Immunohistochemical analysis of neuron types in the mouse small intestine

The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons. Morphological Type II neurons, revealed by anti-neurofilament and anti-calcitonin gene-related peptide antibodies, represented 26% of neurons. The axons of the Type II neurons projected through the circular muscle and submucosa to the mucosa. The cell bodies were immunoreactive for choline acetyltransferase (ChAT), and their terminals were immunoreactive for vesicular acetylcholine transporter (VAChT). Nitric oxide synthase (NOS) occurred in 29% of nerve cells. Most were also immunoreactive for vasoactive intestinal peptide, but they were not tachykinin (TK)-immunoreactive, and only 10% were ChAT-immunoreactive. Numerous NOS terminals occurred in the circular muscle. We deduced that 90% of NOS neurons were inhibitory motor neurons to the muscle (26% of all neurons) and 10% (3% of all neurons) were interneurons. Calretinin immunoreactivity was found in a high proportion of neurons (52%). Many of these had TK immunoreactivity. Small calretinin neurons were identified as excitatory neurons to the longitudinal muscle (about 20% of neurons, with ChAT/calretinin/+/- TK chemical coding). Excitatory neurons to the circular muscle (about 10% of neurons) had the same coding. Calretinin immunoreactivity also occurred in a proportion of Type II neurons. Thus, over 90% of neurons in the myenteric plexus of the mouse small intestine can be currently identified by their neurochemistry and shape.

Hypothalamic Actions of Tumor Necrosis Factor alpha Provide the Thermogenic Core for the Wastage Syndrome in Cachexia

TNF alpha is an important mediator of catabolism in cachexia. Most of its effects have been characterized in peripheral tissues, such as skeletal muscle and fat. However, by acting directly in the hypothalamus, TNF alpha can activate thermogenesis and modulate food intake. Here we show that high concentration TNF alpha in the hypothalamus leads to increased O(2) consumption/CO(2) production, increased body temperature, and reduced caloric intake, resulting in loss of body mass. Most of the thermogenic response is produced by beta 3-adrenergic signaling to the brown adipose tissue (BAT), leading to increased BAT relative mass, reduction in BAT lipid quantity, and increased BAT mitochondria density. The expression of proteins involved in BAT thermogenesis, such as beta 3-adrenergic receptor, peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha, and uncoupling protein-1, are increased. In the hypothalamus, TNF alpha produces reductions in neuropeptide Y, agouti gene-related peptide, proopiomelanocortin, and melanin-concentrating hormone, and increases CRH and TRH. The activity of the AMP-activated protein kinase signaling pathway is also decreased in the hypothalamus of TNF alpha-treated rats. Upon intracerebroventricular infliximab treatment, tumor-bearing and septic rats present a significantly increased survival. In addition, the systemic inhibition of beta 3-adrenergic signaling results in a reduced body mass loss and increased survival in septic rats. These data suggest hypothalamic TNF alpha action to be important mediator of the wastage syndrome in cachexia. (Endocrinology 151: 683-694, 2010)

Participation of peripheral tachykinin NK(1) receptors in the carrageenan-induced inflammation of the rat temporomandibular joint

Temporomandibular disorders represent one of the major challenges in dentistry therapeutics. This study was undertaken to evaluate the time course of carrageenan-induced inflammation in the rat temporomandibular joint (TMJ) and to investigate the role of tachykinin NK(1) receptors. Inflammation was induced by a single intra-articular (i.art.) injection of carrageenan into the left TMJ (control group received sterile saline). Inflammatory parameters such as plasma extravasation, leukocyte influx and mechanical allodynia (measured as the head-withdrawal force threshold) and TNF alpha and IL-1 beta concentrations were measured in the TMJ lavages at selected time-points. The carrageenan-induced responses were also evaluated after treatment with the NK(1) receptor antagonist SR140333. The i.art. injection of carrageenan into the TMJ caused a time-dependent plasma extravasation associated with mechanical allodynia, and a marked neutrophil accumulation between 4 and 24 h. Treatment with SR140333 substantially inhibited the increase in plasma extravasation and leukocyte influx at 4 and 24 h, as well as the production of TNF alpha and IL-1 beta into the joint cavity, but failed to affect changes in head-withdrawal threshold. The results obtained from the present TMJ-arthritis model provide, for the first time, information regarding the time course of this experimental inflammatory process. In addition, our data show that peripheral NK(1) receptors mediate the production of both TNF alpha and IL-1 beta in the TMJ as well as some of the inflammatory signs, such as plasma extravasation and leukocyte influx, but not the nociceptive component. 2008 European Federation of Chapters of the International Association for the Study of Pain. Published by Elsevier Ltd. All rights reserved.

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B(2) and M1 muscarinic acetylcholine receptors

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venom glands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme (ACE) described. Bj-PRO-5a (< EKWAP), a member of this structurally related peptide family, was essential for the development of captopril, the first site-directed ACE inhibitor used for the treatment of human hypertension. Nowadays, more Bj-PROs have been identified with higher ACE inhibition potency compared to Bj-PRO-5a. However, despite its modest inhibitory effect of ACE inhibition, Bj-PRO-5a reveals strong bradykinin-potentiating activity, suggesting the participation of other mechanisms for this peptide. In the present study, we have shown that Bj-PRO-5a induced nitric oxide (NO) production depended on muscarinic acetylcholine receptor M1 subtype (mAchR-M1) and bradykinin B(2) receptor activation, as measured by a chemiluminescence assay using a NO analyzer. Intravital microscopy based on transillumination of mice cremaster muscle also showed that both bradykinin B(2) receptor and mAchR-M1 contributed to the vasodilatation induced by Bj-PRO-5a. Moreover, Bj-PRO-5a-mediated vasodilatation was completely blocked in the presence of a NO synthase inhibitor. The importance of this work lies in the definition of novel targets for Bj-PRO-5a in addition to ACE, the structural model for captopril development. (C) 2011 Elsevier Inc. All rights reserved.

Effects of the antimicrobial peptide gomesin on the global gene expression profile, virulence and biofilm formation of Xylella fastidiosa

In the xylem vessels of susceptible hosts, such as citrus trees, Xylella fastidiosa forms biofilm-like colonies that can block water transport, which appears to correlate to disease symptoms. Besides aiding host colonization, bacterial biofilms play an important role in resistance against antimicrobial agents, for instance antimicrobial peptides (AMPs). Here, we show that gomesin, a potent AMP from a tarantula spider, modulates X. fastidiosa gene expression profile upon 60 min of treatment with a sublethal concentration. DNA microarray hybridizations revealed that among the upregulated coding sequences, some are related to biofilm production. In addition, we show that the biofilm formed by gomesin-treated bacteria is thicker than that formed by nontreated cells or cells exposed to streptomycin. We have also observed that the treatment of X. fastidiosa with a sublethal concentration of gomesin before inoculation in tobacco plants correlates with a reduction in foliar symptoms, an effect possibly due to the trapping of bacterial cells to fewer xylem vessels, given the enhancement in biofilm production. These results warrant further investigation of how X. fastidiosa would respond to the AMPs produced by citrus endophytes and by the insect vector, leading to a better understanding of the mechanism of action of these molecules on bacterial virulence.

Muscle Protein Alterations in LGMD21 Patients With Different Mutations in the Fukutin-related Protein Gene

Fukutin-related protein (FKRP) is a protein involved in the glycosylation of cell surface molecules. Pathogenic mutations in the FKRP gene cause both the more severe congenital muscular dystrophy Type 1C and the milder Limb-Girdle Type 21 form (LGMD21). Here we report muscle histological alterations and the analysis of 11 muscle proteins: dystrophin, four sarcoglycans, calpain 3, dysferlin, telethonin, collagen VI, alpha-DG, and alpha 2-laminin, in muscle biopsies from 13 unrelated LGMD21 patients with 10 different FKRP mutations. In all, a typical dystrophic pattern was observed. In eight patients, a high frequency of rimmed vacuoles was also found. A variable degree of alpha 2-laminin deficiency was detected in 12 patients through immunofluorescence analysis, and 10 patients presented a-DG deficiency on sarcolemmal membranes. Additionally, through Western blot analysis, deficiency of calpain 3 and dystrophin bands was found in four and two patients, respectively. All the remaining proteins showed a similar pattern to normal controls. These results suggest that, in our population of LGMD21 patients, different mutations in the FKRP gene are associated with several secondary muscle protein reductions, and the deficiencies of alpha 2-laminin and alpha-DG on sections are prevalent, independently of mutation type or clinical severity.

Molecular analysis of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) shows an interruption of an inhibitor of apoptosis gene (iap-3) by a new class-II piggyBac-related insect transposon

A new piggyBac-related transposable element (TE) was found in the genome of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus interrupting an inhibitor of apoptosis gene. This mutant virus induces apoptosis upon infection of an Anticarsia gemmatalis cell line, but not in a Trichoplusia ni cell line. The sequence of the new TE (which was named IDT for iap disruptor transposon) has 2531 bp with two DNA sequences flanking a putative Transposase (Tpase) ORF of 1719 bp coding for a protein with 572 amino acids. These structural features are similar to the piggyBac TE, also reported for the first time in the genome of a baculovirus. We have also isolated variants of this new TE from different lepidopteran insect cells and compared their Tpase sequences.

Eag 1, Eag 2 and Kcnn3 gene brain expression of isolated reared rats

The Eag1 and Eag2, voltage-dependent potassium channels, and the small-conductance calcium-activated potassium channel (Kcnn3) are highly expressed in limbic regions of the brain, where their function is still unknown. Eag1 co-localizes with tyrosine hydroxilase enzyme in the substantia nigra and ventral tegmental area. Kcnn3 deficiency leads to enhanced serotonergic and dopaminergic neurotransmission accompanied by distinct alterations in emotional behaviors. As exposure to stress is able to change the expression and function of several ion channels, suggesting that they might be involved in the consequences of stress, we aimed at investigating Eag 1, Eag2 and Kcnn3 mRNA expression in the brains of rats submitted to isolation rearing. As the long-lasting alterations in emotional and behavioral regulation after stress have been related to changes in serotonergic neurotransmission, expressions of serotonin Htr1a and Htr2a receptors in male Wistar rats` brain were also investigated. Rats were reared in isolation or in groups of five for nine weeks after weaning. Isolated and socially reared rats were tested for exploratory activity in the open field test for 5 min and brains were processed for reverse-transcription coupled to quantitative polymerase chain reaction (qRT-PCR). Isolated reared rats showed decreased exploratory activity in the open field. Compared to socially reared rats, isolated rats showed reduced Htr2a mRNA expression in the striatum and brainstem and reduced Eag2 mRNA expression in all examined regions except cerebellum. To our knowledge, this is the first work to show that isolation rearing can change Eag2 gene expression in the brain. The involvement of this channel in stress-related behaviors is discussed.

Elevated CO(2) increases photosynthesis, biomass and productivity, and modifies gene expression in sugarcane

Because of the economical relevance of sugarcane and its high potential as a source of biofuel, it is important to understand how this crop will respond to the foreseen increase in atmospheric [CO(2)]. The effects of increased [CO(2)] on photosynthesis, development and carbohydrate metabolism were studied in sugarcane (Saccharum ssp.). Plants were grown at ambient (similar to 370 ppm) and elevated (similar to 720 ppm) [CO(2)] during 50 weeks in open-top chambers. The plants grown under elevated CO(2) showed, at the end of such period, an increase of about 30% in photosynthesis and 17% in height, and accumulated 40% more biomass in comparison with the plants grown at ambient [CO(2)]. These plants also had lower stomatal conductance and transpiration rates (-37 and -32%, respectively), and higher water-use efficiency (c.a. 62%). cDNA microarray analyses revealed a differential expression of 35 genes on the leaves (14 repressed and 22 induced) by elevated CO(2). The latter are mainly related to photosynthesis and development. Industrial productivity analysis showed an increase of about 29% in sucrose content. These data suggest that sugarcane crops increase productivity in higher [CO(2)], and that this might be related, as previously observed for maize and sorghum, to transient drought stress.

Expression pattern of four storage xyloglucan mobilization-related genes during seedling development of the rain forest tree Hymenaea courbaril L.

During seedling establishment, cotyledons of the rain forest tree Hymenaea courbaril mobilize storage cell wall xyloglucan to sustain growth. The polysaccharide is degraded and its products are transported to growing sink tissues. Auxin from the shoot controls the level of xyloglucan hydrolytic enzymes. It is not yet known how important the expression of these genes is for the control of storage xyloglucan degradation. In this work, partial cDNAs of the genes xyloglucan transglycosylase hydrolase (HcXTH1) and beta-galactosidase (HcBGAL1), both related to xyloglucan degradation, and two other genes related to sucrose metabolism [alkaline invertase (HcAlkIN1) and sucrose synthase (HcSUS1)], were isolated. The partial sequences were characterized by comparison with sequences available in the literature, and phylogenetic trees were assembled. Gene expression was evaluated at intervals of 6 h during 24 h in cotyledons, hypocotyl, roots, and leaves, using 45-d-old plantlets. HcXTH1 and HcBGAL1 were correlated to xyloglucan degradation and responded to auxin and light, being down-regulated when transport of auxin was prevented by N-1-naphthylphthalamic acid (NPA) and stimulated by constant light. Genes related to sucrose metabolism, HcAlkIN1 and HcSUS1, responded to inhibition of auxin transport in consonance with storage mobilization in the cotyledons. A model is proposed suggesting that auxin and light are involved in the control of the expression of genes related to storage xyloglucan mobilization in seedlings of H. courbaril. It is concluded that gene expression plays a role in the control of the intercommunication system of the source-sink relationship during seeding growth, favouring its establishment in the shaded environment of the rain forest understorey.

Multilocus Analyses of Seven Candidate Genes Suggest Interacting Pathways for Obesity-Related Traits in Brazilian Populations

We investigated whether variants in major candidate genes for food intake and body weight regulation contribute to obesity-related traits under a multilocus perspective. We studied 375 Brazilian subjects from partially isolated African-derived populations (quilombos). Seven variants displaying conflicting results in previous reports and supposedly implicated in the susceptibility of obesity-related phenotypes were investigated: beta(2)-adrenergic receptor (ADRB2) (Arg16Gly), insulin induced gene 2 (INSIG2) (rs7566605), leptin (LEP) (A19G), LEP receptor (LEPR) (Gln223Arg), perilipin (PLIN) (6209T > C), peroxisome proliferator-activated receptor-gamma (PPARG) (Pro12Ala), and resistin (RETN) (-420C > G). Regression models as well as generalized multifactor dimensionality reduction (GMDR) were employed to test the contribution of individual effects and higher-order interactions to BMI and waist-hip ratio (WHR) variation and risk of overweight/obesity. The best multilocus association signal identified in the quilombos was further examined in an independent sample of 334 Brazilian subjects of European ancestry. In quilombos, only the PPARG polymorphism displayed significant individual effects (WHR variation, P = 0.028). No association was observed either with the risk of overweight/obesity (BMI >= 25 kg/m(2)), risk of obesity alone (BMI >= 30 kg/m(2)) or BMI variation. However, GMDR analyses revealed an interaction between the LEPR and ADRB2 polymorphisms (P = 0.009) as well as a third-order effect involving the latter two variants plus INSIG2 (P = 0.034) with overweight/obesity. Assessment of the LEPR-ADRB2 interaction in the second sample indicated a marginally significant association (P = 0.0724), which was further verified to be limited to men (P = 0.0118). Together, our findings suggest evidence for a two-locus interaction between the LEPR Gln223Arg and ADRB2 Arg16Gly variants in the risk of overweight/obesity, and highlight further the importance of multilocus effects in the genetic component of obesity.

Gene Expression Profiling of Cultured Cells From Brainstem of Newborn Spontaneously Hypertensive and Wistar Kyoto Rats

The spontaneously hypertensive rat (SHR) is a good model to study several diseases such as the attention-deficit hyperactivity disorder, cardiopulmonary impairment, nephropathy, as well as hypertension, which is a multifactor disease that possibly involves alterations in gene expression in hypertensive relative to normotensive subjects. In this study, we used high-density oligoarrays to compare gene expression profiles in cultured neurons and glia from brainstem of newborn normotensive Wistar Kyoto (WKY) and SHR rats. We found 376 genes differentially expressed between SHR and WKY brainstem cells that preferentially map to 17 metabolic/signaling pathways. Some of the pathways and regulated genes identified herein are obviously related to cardiovascular regulation; in addition there are several genes differentially expressed in SHR not yet associated to hypertension, which may be attributed to other differences between SHR and WKY strains. This constitute a rich resource for the identification and characterization of novel genes associated to phenotypic differences observed in SHR relative to WKY, including hypertension. In conclusion, this study describes for the first time the gene profiling pattern of brainstem cells from SHR and WKY rats, which opens up new possibilities and strategies of investigation and possible therapeutics to hypertension, as well as for the understanding of the brain contribution to phenotypic differences between SHR and WKY rats.

Transcriptional regulation differs in affected facioscapulohumeral muscular dystrophy patients compared to asymptomatic related carriers

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle disorder that has been associated with a contraction of 3.3-kb repeats on chromosome 4q35. FSHD is characterized by a wide clinical inter- and intrafamilial variability, ranging from wheelchair-bound patients to asymptomatic carriers. Our study is unique in comparing the gene expression profiles from related affected, asymptomatic carrier, and control individuals. Our results suggest that the expression of genes on chromosome 4q is altered in affected and asymptomatic individuals. Remarkably, the changes seen in asymptomatic samples are largely in products of genes encoding several chemokines, whereas the changes seen in affected samples are largely in genes governing the synthesis of GPI-linked proteins and histone acetylation. Besides this, the affected patient and related asymptomatic carrier share the 4qA161 haplotype. Thus, these polymorphisms by themselves do not explain the pathogenicity of the contracted allele. Interestingly, our results also suggest that the miRNAs might mediate the regulatory network in FSHD. Together, our results support the previous evidence that FSHD may be caused by transcriptional dysregulation of multiple genes, in cis and in trans, and suggest some factors potentially important for FSHD pathogenesis. The study of the gene expression profiles from asymptomatic carriers and related affected patients is a unique approach to try to enhance our understanding of the missing link between the contraction in D4Z4 repeats and muscle disease, while minimizing the effects of differences resulting from genetic background.

Dipeptidyl peptidase IV in the hypothalamus and hippocampus of monosodium glutamate obese and food-deprived rats

Proline-specific dipeptidyl peptidases are emerging as a protease family with important roles in the regulation of signaling by peptide hormones related to energy balance. The treatment of neonatal rats with monosodium glutamate (MSG) is known to produce a selective damage on the arcuate nucleus with development of obesity. This study investigates the relationship among dipeptidyl peptidase IV (DPPIV) hydrolyzing activity, CD26 protein, fasting, and MSG model of obesity in 2 areas of the central nervous system. Dipeptidyl peptidase IV and CD26 were, respectively, evaluated by fluorometry, and enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction in soluble (SF) and membrane-bound (MF) fractions from the hypothalamus and hippocampus of MSG-treated and normal rats, submitted or not to food deprivation (FD). Dipeptidyl peptidase IV in both areas was distinguished kinetically as insensitive (DI) and sensitive (DS) to diprotin A. Compared with the controls, MSG and/or FD decreased the activity of DPPIV-DI in the SF and MF from the hypothalamus, as well as the activity of DPPIV-DS in the SF from the hypothalamus and in the MF from the hippocampus. Monosodium glutamate and/or FD increased the activity of DPPIV-DI in the MF from the hippocampus. The monoclonal protein expression of membrane CD26 by enzyme-linked immunosorbent assay decreased in the hypothalamus and increased in the hippocampus of MSG and/or FD relative to the controls. The existence of DPPIV-like activity with different sensitivities to diprotin A and the identity of insensitive with CD26 were demonstrated for the first time in the central nervous system. Data also demonstrated the involvement of DPPIV-DI/CD26 hydrolyzing activity in the energy balance probably through the regulation of neuropeptide Y and beta-endorphin levels in the hypothalamus and hippocampus. (C) 2011 Elsevier Inc. All rights reserved.