Opposite effects of fasting on TGF-beta 3 and T beta RI distribution in the gastric mucosa of suckling and early weanling rats

Objective: Our aim was to evaluate the effects of a dietary regimen (suckling or early weaning) and feeding status (fed or fasted) on the distribution of transforming growth factor-beta 3 (TGF-beta 3) and TGF receptor-I (T beta RI) in the gastric epithelium of pups Methods: Wistar rats were used At 15 d, half of the pups were separated from dams and fed with hydrated powered chow On day 17, suckling and early weanling rats were subjected to fasting (17 h). Four different conditions were established. suckling fed and fasted and early weanling fed and fasted At 18 d stomachs were collected under anesthesia and were fixed in 4% formaldehyde for immunohistochemistry The number of immunostained epithelial cells per microscopic field was determined for TGF-beta 3 and T beta RI in longitudinal sections from the gastric mucosa Results: We found that during suckling, fasting reduced the number of immunolabeled cells per field of both molecules when compared with the fed group (P < 0.05), whereas in early weaning, food restriction increased TGF-beta 3 and T beta RI distributions (P < 0.05) We also observed that TGF-beta 3 and T beta RI were more concentrated in parietal cells in the upper gland in suckling pups, whereas after early weaning these were displaced to parietal and chief cells at the bottom of the gland Conclusion: Suckling and early weaning directly influence TGF-beta 3 and T beta RI distributions in the gastric epithelium in response to fasting, such that early weaning anticipates the effects observed in adult rats. Furthermore, the differential concentrations of TGF-beta 3 and T beta RI indicate that they might be important for cell proliferation events in growth control (C) 2010 Elsevier Inc. All rights reserved

Fasting differentially regulates plasma corticosterone-binding globulin, glucocorticoid receptor, and cell cycle in the gastric mucosa of pups and adult rats

Ogias D, de Andrade Sa ER, Kasai A, Moisan M, Alvares EP, Gama P. Fasting differentially regulates plasma corticosterone-binding globulin, glucocorticoid receptor, and cell cycle in the gastric mucosa of pups and adult rats. Am J Physiol Gastrointest Liver Physiol 298: G117-G125, 2010. First published October 15, 2009; doi:10.1152/ajpgi.00245.2009.-The nutritional status influences gastric growth, and interestingly, whereas cell proliferation is stimulated by fasting in suckling rats, it is inhibited in adult animals. Corticosterone takes part in the mechanisms that govern development, and its effects are regulated in particular by corticosterone-binding globulin (CBG) and glucocorticoid receptor (GR). To investigate whether corticosterone activity responds to fasting and how possible changes might control gastric epithelial cell cycle, we evaluated different parameters during the progression of fasting in 18- and 40-day-old rats. Food restriction induced higher corticosterone plasma concentration at both ages, but only in pups did CBG binding increase after short-and long-term treatments. Fasting also increased gastric GR at transcriptional and protein levels, but the effect was more pronounced in 40-day-old animals. Moreover, in pups, GR was observed in the cytoplasm, whereas, in adults, it accumulated in the nucleus after the onset of fasting. Heat shock protein (HSP) 70 and HSP 90 were differentially regulated and might contribute to the stability of GR and to the high cytoplasmic levels in pups and elevated shuttling in adult rats. As for gastric epithelial cell cycle, whereas cyclin D1 and p21 increased during fasting in pups, in adults, cyclin E slowly decreased, concomitant with higher p27. In summary, we demonstrated that corticosterone function is differentially regulated by fasting in 18-and 40-day-old rats, and such variation might attenuate any possible suppressive effects during postnatal development. We suggest that this mechanism could ultimately increase cell proliferation and allow regular gastric growth during adverse nutritional conditions.

Exhaustive Exercise Increases Inflammatory Response via Toll Like Receptor-4 and NF-kappa Bp65 Pathway in Rat Adipose Tissue

Cytokines (IL-6, IL-10, and TNF-alpha) are increased after exhaustive exercise in the retroperitoneal adipose tissue (RPAT) and mesenteric adipose tissue (MEAT). An exhaustive acute exercise protocol induces inflammation in adipose tissue that lasts 6 h after the exercise has ended. It is well-established that this protocol increases circulating plasma levels of non-esterified fatty acids (NEFAs) and lipopolysaccharides (LPS), compounds that are important in stimulating signaling via toll like receptor-4 (TLR-4) in different type cells. In the present study, we investigated the regulation of TLR-4 and DNA-binding of nuclear factor-kappa Bp65 (NF-kappa Bp65) in different depots of adipose tissue in rats after exhaustive exercise. Rats were killed by decapitation immediately (E0 group, n = 6), 2 (E2 group, n = 6), and 6 h (E6 group, n = 6) after the exhaustive exercise, which consisted of running on a treadmill (approximately 70% V(O2max)) for 50 min and then running at an elevated rate that increased at 1 m/min, until exhaustion. The control group (C group, n = 6) was not subjected to exercise. In RPAT, TLR-4, MYD-88, and IkB alpha increased in the E2 group after exercise. MYD-88 and TRAF6 remained increased in the E6 group in comparison with the control group. DNA-binding of NF-kappa Bp65 was not altered. In MEAT, TLR-4, MYD-88, TRAF6, and DNA-binding of NF-kappa Bp65 were increased only in the E6 group. In conclusion, we have shown that increases in pro-inflammatory cytokines in adipose tissue pads after exhaustive exercise may be mediated via TLR-4 signaling, leading to increases in NF-kappa Bp65 binding to DNA in MEAT. J. Cell. Physiol. 226: 1604-1607, 2011. (C) 2010 Wiley-Liss, Inc.

Effects of let-7 microRNA on Cell Growth and Differentiation of Papillary Thyroid Cancer

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy and RET/PTC rearrangements represent key genetic events frequently associated to this cancer, enhancing proliferation and dedifferentiation by activation of the RET/PTC-RAS-BRAF-mitogen-activated protein kinase (MAPK) pathway. Recently, let-7 microRNA was found to reduce RAS levels in lung cancer, acting as a tumor suppressor gene. Here, we report that RET/PTC3 oncogenic activation in PCCL3 rat thyroid cells markedly reduces let-7f expression. Moreover, stable transfection of let-7 microRNA in TPC-1 cells, which harbor RET/PTC1 rearrangement, inhibits MAPK activation. As a result, let-7f was capable of reducing TPC-1 cell growth, and this might be explained, at least in part, by decreased messenger RNA (mRNA) expression of cell cycle stimulators such as MYC and CCND1 (cyclin D1) and increased P21 cell cycle inhibitor mRNA. In addition, let-7 enhanced transcriptional expression of molecular markers of thyroid differentiation such as TITF1 and TG. Thus, reduced expression of let-7f might be an essential molecular event in RET/PTC malignant transformation. Moreover, let-7f effects on thyroid growth and differentiation might attenuate neoplastic process of RET/PTC papillary thyroid oncogenesis through impairment of MAPK signaling pathway activation. This is the first functional demonstration of an association of let-7 with thyroid cancer cell growth and differentiation.

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.

Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca(2+), with an estimated K(d) value of 0.52 mu m. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells.

Rod bipolar cells in the retina of the capuchin monkey (Cebus apella): Characterization and distribution

Rod bipolar cells in Cebus apella monkey retina were identified by an antibody against the alpha isoform of protein kinase C (PKC alpha). which has been shown to selectively identify rod bipolars in two other primates and various mammals. Vertical sections were used to confirm the identity of these cells by their characteristic morphology of dendrites and axons. Their topographic distribution was assessed in horizontal sections; counts taken along the dorsal, ventral, nasal, and temporal quadrants. The density of rod bipolar cells increased from 500 to 2900 cells/mm(2) at 1 mm from the fovea to reach a peak of 10,000-12,000 cellss/mm(2) at 4 mm, approximately 5 deg of eccentricity, and then gradually decreased toward retinal periphery to values of 5000 cells/mm(2) or less. Rod to rod bipolar density ratio remained between 10 and 20 across most of the retinal extension. The number of rod bipolar cells per retina was 6,360,000 +/- 387,433 (mean +/- S.D., n = 6). The anti-PKC alpha antibody has shown to be a good marker of rod bipolar cells of Cebus, and the cell distribution is similar to that described for other primates. In spite of the difference in the central retina, the density variation of rod bipolar cells in the Cebus and Macaca as well as the convergence from rod to rod bipolar cells are Generally similar, suggesting that both retinae stabilize similar sensitivity (as measured by rod density) and convergence.

Effects of dietary lipid composition bioavailability and inulin-type fructans on mineral in growing rats

Objective: This study reports the effects of feeding with a combination of inulin-type fructans (ITF) and fish oil (FO) on mineral absorption and bioavailability as part of a semipurified diet offered to rats. Methods: Male Wistar rats (n = 24) were fed a 15% lipid diet (soybean oil [SO] or a 1:0.3 fish:soybean oil mixture [FSO]) and diets containing the same sources of lipids supplemented with 10% ITF (Raftilose Synergy 1) ad libitum for 15 d. Feces and urine were collected for mineral analyses during the last 5 d of the test period. Fatty acid composition was determined in liver and cecal mucosa homogenates. Liver and bone mineral analyses were performed by atomic absorption spectrophotometry. Bone biomechanical analyses were evaluated by a 3-point bending test. Results: Compared with the controls, ITF-fed rats had enlarged ceca and a significant decrease in cecal content pH (P < 0.001). The apparent mineral absorption was improved in these rats, and this effect was enhanced by dietary combination with FO for all minerals except for magnesium. Addition of ITF to the diet resulted in higher bone mineral content (calcium and zinc) and bone strength, but increased bone mineral content was only statistically significant in FO-fed animals. A decrease in liver iron stores (P = 0.015) was observed in rats fed FO, considering that ITF consumption returned to levels comparable to the SO control group. Conclusion: These findings confirm the positive influence of ITF on mineral bioavailability, which was potentiated by addition of FO to the diet. (C) 2009 Published by Elsevier Inc.

Neurofilament heavy subunit in cerebrospinal fluid: A biomarker of amyotrophic lateral sclerosis?

The objectives of this study were to investigate the presence of the three neurofilament subunits, ubiquitin, proteasome and 3-nitrotyrosine, in CSF samples of ALS patients. CSF samples were obtained by lumbar puncture from 10 ALS patients and six controls. All samples were analysed by Western blotting. Results revealed that neurofilament heavy subunit was identified in 70% of ALS cases and we conclude that this subunit may be a promising biomarker for clinical diagnosis of ALS.

Relative contribution of pre- and post-synaptic effects to the neostigmine-induced recovery of neuromuscular transmission blocked by vecuronium

The relative contribution of the pre- and post-synaptic effects to the neostigmine-induced recovery of neuromuscular transmission blocked by vecuronium was studied. A conjunction of myographical and electrophysiological techniques was employed. The preparation was the sciatic nerve-extensor digitorum longus muscle of the rat, in vitro. The physiological variables recorded were nerve-evoked twitches (generated at 0.1 Hz), tetanic contractions (generated at 50 Hz) and end-plate potentials (epps), generated in trains of 50 Hz. The epps were analyzed in: amplitude of first epp in the train; mean amplitude of the 30th to the 59th epp in the train (epps-plateau); half-decay time of the epp; early tetanic rundown of epps in the train; plateau tetanic rundown of epps in the train; quantal content of the epps and quantal size. In myographical experiments, a concentration of vecuronium was found (0.8 mu m) that affected both twitches and tetanic contractions and a concentration of neostigmine was found (0.048 mu m) that completely restored the twitch affected by vecuronium. The cellular effects of vecuronium and neostigmine, studied alone or in association, in the above-mentioned concentrations, were scrutinized by means of electrophysiological techniques. These showed that vecuronium alone decreased the peak amplitude, the quantal content of epps and the quantal size and reinforced the tetanic rundown of epps. Neostigmine alone increased the peak amplitude, the quantal content and the half-decay time of the epps. When employed in the presence of vecuronium, neostigmine increased both the quantal content of the epps (via a presynaptic effect) and the half-decay time of the epps (via a postsynaptic effect). Seeing the pre- and the post-synaptic effects of neostigmine were of similar magnitude, they permit to conclude that both these effects contributed significantly to the restoration by neostigmine of the neuromuscular transmission blocked by vecuronium.

PAR(2) and Temporomandibular Joint Inflammation in the Rat

The proteinase-activated receptor 2 (PAR(2)) is a putative therapeutic target for arthritis. We hypothesized that the early pro-inflammatory effects secondary to its activation in the temporomandibular joint (TMJ) are mediated by neurogenic mechanisms. Immunofluorescence analysis revealed a high degree of neurons expressing PAR(2) in retrogradely labeled trigeminal ganglion neurons. Furthermore, PAR(2) immunoreactivity was observed in the lining layer of the TMJ, co-localizing with the neuronal marker PGP9.5 and substance-P-containing peripheral sensory nerve fibers. The intra-articular injection of PAR(2) agonists into the TMJ triggered a dose-dependent increase in plasma extravasation, neutrophil influx, and induction of mechanical allodynia. The pharmacological blockade of natural killer 1 (NK(1)) receptors abolished PAR(2)-induced plasma extravasation and inhibited neutrophil influx and mechanical allodynia. We conclude that PAR(2) activation is proinflammatory in the TMJ, through a neurogenic mechanism involving NK(1) receptors. This suggests that PAR(2) is an important component of innate neuro-immune response in the rat TMJ.

Effect of plant neutrophil elastase inhibitor on leucocyte migration, adhesion and cytokine release in inflammatory conditions

BACKGROUND AND PURPOSE The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (K(iapp) 5.3 nM) and cathepsin G (K(iapp) 160.0 nM), and the cysteine peptidase cathepsin L (K(iapp) 0.2 nM). These three peptidases play important roles in the inflammatory process. EXPERIMENTAL APPROACH We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte-endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by ELISA. KEY RESULTS Pretreatment of the animals with BbCI (2.5 mg.kg(-1)), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1 beta or tumour necrosis factor-alpha, however, did not change. CONCLUSIONS AND IMPLICATIONS Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.

Anabolic-androgenic steroid treatment induces behavioral disinhibition and downregulation of serotonin receptor messenger RNA in the prefrontal cortex and amygdala of male mice

Nandrolone is an anabolic-androgenic steroid (AAS) that is highly abused by individuals seeking enhanced physical strength or body appearance. Supraphysiological doses of this synthetic testosterone derivative have been associated with many physical and psychiatric adverse effects, particularly episodes of impulsiveness and overt aggressive behavior. As the neural mechanisms underlying AAS-induced behavioral disinhibition are unknown, we investigated the status of serotonergic system-related transcripts in several brain areas of mice receiving prolonged nandrolone administration. Male C57BL/6J mice received 15 mg/kg of nandrolone decanoate subcutaneously once daily for 28 days, and different sets of animals were used to investigate motor-related and emotion-related behaviors or 5-HT-related messenger RNA (mRNA) levels by real-time quantitative polymerase chain reaction. AAS-injected mice had increased body weight, were more active and displayed anxious-like behaviors in novel environments. They exhibited reduced immobility in the forced swim test, a higher probability of being aggressive and more readily attacked opponents. AAS treatment substantially reduced mRNA levels of most investigated postsynaptic 5-HT receptors in the amygdala and prefrontal cortex. Interestingly, the 5-HT(1B) mRNA level was further reduced in the hippocampus and hypothalamus. There was no alteration of 5-HT system transcript levels in the midbrain. In conclusion, high doses of AAS nandrolone in male mice recapitulate the behavioral disinhibition observed in abusers. Furthermore, these high doses downregulate 5-HT receptor mRNA levels in the amygdala and prefrontal cortex. Our combined findings suggest these areas as critical sites for AAS-induced effects and a possible role for the 5-HT(1B) receptor in the observed behavioral disinhibition.

Impaired Vasodilator Activity in Deoxycorticosterone Acetate-Salt Hypertension Is Associated With Increased Protein O-GlcNAcylation

Hyperglycemia, which increases O-linked beta-N-acetylglucosamine (O-GlcNAc) proteins, leads to changes in vascular reactivity. Because vascular dysfunction is a key feature of arterial hypertension, we hypothesized that vessels from deoxycorticosterone acetate and salt (DOCA-salt) rats exhibit increased O-GlcNAc proteins, which is associated with increased reactivity to constrictor stimuli. Aortas from DOCA rats exhibited increased contraction to phenylephrine (E(max) [mN] = 17.6 +/- 4 versus 10.7 +/- 2 control; n = 6) and decreased relaxation to acetylcholine (47.6 +/- 6% versus 73.2 +/- 10% control; n = 8) versus arteries from uninephrectomized rats. O- GlcNAc protein content was increased in aortas from DOCA rats (arbitrary units = 3.8 +/- 0.3 versus 2.3 +/- 0.3 control; n = 5). PugNAc (O- GlcNAcase inhibitor; 100 mu mol/L; 24 hours) increased vascular O- GlcNAc proteins, augmented phenylephrine vascular reactivity (18.2 +/- 2 versus 10.7 +/- 3 vehicle; n = 6), and decreased acetylcholine dilation in uninephrectomized (41.4 +/- 6 versus 73.2 +/- 3 vehicle; n = 6) but not in DOCA rats (phenylephrine, 16.5 +/- 3 versus 18.6 +/- 3 vehicle, n = 6; acetylcholine, 44.7 +/- 8 versus 47.6 +/- 7 vehicle, n = 6). PugNAc did not change total vascular endothelial nitric oxide synthase levels, but reduced endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) phosphorylation (P < 0.05). Aortas from DOCA rats also exhibited decreased levels of endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) (P < 0.05) but no changes in total endothelial nitric oxide synthase or Akt. Vascular O-GlcNAc-modified endothelial nitric oxide synthase was increased in DOCA rats. Blood glucose was similar in DOCA and uninephrectomized rats. Expression of O- GlcNAc transferase, glutamine: fructose-6-phosphate amidotransferase, and O- GlcNAcase, enzymes that directly modulate O-GlcNAcylation, was decreased in arteries from DOCA rats (P < 0.05). This is the first study showing that O-GlcNAcylation modulates vascular reactivity in normoglycemic conditions and that vascular O- GlcNAc proteins are increased in DOCA-salt hypertension. Modulation of increased vascular O-GlcNAcylation may represent a novel therapeutic approach in mineralocorticoid hypertension. (Hypertension. 2009; 53: 166-174.)

Environmental modulation of ethanol-induced locomotor activity: Correlation with neuronal activity in distinct brain regions of adolescent and adult Swiss mice

Drug abuse is a concerning health problem in adults and has been recognized as a major problem in adolescents. induction of immediate-early genes (IEG), such as c-Fos or Egr-1, is used to identify brain areas that become activated in response to various stimuli, including addictive drugs. It is known that the environment can alter the response to drugs of abuse. Accordingly, environmental cues may trigger drug-seeking behavior when the drug is repeatedly administered in a given environment. The goal of this study was first to examine for age differences in context-dependent sensitization and then evaluate IEG expression in different brain regions. For this, groups of mice received i.p. ethanol (2.0 g/kg) or saline in the test apparatus, while other groups received the solutions in the home cage, for 15 days. One week after this treatment phase, mice were challenged with ethanol injection. Acutely, ethanol increased both locomotor activity and IEG expression in different brain regions, indistinctly, in adolescent and adult mice. However, adults exhibited a typical context-dependent behavioral sensitization following repeated ethanol treatment, while adolescent mice presented gradually smaller locomotion across treatment, when ethanol was administered in a paired regimen with environment. Conversely, ethanol-treated adolescents expressed context-independent behavioral sensitization. Overall, repeated ethanol administration desensitized IEG expression in both adolescent and adult mice, but this effect was greatest in the nucleus accumbens and prefrontal cortex of adolescents treated in the context-dependent paradigm. These results suggest developmental differences in the sensitivity to the conditioned and unconditioned locomotor effects of ethanol. (C) 2008 Elsevier B.V. All rights reserved.