether leptin would similarly potentiate the feeding inhibitory actions of another putative satiety peptide, we evaluated the effects of third ventricular leptin administration on food intake and c-Fos activation in response to systemically administered bombesin (BN). Leptin (3.5 microg) was administered 1 h before either 0.9% saline or BN (0.32 and 1.0 nmol/kg) followed by 30-min access to Ensure liquid diet. Although neither leptin nor 0.32 nmol/kg BN alone suppressed Ensure intake, the combination reduced intake by 28%. The higher BN dose (1.0 nmol/kg) produced a significant suppression by itself but was further enhanced in the presence of leptin. Consistent with the behavioral results, c-Fos activation in the nucleus of the solitary tract was increased by combined dosages of leptin and 0.32 nmol/kg BN beyond the individual response to either peptide. In the presence of leptin, BN produced a 3.4- to 5.2-fold increase in the number of c-Fos-positive cells in the nucleus of the solitary tract compared with when BN was given alone. These data provide further support for the hypothesis that the effect of leptin on food intake may be mediated, in part, by modulating meal-related satiety signals.     

Expression of the Arabidopsis metallothionein 2b enhances arsenite sensitivity and root to shoot translocation in tobacco

We expressed the AtMT2b gene under the 35 S cauliflower mosaic virus promoter in Nicotiana tabacum (Sr1), using leaf disc transformation. Arsenite tolerance and uptake, as well as arsenite-induced phytochelatin (PC) accumulation in roots were measured in transgenic lines, and compared to untransformed (‘wild type’) tobacco. Measured after 5 days of exposure, arsenite tolerance was slightly but significantly decreased in the transgenic lines compared to wild type. The highest AtMT2b expressing line exhibited a significantly decreased arsenic accumulation in roots, but an increased accumulation in shoots, while the total amount of arsenic taken up remained unchanged, suggesting that AtMT2b expression enhanced the arsenic root to shoot transport. The same transformant line also exhibited a decreased rate of phytochelatin accumulation in the roots, but the phytochelatin-SH to As molar ratio was higher than in wild type, suggesting that the lower arsenite tolerance in the transformant lines was not due to a potential shortage of cysteine for PC synthesis, imposed by expression of the transgene.     

Leptin deficiency enhances myocardial necrosis and lethality in a murine model of viral myocarditis

To investigate the role of leptin in the development of viral myocarditis and cardiac necrosis, we used a murine model of viral myocarditis. We intraperitoneally injected encephalomyocarditis virus (500 plaque-forming units/mouse) for wild type C57 BL/6 mice (WT) and leptin-deficient ob/ob mice (OB) (n = 20 for each). Ten-day survival rate was 25% in OB, whereas it was 95% in WT. Heart weights on day 10 were significantly elevated in OB compared with those in WT (107.2 +/- 9.4 vs. 96.6 +/- 7.9 mg, n = 4 for each). Thymus weights were significantly diminished in OB compared with those in WT on days 6 and 10. Histological score (grade 1 to 4 according to the size of involved area) for myocardial necrosis were significantly higher in OB than in WT (1.5 +/- 0.5 vs. 0.8 +/- 0.5, n = 4 for each). On day 4, viral titer in hearts was significantly elevated in OB compared with that in WT (3.3 +/- 0.5 vs. 1.9 +/- 0.2 TCID50/mg, n = 3 for each). Comparative expression of TNF-alpha mRNA in hearts from OB were significantly increased compared with those in WT on day 7 (n = 3 for each). Natural killer cell activities in spleens from OB were significantly lower than from WT on day 4 (27 +/- 5 vs. 42 +/- 8%, n = 4 for each). Thus, leptin deficiency could enhance severity of myocardial necrosis and mortality due to viral myocarditis.     

Feeding a corn oil/sucrose-enriched diet enhances steatohepatitis in sedentary rats

The current study investigated the combined effects of feeding a high-fat/high-sucrose (HF/HS) diet to rodents rendered sedentary via hindlimb unloading (HU). For 3 wk before HU, male Wistar rats were fed chow or a diet in which 32% of calories were derived from corn oil fat and 48% of calories from sucrose. Feeding continued during an additional 3-wk period of HU. Subsequently, blood samples were collected for determination of circulating leukocyte counts, insulin levels, and portal vein endotoxin. Inflammation, necrosis, and steatosis were assessed in formalin-fixed liver sections. No biochemical or histological evidence of injury was observed in control rats fed chow or HF/HS. HU increased circulating neutrophils and resulted in hyperinsulinemia. Mild hepatic fat accumulation and minimal focal necroinflammation were observed in this group. Feeding HF/HS during HU exacerbated hyperinsulinemia, hepatic steatosis, Kupffer cell content, and cytokine expression. Significant portal endotoxemia was noted in HU rats but was not influenced by HF/HS diet. On the other hand, feeding HF/HS significantly enhanced lipid peroxidation end products in liver of HU rats by approximately threefold compared with chow-fed rats. In summary, these findings demonstrate that feeding a high-calorie diet potentiates steatosis and injury in sedentary HU rats. Mechanisms underlying enhanced injury most likely involved lipid peroxidation. Importantly, these findings suggest that dietary manipulation combined with physical inactivity can be used to model steatohepatitis.     

Sodium deficiency enhances the behavioral responses to centrally administered vasopressin in rats

The ability of sodium deficiency to stimulate vasopressin (VP) release was examined by determining if sodium deficiency sensitizes the animal to the behavioral disruption caused by intraventricular injections of VP. In sodium-replete rats, intraventricular injections of 50 ng VP on Day 1 had no effect on behavior, but this dose elicited abnormal behaviors (barrel rolls, hind-limb extensions) when administered on Day 2, indicating a sensitization phenomenon. In separate experiments, the first intraventricular injection of 50 ng VP in sodium-deficient but not in sodium-replete rats also elicited barrel rotations followed by hind-limb extension. Intraventricular injection of VP also disrupted motor behavior in sodium-replete rats that had multiple prior experiences with sodium deficiency but not in naive rats. These results show that sodium deficiency can mimic the effect of central injections of VP in sensitizing the brain to the behavioral effects of exogenous VP. This suggests that sodium deficiency induces the central release of VP.     

Zeaxanthin deficiency enhances the high light sensitivity of an ascorbate-deficient mutant of Arabidopsis

The ascorbate content of plants is usually increased in high light (HL), implying a function for ascorbate in the acclimation of plants to HL. Nevertheless, the importance of ascorbate in HL acclimation has not yet been tested directly. Here, we report on the acclimation process of an ascorbate-deficient Arabidopsis mutant to HL. The mutant vtc2 has only 10% to 30% of wild-type levels of ascorbate, and it is also slightly deficient in feedback de-excitation (qE), a photoprotective mechanism that causes the dissipation of excess light as heat. The vtc2 mutant was unable to acclimate to HL, when transferred from low light to HL. Its mature leaves bleached, and it showed an increased degree of lipid peroxidation and photoinhibition. In parallel, we tested the photosensitivity of an ascorbate-deficient xanthophyll cycle mutant, vtc2npq1, which also lacks zeaxanthin and nearly all qE. The double mutant bleached sooner and had higher degrees of lipid peroxidation and photoinhibition than the vtc2 mutant. This was in contrast to the npq1 single mutant that showed only slight deviations from the wild-type phenotype under the conditions used. These results demonstrate the antioxidant role of ascorbate in the acclimation process to HL and point to the relative importance of ascorbate in comparison with other photoprotective processes, such as specific xanthophylls or feedback de-excitation. The results also provide further support for the proposed role of zeaxanthin as an antioxidant and lipid stabilizer.     

Influence of maternal mineral deficiency on the hepatic metallothionein and zinc in newborn rats

The effects of maternal Zn, Cu, or Fe deficiencies during late gestation on hepatic levels of metals and metallothionein (MT) and the binding of Zn and Cu to protein fractions were investigated in newborn rats. Timed pregnant rats were fed one of the following diets: Zn deficient (Zn-D), Cu-D, Fe-D, or control from day 12 of gestation until birth. The specific nutritional deficiency status of the dams was confirmed by low plasma levels of the deficient metal. Livers from pups were analyzed for MT, metal content, and metal-protein binding. Maternal Zn-D resulted in a greater than 50% reduction of hepatic MT levels in pups, whereas Cu-D and Fe-D had no significant effects. Pups in each deficient group showed a significant decrease in the hepatic levels of the respective metals. Fractionation of hepatic cytosols from the pups by Sephadex G-75 gel filtration showed that in both Fe-D and Cu-D pups the respective metals were depleted from the high molecular weight protein fraction, whereas in Zn-D pups the Zn was depleted mainly from the MT fraction (Ve/V0 approximately 2). Incorporation of [35S] cysteine into MT fractions was significantly lower in Zn-D pups as compared with control pups. These results indicate that there is a specific effect of the maternal Zn-D on the hepatic storage of Zn as MT in newborn rats.     

Induced suppression of the efferent phase of contact sensitivity in rats

A study of the immunosuppressive systems of rats has been conducted with special attention to whether suppressor cells can be induced to down-regulate the efferent limb of contact sensitivity. Contact sensitivity (CS)1 was induced in DA rats 5 days after immunization with trinitrochlorobenzene (TNCB). Intravenous pretreatment of naive rats with TNP-coupled syngeneic spleen cells 7 days before sensitization suppressed the induction of CS by 60%. Suppression of the inductive phase of CS could be transferred adoptively into syngeneic rats with spleen cells of such tolerized animals. Cell fractionation studies showed the OX8+ (CD8) T cell population (cytotoxic/suppressor) was responsible for the suppression in the afferent phase of CS. Such cells were incapable of suppressing preexisting CS. To investigate whether suppression could be induced for the efferent phase, spleen and peritoneal exudate cells (PEC) from rats tolerized by administering TNP-spleen cells iv plus epidermal paintings with TNCB were adoptively transferred into recipients sensitized 4 days earlier. Both spleen cells and PEC suppressed the efferent phase of CS but PEC did so more efficiently. Separation of splenic cells revealed the suppressors to be CD8+ T cells. Furthermore, separation of PEC into plastic adherent and nonadherent cells showed the nonadherent (T cell enriched) cells to be noneffective alone. The adherent subpopulation conveyed suppression but did so more effectively upon addition of the T cells. Thus, T cells and macrophages may operate in concert to achieve suppression of the efferent limb of CS. PEC from tolerized rats suppressed performed CS of any specificity but only after the suppressor cells were triggered with the same antigen that induced them. Since both the afferent and efferent phases of CS have now been shown to be suppressable, two separate suppressor mechanisms may be operable in rats.     

Leptin responses to glucose infusions in obesity-prone rats

The secretion of leptin is dually regulated. In fasting animals, plasma leptin concentrations reflect body fat stores, whereas the incremental leptin response to fasting or refeeding most likely reflects insulin-mediated energy flux and metabolism within adipocytes. Impaired secretion of leptin in either pathway could result in obesity. We therefore measured plasma leptin concentrations in fasted animals and plasma leptin concentrations after an intravenous glucose infusion in a rat model of obesity. Young Sprague-Dawley (S-D) and Fischer 344 (F344) rats had similar percent body fat and fasting glucose and fasting leptin concentrations. However, F344 animals had higher insulin concentrations and leptin responses to intravenous glucose than did the S-D animals. The animals were then fed a control or high-fat diet for 6 wk. High-fat fed animals gained more weight and body fat than did the control fed animals. Control and high-fat fed F344 animals gained approximately 40% (P < 0.0001) more weight and >100% (P < 0.01) more body fat than did the S-D animals. Fasting leptin concentrations and leptin concentrations after intravenous glucose infusions and feeding were more than double (P < 0.05) in F344 animals compared with S-D animals. Whether an animal is fed a control or high-fat diet had little effect on the leptin response to intravenous glucose. In conclusion, young, lean F344 animals, before the onset of obesity, demonstrated a greater acute leptin response to intravenous glucose than similarly lean S-D animals. After a 6-wk diet, F344 animals had a greater percent increase in body weight and insulin resistance and exhibited higher fasting leptin concentrations and a greater absolute leptin response to intravenous glucose compared with the S-D animals. The chronic diet (control or high fat) had little impact on the acute leptin response to intravenous glucose. F344 animals exhibit leptin resistance in young, lean animals and after aging and fat accumulation.     

Interferon-gamma enhances target cell sensitivity to monocyte killing

The mechanism of human peripheral blood monocyte-mediated cytotoxicity was investigated using the HT-29 human colon adenocarcinoma line, A673 human rhabdomyosarcoma line, and A375 human melanoma line as target cells. Pretreatment of these target cells with 100 U/ml of recombinant human interferon (IFN)-gamma for 48 hr increased their susceptibility to monocyte killing. Increased susceptibility to the lytic action was particularly pronounced at low effector/target cell ratios. Unlike IFN-gamma human IFN-alpha did not potentiate monocyte cytotoxicity, and pretreatment of HT-29 with IFN-alpha also had virtually no effect on their susceptibility to monocyte killing. However, IFN-gamma appeared to prime either monocytes or target cells to become responsive to IFN-alpha. Our data suggest that IFN-gamma can promote the killing of tumor cells by monocytes through two separate actions, one on the monocyte and one on the target cell.     

Effect of dietary zinc deficiency on metallothionein concentration of epididymal luminal fluids of weanling Wistar albino rats

Metallothionein (NIT) and zinc concentrations have been estimated in luminal fluids of caput/corpus and cauda epididymis and serum of zinc deficient (ZD), pairfed (PF) and control--ad libitum fed (ZC) groups of Wistar rats. MT decreased significantly in luminal fluids of caput corpus and cauda epididymis and serum of zinc deficient rats as compared to their respective controls. However, the decrease was non-significant in luminal fluids of corpus epididymis and serum of 4-weeks zinc deficient animals as compared to their control. Zinc levels also declined significantly in luminal fluids of epididymis and serum of zinc deficient rats as compared to their respective pairfed and control groups. Thus zinc deficiency state reduces zinc and MT concentrations in luminal fluid of epididymis and serum.     

Leptin deficiency leads to the regulation of kinin receptors expression in mice

Kinins are vasoactive and pro-inflammatory peptides generated by the cleavage of the kininogen by kallikreins. Two kinin receptors have been described and denominated B1 and B2. Obesity frequently accompanies other pathologies, such as diabetes and hypertention. The clustering of these pathologies is usually known as "metabolic syndrome". Mice lacking leptin gene (ob/ob) are severely obese and hyperphagic. Using quantitative RT-PCR analysis of B1 and B2 mRNAs expression, we described for the first time a correlation between the kallikrein-kinin system (KKS) and severe obesity in mice. The ob/ob mice presented lower expression of B2 mRNA in the white adipose tissue (WAT) and hypothalamus, both primary sites for neuroendocrine regulation of the energetic metabolism. B1 mRNA, however, is overexpressed in these tissues of ob/ob mice. An upregulation of the B1 mRNA has also been seen in liver, abdominal aorta and stomach fundus. However, different from the lean mice, the expression of the B1 mRNA in brown adipose tissue (BAT) and heart is completely abolished. Our data show that kinin receptors are differently modulated in distinct tissues in obesity. These findings suggest a connection between the KKS and obesity, and suggest that kinin receptors could be involved in the ethiopathogenesis of the metabolic syndrome.     

Quercetin cumulatively enhances copper induction of metallothionein in intestinal cells

Wilson’s disease, a genetic copper-overload condition, is currently treated with zinc because of the ability of zinc to induce metallothionein. We are interested in nonmetal chemicals that may alter intestinal copper metabolism and thus help to alleviate copper toxicity. Previously, we have shown that quercetin, a dietary flavonoid, can chelate copper. This study further examined the interaction of quercetin and copper in intestinal epithelial cells. We found that quercetin enhanced metallothoinein induction by copper and the effect was dose dependent. Quercetin also exerted a cumulative effect after repeated exposure. Repeated low-dose treatment (3–10 μM) of cells with quercetin can lead to the same effect on metallothoinein as one higher concentration treatment (100 μM). This property of quercetin is distinct from its chemical interaction with copper, but both can contribute to a reduction of copper toxicity. Among other flavonoids tested, two other copper chelators, catechin and rutin, did not increase copper induction of metallothionein, whereas genistein, an isoflavone that does not interact with copper chemically, increased copper induction of metallothionein. The effect of quercetin on copper metabolism is unique. Quercetin decreased zinc-stimulated metallothionein expression and had no effect on the cadmium induction of metallothionein. The clinical application of our observation needs to be explored.     

Further studies of diabetes-prone BHE/Cdb rats: increased sensitivity to calcium ion suppression of oxidative phosphorylation

The responsiveness of hepatic mitochondria isolated from hyperthyroid and control Sprague-Dawley (SD) and diabetes-prone BHE/Cdb rats was studied. Hyperthyroidism was induced through the addition of thyroxine (T(4)) to the diet (2 mg/kg of diet). Oxidative phosphorylation (OXPHOS) with the addition of adenosine diphosphate (ADP) or an 8:1 mixture of adenosine monophosphate (AMP):ADP was studied. Dose response curves of state 3 and state 4 respiration, respiratory control (RC) ratio, and ADP:O ratio to calcium levels (0-7.5 microm) were generated. Mitochondria from BHE/Cdb rats were more sensitive to the addition of calcium than mitochondria from SD rats, as judged by losses in OXPHOS. T(4) treatment potentiated this strain difference and we conclude that the diabetes phenotype in the BHE/Cdb rat is probably related not only to the previously described mutation in the F(O)ATPase but also to a defect in the efflux of the calcium ion that, in turn, affected the regulation of OXPHOS.     

Gastrin enhances gastric mucosal integrity through cyclooxygenase-2 upregulation in rats

Gastrin, PGs, and growth factors have important roles in maintaining gastrointestinal mucosal integrity. Cyclooxygenases (COX-1 and COX-2) are the key enzymes involved in PG synthesis. This study aimed to clarify the mechanisms of gastric mucosal protection by gastrin. Fasted rats were administered subcutaneous gastrin 17 with or without gastrin receptor antagonist YM022 pretreatment. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) and COX-2 expression were examined using Western blot analysis. Another series of experiments investigated 1) PGE(2) levels in gastric mucosa, 2) the protective action of gastrin against gastric damage by acidified ethanol, 3) the effects of a specific HB-EGF-neutralizing antibody on gastrin-induced COX-2 expression, and 4) the effects of a specific COX-2 inhibitor NS-398 on PGE(2) synthesis and the mucosal protection afforded by gastrin. Gastrin dose-dependently increased HB-EGF, COX-2 expression, and PGE(2) levels and reduced gastric damage. However, pretreatment with YM022 dose-dependently abolished such effects of gastrin. A specific HB-EGF- neutralizing antibody and an EGF receptor inhibitor decreased gastrin-induced COX-2 expression. NS-398 blocked gastrin-induced PGE(2) synthesis and mucosal protection. In conclusion, this study demonstrates that gastrin enhances gastric mucosal integrity through COX-2, which is partially mediated by HB-EGF, and PGE(2) upregulation in rats.     

Marginal iron deficiency enhances liver triglyceride accumulation in rats fed a high-sucrose diet

ABSTRACT

We investigated whether marginal iron-deficiency (MID) without anemia influences liver lipid accumulation in rats. Ingestion of a MID diet in which the iron concentration was half of AIN-93 formulation (iron-adequate, IA) for 3 weeks decreased liver iron concentration without anemia. We then evaluated the influence of the MID diet on liver lipid accumulation in combination with a high-sucrose (HS) diet and confirmed that the HS-MID diet successfully decreased liver iron concentration without anemia. Additionally, a significant increase in liver triglyceride concentration was found, accompanied by upregulation of hepatic fatty acid synthase expression in the rats fed the HS-MID diet compared to those in the rats fed an HS-IA diet, although no difference was observed in plasma transaminase activity and hepatic interleukin-1β expression. These results suggest that MID enhances de novo lipid synthesis via upregulation of lipogenic gene expression in combination with sucrose in the diet.

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; HS, high sucrose; IA, iron adequate; ID, iron deficiency; MID, marginal irondeficiency; NAFLD, non-alcoholic fatty liver disease