Escherichia coli K12 does not colonize the gastrointestinal tract of Fischer-344 rats

Summary The colonizing potential ofEscherichia coli K12 containing a vector coding for somidobove (bovine somatotropin) was determined. Treated male and female Fischer-344 rats were given a single oral gavage inoculum of sucrose with/without tetracycline (15 g/ml). Untreated control animals received similar drinking water regimes. All animals survived until termination. There were no clinical signs of toxicity observed and no treatment-related effect upon body weight, food consumption, or efficiency of food utilization. Fresh fecal samples were collected from each rat every 24 h following inoculation and the population of the marked strain was quantitated until no bacterial colonies were observed for two consecutive days. While all inoculated rats were positive at 24 h, by 72 and 96 h all had become negative for the test (marked) strain, as were the corresponding control group throughout the test. The frozen stock of the marked strain used as the positive control demonstrated that the agar plates were selective for the test strain. Fourteen days following inoculation, all groups of rats were killed and the gastrointestinal tracts removed and treated to recover the marked strain. There was no evidence of the marked strain in the gastrointestinal tract of any rat from any group. Thus, theE. coli K12 host/vector system used in this experiment does not colonize the gastrointestinal tract of Fischer-344 rats.     

Behavioral and electrophysiological responses to NaCl in young and old Fischer-344 rats

This study compared both behavioral and electrophysiologicalresponses to NaCl in young and old Fischer-344 rats. These comparisonswere made in the same individuals. Preference for NaCl solutionsversus water was assessed using two-bottle preference tests.The integrated response of the chorda tympani nerve to NaClwas recorded. NaCl neural-response magnitude and correspondingbehavioral sensitivity appear to decrease with age in the Fischer-344rat at concentrations > 0.15 M and neural-response magnitudesincrease at lower concentrations.     

Catecholamine-synthesizing enzymes in paraganglia of aged Fischer-344 rats

Summary The catecholamine-synthesizing enzymes, tyrosine hydroxylase, dopamine--hydroxylase and phenylethanolamine-N-methyltransferase were examined by immunohistochemistry in hypertrophied paraganglia of aged male Fischer-344 rats. All paraganglionic cells reacted with antibodies against tyrosine hydroxylase. Dopamine -hydroxylase was identified in most paraganglionic cells, indicating that they synthesized norepinephrine. A variable number of paraganglia were positive for phenylethanolamine-N-methyltransferase, which suggested that they synthesized epinephrine. The formaldehyde-induced fluorescence method demonstrated greenish-yellow fluorescence or yellowish-brown fluorescence. The intensity of the fluorescence was in the same range as in adrenal medullary cells. The observations indicate that paraganglia are capable of synthesizing epinephrine.Dr. J.M. Stolk is the recipient of RSDA K2-00018 from the National Institute of Health.     

Hepatic macromolecular covalent binding of mononitrotoluenes in Fischer-344 rats

The mononitrotoluenes are important industrial chemicals which display isomeric specificity in their ability to induce hepatic DNA excision repair in Fischer-344 rats. Covalent binding of the structurally related hepatocarcinogen, 2,6-dinitrotoluene, to hepatic DNA is markedly decreased by prior administration of the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The objectives of this study were to determine whether hepatic macromolecular covalent binding of the mononitrotoluene isomers differed and to determine whether covalent binding of the mononitrotoluenes to hepatic DNA in vivo was decreased by inhibitors of sulfotransferase. Male Fischer-344 rats were given a single oral dose of [ring-U-14C]-2-, 3- or 4-nitrotoluene (2-, 3- or 4-NT) and killed at various times thereafter. Livers were removed and analyzed for total and covalently bound radiolabel. Maximal concentrations of total radiolabel were observed between 3 and 12 h after the dose, and there were no large differences among the 3 isomers in peak concentrations achieved. Covalent binding to hepatic macromolecules was maximal 12 h after administration for all three isomers. Thereafter, concentrations of covalently bound 2-NT-derived material were always 2-6 times higher than those of 3- or 4-NT-derived material. When DNA was isolated from livers of rats given the mononitrotoluenes 12 h previously, only 2-NT was observed to covalently bind at concentrations above the limits of detection of the assay. The covalent binding of 2-NT, but not that of 3- or 4-NT, to both total hepatic macromolecules and DNA was markedly decreased by prior administration of either PCP or DCNP. Covalent binding to hepatic DNA was decreased by greater than 96%. The results of this study correlate well with studies which have demonstrated that 2-NT, but not 3- or 4-NT, induces DNA excision repair. Furthermore, they suggest that 2-NT, like the hepatocarcinogen 2,6-dinitrotoluene, requires the action of sulfotransferase for its conversion to a species capable of covalently binding to hepatic DNA.     

Exercise in rats does not alter hypothalamic AMP-activated protein kinase activity

Recent studies have demonstrated that AMP-activated protein kinase (AMPK) in the hypothalamus is involved in the regulation of food intake. Because exercise is known to influence appetite and cause substrate depletion, it may also influence AMPK in the hypothalamus. Male rats that either rested or ran for 30 or 60 min on a treadmill (22 m/min, 10% slope) were sacrificed immediately after exercise or after 60 min recovery either in the fasted state or after oral gavage with glucose (3g/kg body weight). Exercise decreased muscle and liver glycogen substantially. Hypothalamic total or alpha2-associated AMPK activity and phosphorylation state of the AMPK substrate acetyl-CoA carboxylase were not changed significantly immediately following treadmill running or during fed or fasted recovery. Plasma ghrelin increased (P<0.05) by 40% during exercise whereas the concentration of PYY was unchanged. In recovery, glucose feeding increased plasma glucose and insulin concentrations whereas ghrelin and PYY decreased to (ghrelin) or below (PPY) resting levels. It is concluded that 1h of strenuous exercise in rats does not elicit significant changes in hypothalamic AMPK activity despite an increase in plasma ghrelin. Thus, changes in energy metabolism during or after exercise are likely not coordinated by changes in hypothalamic AMPK activity.     

Amiloride alters lick rate responses to NaCl and KCl in rats

The role-of cation channels on taste cell membranes to salttaste sensation was assessed in rats. We measured the numberof licks during multiple 10-s presentations of NaCl and KClconcentrations (0.05, 0.09, 0.16, 0.28, 0.5 M) dissolved ineither water or in 100 µM amiloride, a sodium-channelblocker. The number of licks to water and 0.3 M sucrose wasalso measured. The number of licks to NaCl was significantlylower and the number of licks to KCl was significantly higherwhen these test solutions were dissolved in amiloride than inwater. There were no differences in lick responses to waterand sucrose. These results suggest that amiloride may have alteredthe taste of NaCl and KCl. The results are discussed in relationshipto prior electrophysiological studies characterizing the effectof amiloride in blocking salt responses of the chorda tympaninerve.     

Protective effects of wheat bran against diquat toxicity in male Fischer-344 rats

After injection with 0.1 mmol diquat/kg body weight, survival time was markedly shorter in Fischer-344 rats fed a purified diet than in rats fed a regular diet, and much more severe hepatotoxicity and nephrotoxicity were observed in the former than in the latter. The longer the feeding period on the purified diet, the shorter the survival time after diquat administration. These results indicate that the purified diet lacked components present in the regular diet that had protective effects against diquat toxicity. These two diets had nearly the same composition and content of vitamins and minerals. We tested the ingredients of the regular diet to determine which ones reduce diquat toxicity. We found that wheat bran had a protective effect, but that rice bran and bean-curd refuse (okara) did not.     

Effect of diquat-induced oxidative stress on iron metabolism in male Fischer-344 rats

Diquat toxicity causes iron-mediated oxidative stress; however, it remains unclear how diquat affects iron metabolism. Here, we examined the effect of diquat-induced oxidative stress on iron metabolism in male Fischer-344 rats, with particular focus on gene expression. Hepatic nonheme iron content was unchanged until 20?h after diquat treatment. Hepatic free iron levels increased markedly in the early stages following treatment and remained elevated for at least 6?h, resulting in severe hepatotoxicity, until returning to control levels at 20?h. The level of hepatic ferritin, especially the H-subunit, increased 20?h after diquat treatment due to elevated hepatic ferritin-H mRNA expression. These results indicate that early elevated levels of free iron in the liver of diquat-treated rats cause hepatotoxicity, and that this free iron is subsequently sequestered by ferritin synthesized under conditions of oxidative stress, thus limiting the pro-oxidant challenge of iron. The plasma iron concentration decreased at 6 and 20?h after diquat treatment, whereas the level of plasma interleukin-6 increased markedly at 3?h and remained high until 20?h. In the liver of diquat-treated rats, expression of hepcidin mRNA was markedly upregulated at 3 and 6?h, whereas ferroportin mRNA expression was downregulated slightly at 20?h. Transferrin receptor 1 mRNA expression was significantly upregulated at 3, 6, and 20?h. These results indicate that inhibition of iron release from iron-storage tissues, through stimulation of the interleukin-6-hepcidin-ferroportin axis, and enhanced iron uptake into hepatocytes, mediated by transferrin receptor 1, cause hypoferremia.     

Fire does not alter vegetation in infertile prairie

The paradigm in prairie ecology is that fire is one of the key factors determining vegetation composition. Fire can impact grassland ecosystems in various ways, including changing plant species composition and inducing nitrogen loss. I found that 17 years of different burning frequencies in infertile grassland had only a minor impact on the vegetation composition and diversity. The only major impact from increasing the frequency of fires was a decrease of Poa pratensis abundance. However, other plant species did not respond to the change in Poa abundance. This result contrasts with previous studies in savannas and more productive grasslands, where the balance between trees, grasses, and the elimination of the litter layer can result in large vegetation changes. However, in this system primary productivity was low, litter did not accumulate and no major vegetation shifts occurred. Thus, the long-term vegetation impacts of burning in an infertile, low-productivity prairie were minimal.     

Luciferase does not alter metabolism in cancer cells

Luciferase transfected cell lines are used extensively for cancer models, revealing valuable biological information about disease mechanisms. However, these genetically encoded reporters, while useful for monitoring tumor response in cancer models, can impact cell metabolism. Indeed firefly luciferase and fatty acyl-CoA synthetases differ by a single amino acid, raising the possibility that luciferase activity might alter metabolism and introduce experimental artifacts. Therefore knowledge of the metabolic response to luciferase transfection is of significant importance, especially given the thousands of research studies using luciferase as an in vivo bioluminescence imaging reporter. Untargeted metabolomics experiments were performed to examine three different types of lymphoblastic leukemia cell lines (Ramos, Raji and SUP-T1) commonly used in cancer research, each were analyzed with and without vector transduction. The Raji model was also tested under perturbed starvation conditions to examine potential luciferase-mediated stress responses. The results showed that no significant metabolic differences were observed between parental and luciferase transduced cells for each cell line, and that luciferase overexpression does not alter cell metabolism under basal or perturbed conditions.     

Hyperglycemia does not alter state 3 respiration in cardiac mitochondria from type-I diabetic rats

The possible role of nitric oxide on the exercise-induced changes in bleomycin-detectable iron (BDI) in the liver, spleen, bone marrow cells and heart was investigated. Female Sprague-Dawley rats were randomly assigned to four groups: S1 (Sedentary), S2 (Sedentary + L-NAME [N-nitro-L-arginine methyl ester]), E1 (Exercise) and E2 (Exercise + L-NAME). Animals in the E1 and E2 swam for 2 h/day for 3 months. L-NAME in the drinking water (1 mg/ml) was administrated to rats in the S2 and E2 groups for the same period. At the end of the 3rd month, nitrite and nitrate (NOx), BDI and non-heme iron (NHI) contents in the liver, spleen, bone marrow cells and heart were measured. The ratio of BDI/NHI was calculated. The exercise induced a significant increase in NOx and BDI contents and/or BDI/NHI ratio in the spleen, bone morrow cells and heart. Treatment with L-NAME, an inhibitor of NOS, led to a significant decrease in NOx and an increase in BDI levels and BDI/NHI ratios in these tissues. The correlative analysis showed that there is significantly positive correlation between NOx levels and BDI contents and/or BDI/NHI ratios in the spleen, bone marrow cells and heart. These results suggest that the increased nitric oxide might be one of the reasons leading to the increased BDI levels in these tissues in the exercised rats. In contrast to the above tissues, in the liver, exercise led to a significant decrease rather than increase in BDI levels and BDI/NHI ratios with a significant increase in NOx contents. Treatment with L-NAME led to a significant increase in BDI levels and BDI/NHI ratios and a decrease in NOx contents in the tissue. These findings plus the results reported by others imply that nitric oxide might have an inhibitory effect on BDI in the liver.     

Amiloride does not block taste transduction in the mudpuppy, Necturus maculosus

Activity of the glossopharyngeal nerve was recorded with bipolarsilver wire electrodes while taste stimuli were applied to thelingual surface in anesthetized mudpuppies. Taste stimuli wereinjected into a continuous stream of distilled water which wasrunning over the tongue, KCl, CaCl₂ and LiCl₂ at 0.4 M elicitedbrisk responses, as did HCl at 0.2 M and quinine at 6 x 10^–4M. Sucrose, glucose and saccharin did not elicit responses.Twenty amino acids were surveyed for their ability to evokea response at 0.04 M: 1-arginine, 1-valine, 1-phenylalanine,1-tryptophan, 1-tyrosine, 1-glutamic acid, 1-lysine and histidinealways evoked responses, whereas other amino acids either didnot evoke responses or only occasionally evoked responses. Thesupernatants from solutions of minced worms and minnows andPurina Trout Chow were effective taste stimuli. Pre-adaptingthe tongue to Ringer's solution by running a continuous streamof Ringer's solution over it eliminated responses to quinineand decreased responses to NaCl. Pre-adapting the tongue to10^–4 to 10^–3 M amiloride, a potent sodium channelblocker, did not alter the responses to NaCl, LiCl, or othertaste stimuli.     

Quercetin supplementation does not alter antioxidant status in humans

Abstract

This study measured the influence of ingesting quercetin on plasma measures for oxidative stress and antioxidant capacity. Male and female subjects (n = 1002) varying in age (18–85 years) and body mass index (BMI) (16.7–52.7 kg/m²) were studied. Subjects were randomized to one of three groups using double-blinded methods: placebo, 500 mg or 1000 mg quercetin/day with 125 mg or 250 mg vitamin C/day, respectively. Pre- and post-study fasting blood samples show that plasma quercetin increased in a dose-responsive manner. The pattern of change in plasma F₂-isoprostanes, oxidized low density lipoprotein, reduced glutathione, ferric reducing ability of plasma (FRAP) and oxygen radical absorbance capacity (ORAC) did not differ between supplementation groups or after adjustment for gender, age, BMI and disease status. In summary, quercetin supplementation over 12 weeks in doses of 500 mg or 1000 mg/day significantly increased plasma quercetin levels, but had no influence on several measures of oxidative stress and antioxidant capacity.     

Restraint stress depresses prolactin surges in pseudopregnant rats and adrenalectomy does not alter the response

Experiments were performed to determine whether restraint stress decreases the two prolactin (PRL) surges in pseudopregnant (PSP) rats in a manner similar to the stress-induced decrease of the proestrous PRL surge. Adrenal involvement as well as adaptation of the response was also investigated. Vaginal cycles were followed and animals exhibiting 2-3 normal cycles were cervically stimulated (CS) electromechanically to induce PSP. In one experiment the effect of adrenalectomy (ADX) on the nocturnal surge (NS) was investigated and was found to have no effect. In another set of experiments the effect of restraint stress was investigated. Immediately following an initial sample, the animals to be stressed had their hind legs tied together with plastic coated bell wire. Subsequent samples were taken for 3 hours. Restraint stress decreased the NS to 15% of the initial value within 30 minutes. ADX did not alter this response. Furthermore, 6-9 days of 3 hours of restraint stress did not attenuate the stress-induced decrease of the NS. Restraint stress also depressed the diurnal surge in PSP rats. These results indicate that restraint stress applied during the two PRL surges of PSP results in significant decreases in plasma PRL and that this response is not altered by ADX or by habituation to the stimulus.     

Long-term cimetidine does not alter serum prolactin.

The effect of repeated cimetidine ingestion on serum prolactin values was studied prospectively in 17 men with proven duodenal ulcers. These patients received 400 mg of cimetidine twice daily for 12 weeks but showed no alteration in their mean serum prolactin levels. Cimetidine-induced hyperprolactinaemia is not the explanation for the development of gynaecomastia in men exposed to this drug.     

Long-term AT1 receptor blockade improves metabolic function and provides renoprotection in Fischer-344 rats

Fischer-344 (F344) rats exhibit proteinuria and insulin resistance in the absence of hypertension as they age. We determined the effects of long-term (1 yr) treatment with the angiotensin (ANG) II type 1 (AT(1)) receptor blocker L-158,809 on plasma and urinary ANG peptide levels, systolic blood pressure (SBP), and indexes of glucose metabolism in 15-mo-old male F344 rats. Young rats at 3 mo of age (n = 8) were compared with two separate groups of older rats: one control group (n = 7) and one group treated with L-158,809 (n = 6) orally (20 mg/l) for 1 yr. SBP was not different between control and treated rats but was higher in young rats. Serum leptin, insulin, and glucose levels were comparable between treated and young rats, whereas controls had higher glucose and leptin with a similar trend for insulin. Plasma ANG I and ANG II were higher in treated than untreated young or older rats, as evidence of effective AT(1) receptor blockade. Urinary ANG II and ANG-(1-7) were higher in controls compared with young animals, and treated rats failed to show age-related increases. Protein excretion was markedly lower in treated and young rats compared with control rats (young: 8 +/- 2 mg/day vs. control: 129 +/- 51 mg/day vs. treated: 9 +/- 3 mg/day, P < 0.05). Long-term AT(1) receptor blockade improves metabolic parameters and provides renoprotection. Differential regulation of systemic and intrarenal (urinary) ANG systems occurs during blockade, and suppression of the intrarenal system may contribute to reduced proteinuria. Thus, insulin resistance, renal injury, and activation of the intrarenal ANG system during early aging in normotensive animals can be averted by renin-ANG system blockade.     

32P-postlabelling analysis of DNA adducts from Fischer-344 rats administered 2,4-diaminotoluene.

Using 32P-postlabelling and thin layer chromatography, DNA adduct formation by the potent animal carcinogen 2,4-diaminotoluene in Fischer-344 rats was investigated. DNA from four different organs, liver, mammary gland, kidney and lung, were examined for adducts following single administration of this compound. DNA binding was detected in all four organs, with each producing one major and two minor adduct spots on autoradiograms. The adducts induced were qualitatively identical among the different organs, but quantitative differences were observed. The two target organs of 2,4-diaminotoluene induced carcinogenesis, the liver and mammary gland produced higher adduct yields, with levels up to 30-times higher than those for the two non-target organs. Since the liver is the principal target for 2,4-diaminotoluene induced carcinogenesis, we further examined DNA adducts from this site for the effects of different doses and time points. DNA binding in liver was detected following doses as low as 4.1 mumol/kg. At the highest concentration examined (2046 mumol/kg), the level of the major adduct was 29.2 adducted nucleotides per 10(7) total nucleotides. The yields for the two minor adducts were approximately one-tenth that for the major adduct. Following a 410 mumol/kg dose, DNA adduct removal over time was examined. DNA adduct removal exhibited biphasic kinetics, with a rapid initial phase followed by a slower rate of elimination. Up to 60% of maximum adduct levels persisted after 2 weeks. DNA binding by 2,4-diaminotoluene was also compared to that by its weakly carcinogenic analog, 2,4-dinitrotoluene. The two compounds produced identical adduct patterns, suggesting that they share common metabolites and adducts. Adduct yields from 2,4-dinitrotoluene, however, were lower. The results of our studies suggest that the differences in carcinogenic potency between 2,4-diaminotoluene and 2,4-dinitrotoluene, as well as the organotropic effects of 2,4-diaminotoluene may be explained, in part, by quantitative differences in the extent of DNA adduct formation.     

Short-term androgen deprivation does not alter CaR and VDR mRNA expression in duodenal mucosa in male rats

Androgen deprivation is associated with decline in intestinal calcium absorption. The effect of androgen on CaR and VDR intestinal mucosa has not yet been studied. Calcium homeostasis, a real bone mineral density (aBMD, dual energy X-ray absorptiometry) and expression of CaR and VDR mRNA in duodenal mucosa of orchidectomized (ORX) and sham operated (Sham) adult Sprague Dawley rats at 4 week have been studied. There was no significant difference in serum calcium, alkaline phosphatase, calcidiol and calcitriol levels between both the groups. Serum testosterone (T) (ng/dl) and inorganic phosphorous (iP) (mg/dl) levels were significantly lower in ORX rats. As compared to sham rats, ORX rats had significant decline in in-vitro aBMD at proximal, middle and distal tibia, proximal, mid and distal femur and femoral neck (P < 0.05). Northern blot analysis revealed no significant alteration in the CaR and VDR mRNA expression in duodenal mucosa in ORX rats. CaR and VDR mRNA expression in duodenal mucosa is therefore, not affected by physiological concentrations of testosterone in rats.     

Amiloride does not block taste transduction in the mouse (Slc:ICR)

1. The receptor potential of the mouse taste cell was recorded with an intracellular microelectrode while taste stimuli were applied to the tongue surface of the anesthetized mouse. 2. A membrane depolarization accompanied by an increase in membrane resistance was observed after a sucrose stimulus. 3. A sodium-chloride stimulus initiated a membrane depolarization accompanied by a decrease in membrane resistance. 4. Amiloride elicits a depolarization of the membrane and is accompanied by an increase in membrane resistance. 5. Pre-adapting the tongue to amiloride, which is known as a potent sodium channel blocker, did not alter the responses to sodium-chloride and other taste stimuli.     

Cyclic GMP-phosphodiesterase inhibition does not alter cerebral oxygen consumption

The effect of zaprinast, a cyclic guanosine monophosphate inhibitor, on the level of cyclic GMP and cerebral O₂ consumption was determined. Anesthetized male Long-Evans rats were divided into a control group (n=15) and a zaprinast treated group (n=15). Vehicle was applied topically to the left cortex and 3·10⁻³ M zaprinast was applied to the right cortex. A saline treated control group was also studied. Regional cerebral blood flow was determined by [¹⁴C]-iodoantipyrine and regional O₂ extraction was determined by microspectrophotometry. The level of cyclic GMP was measured by radioimmunoassay. There were no hemodynamic or blood gas differences between groups. The level of cyclic GMP was not significantly different between the right and left cerebral cortex of the control group (17.0±4.3 and 17.7±4.6 pmol/g). In the zaprinast treated group, there was a significant (46%) increase in the level of cyclic GMP in the zaprinast treated cortex (20.5±8.1) in comparison to the vehicle treated cortex (14.0±5.7). Zaprinast did not significantly alter cerebral blood flow. There were no significant differences in regional O₂ extraction. The O₂ consumption of the zaprinast treated cortex (8.0±3.3 ml O₂·min⁻¹·100 g⁻¹) was not different from that of the vehicle treated cortex (7.0±2.9) or those of the control group. Thus, our data indicated that the increased level of cyclic GMP had no significant effect on cerebral oxygen consumption.