Early motor and electrophysiological changes in transgenic mouse model of amyotrophic lateral sclerosis and gender differences on clinical outcome

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disorder affecting motoneurons and the SOD1(G93A) transgenic mice are widely employed to study disease physiopathology and therapeutic strategies. Despite the cellular and biochemical evidences of an early motor system dysfunction, the conventional behavioral tests do not detect early motor impairments in SOD1 mouse model. We evaluated early changes in motor behavior of ALS mice by doing the analyses of tail elevation, footprint, automatic recording of motor activities by means of an infrared motion sensor activity system and electrophysiological measurements in male and female wild-type (WT) and SOD1(G93A) mice from postnatal day (P) 20 up to endpoint. The classical evaluations of mortality, weight loss, tremor, rotometer, hanging wire and inclined plane were also employed. There was a late onset (after P90) of the impairments of classical parameters and the outcome varied between genders of ALS mice, being tremor, cumulative survival, weight loss and neurological score about 10 days earlier in male than female ALS mice and also about 20 days earlier in ALS males regarding rotarod and hanging wire performances. While diminution of hindpaw base was 10 days earlier in ALS males (P110) compared to females, the steep length decreased 40 days earlier in ALS females (P60) than ALS males. The automatic analysis of motor impairments showed substantial late changes (after P90) of motility and locomotion in the ALS females, but not in the ALS males. It was surprising that the scores of tail elevation were already decreased in ALS males and females by P40, reaching the minimal values at the endpoint. The electrophysiological analyses showed early changes of measures in the ALS mouse sciatic nerve, i.e., decreased values of amplitude (P40) and nerve conduction velocity (P20), and also an increased latency (P20) reaching maximal level of impairments at the late disease phase. The early changes were not accompanied by reductions of neuronal protein markers of neurofilament 200 and ChAT in the ventral part of the lumbar spinal cord of P20 and P60 ALS mice by means of Western blot technique, despite remarkable decreases of those protein levels in P120 ALS mice. In conclusion, early changes of motor behavior and electrophysiological parameters in ALS mouse model must be taken into attention in the analyses of disease mechanisms and therapeutic effects. (C) 2011 Published by Elsevier B.V.

Effects of protein deprivation and re-feeding on P2X(2) receptors in enteric neurons

AIM: To investigate the effects of malnutrition and refeeding on the P2X(2) receptor, nitric oxide synthase (NOS), calretinin, calbindin and choline acetyltransferase (ChAT) in neurons of the rat ileum. METHODS: We analyzed the co-localization, numbers and sizes of P2X(2)-expressing neurons in relation to NOS-IR (immunoreactive), calbindin-IR, ChAT-IR, and calretinin-IR neurons of the myenteric and submucosal plexus. The experimental groups consisted of: (1) rats maintained on normal feed throughout pregnancy until 42 d post-parturition (N); (2) rats deprived of protein throughout pregnancy and 42 d post-parturition (D); and (3) rats undernourished for 21 d post-parturition and then given a protein diet from days 22 to 42 (DR). The myenteric and submucosal plexuses were evaluated by double labeling by immunohistochemical methods for P2X(2) receptor, NOS, ChAT, calbindin and calretinin. RESULTS: We found similar P2X(2) receptor immunoreactivity in the cytoplasm and surface membranes of myenteric and submucosal neurons from the N, D and DR groups. Double labeling of the myenteric plexus demonstrated that approximately 100% of NOS-IR, calbindin-IR, calretinin-IR and ChAT-IR neurons in all groups also expressed the P2X(2) receptor. In the submucosal plexus, the calretinin-IR, ChAT-IR and calbindinIR neurons were nearly all immunoreactive for the P2X(2) receptor. In the myenteric plexus, there was a 19% increase in numbers per cm(2) for P2X(2) receptor-IR neurons, 64% for NOS-IR, 84% for calretinin-IR and 26% for ChAT-IR neurons in the D group. The spatial density of calbindin-IR neurons, however, did not differ among the three groups. The submucosal neuronal density increased for calbindin-IR, calretinin-IR and ChAT-IR neurons. The average size of neurons in the myenteric plexus neurons in the D group was less than that in the controls and, in the re-fed rats; there was a 34% reduction in size only for the calretinin-IR neurons. CONCLUSION: This work demonstrates that expression of the P2X(2) receptor is present in inhibitory, intrinsic primary afferent, cholinergic secretomotor and vasomotor neurons. Undernutrition affected P2X(2) receptor expression in the submucosal plexus, and neuronal and size. These changes were rescued in the re-fed rats. (C) 2010 Baishideng. All rights reserved.

Neurodegeneration and Increased Production of Nitrotyrosine, Nitric Oxide Synthase, IFN-gamma and S100 beta Protein in the Spinal Cord of IL-12p40-Deficient Mice Infected with Trypanosoma cruzi

Background/Aim: Chagas` disease is caused by Trypanosoma cruzi and occurs in most Latin American countries. The protozoan may colonize the central nervous system (CNS) of immune-compromised human hosts, thus causing neuronal disorders. Systemic control of the intracellular forms of the parasite greatly depends on the establishment of a TH1 response and subsequent nitric oxide (NO) release. At the CNS, it is known that low concentrations of NO promote neuronal survival and growth, while high concentrations exert toxic effects and neuron death. Accounting for NO production by astrocytes is the glia-derived factor S100 beta, which is overproduced in some neurodegenerative diseases. In the current work, we studied the expression of NO, interferon (IFN)-gamma and S100 beta in the spinal cord tissue of IL-12p40KO mice infected with T. cruzi, a model of neurodegenerative process. Methods: IL-12p40KO and wild-type (WT) female mice infected with T. cruzi Sylvio X10/4 (10(5) trypomastigotes, intraperitoneally) were euthanized when IL-12p40KO individuals presented limb paralysis. Spinal cord sections were submitted to immunohistochemical procedures for localization of neurofilament, laminin, nitrotyrosine, NO synthases (NOS), IFN-gamma and S100 beta. The total number of neurons was estimated by stereological analysis and the area and intensity of immunoreactivities were assessed by microdensitometric/morphometric image analysis. Results: No lesion was found in the spinal cord sections of WT mice, while morphological disarrangements, many inflammatory foci, enlarged vessels, amastigote nests and dying neurons were seen at various levels of IL-12p40KO spinal cord. Compared to WT mice, IL-12p40KO mice presented a decrement on total number of neurons (46.4%, p<0.05) and showed increased values of immunoreactive area for nitrotyrosine (239%, p<0.01) and NOS (544%, p<0.001). Moreover, the intensity of nitrotyrosine (16%, p<0.01), NOS (38%, p<0.05) and S100 beta (21%, p<0.001) immunoreactivities were also augmented. No IFN-gamma labeled cells were seen in WT spinal cord tissue, contrary to IL-12p40KO tissue that displayed inflammatory infiltrating cells and also some parenchymal cells positively labeled.Conclusion: We suggest that overproduction of NO may account for neuronal death at the spinal cord of T. cruzi-infected IL-12p40KO mice and that IFN-gamma and S100 beta may contribute to NOS activation in the absence of IL-12. Copyright (C) 2009 S. Karger AG, Basel

Enhanced Neural Progenitor/Stem Cells Self-Renewal via the Interaction of Stress-Inducible Protein 1 with the Prion Protein

Prion protein (PrPC), when associated with the secreted form of the stress-inducible protein 1 (STI1), plays an important role in neural survival, neuritogenesis, and memory formation. However, the role of the PrP(C)-STI1 complex in the physiology of neural progenitor/stem cells is unknown. In this article, we observed that neurospheres cultured from fetal forebrain of wild-type (Prnp(+/+)) and PrP(C)-null (Prnp(0/0)) mice were maintained for several passages without the loss of self-renewal or multipotentiality, as assessed by their continued capacity to generate neurons, astrocytes, and oligodendrocytes. The homogeneous expression and colocalization of STI1 and PrP(C) suggest that they may associate and function as a complex in neurosphere-derived stem cells. The formation of neurospheres from Prnp(0/0) mice was reduced significantly when compared with their wild-type counterparts. In addition, blockade of secreted STI1, and its cell surface ligand, PrP(C), with specific antibodies, impaired Prnp(+/+) neurosphere formation without further impairing the formation of Prnp(0/0) neurospheres. Alternatively, neurosphere formation was enhanced by recombinant STI1 application in cells expressing PrP(C) but not in cells from Prnp(0/0) mice. The STI1-PrP(C) interaction was able to stimulate cell proliferation in the neurosphere-forming assay, while no effect on cell survival or the expression of neural markers was observed. These data suggest that the STI1-PrP(C) complex may play a critical role in neural progenitor/stem cells self-renewal via the modulation of cell proliferation, leading to the control of the stemness capacity of these cells during nervous system development. STEM CELLS 2011;29:1126-1136

Two-dimensional gel electrophoretic protein profile analysis during seed development of Ocotea catharinensis: a recalcitrant seed species

The aim of the present work was to characterize changes in the protein profile throughout seed development in O. catharinensis, a recalcitrant species, by two-dimensional gel electrophoresis. Protein extraction was undertaken by using a thiourea/urea buffer, followed by a precipitation step with 10% TCA. Comparative analysis during seed development showed that a large number of proteins were exclusively detected in each developmental stage. The cotyledonary stage, which represents the transition phase between embryogenesis and the beginning of metabolism related to maturation, presents the highest number of stage-specific spots. Protein identification, through MS/MS analysis, resulted in the identification of proteins mainly related to oxidative metabolism and storage synthesis. These findings contribute to a better understanding of protein metabolism during seed development in recalcitrant seeds, besides providing information on established markers that could be useful in defining and improving somatic embryogenesis protocols, besides monitoring the development of somatic embryos in this species.

Lisina digestível para frangos de corte machos entre 12 e 22 dias de idade

Avaliou-se nível de lisina digestível para 1050 frangos de corte dos 12 aos 22 dias de idade. O delineamento experimental foi inteiramente ao acaso, com cinco tratamentos, sete repetições e 30 aves por unidade experimental. Os tratamentos foram: 1,05; 1,10; 1,15; 1,20 e 1,25% de lisina digestível. Avaliaram-se ganho de peso, consumo de ração, conversão alimentar, composição e deposição de nutrientes corporais. Foram constatados efeitos quadráticos de lisina digestível no consumo de ração e resposta linear ascendente no peso da carcaça. Na composição química da carcaça, houve resposta quadrática do nível de lisina na concentração de proteína. As taxas de deposição proteica, deposição de água, da carcaça e do corpo total tiveram aumento linear em resposta ao acréscimo de lisina na dieta. O aumento da concentração de lisina, todavia, coincidiu com a redução da matéria mineral nas vísceras e sangue e no corpo total. Considerado o desempenho, o nível 1,1% de lisina digestível atendeu às necessidades do frango de corte entre o 12º e o 22º dia de idade. Consideradas a composição química e as taxas de deposição dos nutrientes corporais, a demanda pelo aminoácido digestível torna-se igual ou maior que 1,25%

Lisina digestível e zinco orgânico para frangos de corte machos na fase de 22 a 42 dias de idade

Os efeitos dietéticos de lisina digestível e zinco quelato para frangos de corte machos entre 22 e 42 dias de idade foram avaliados em dois ensaios experimentais. Adotou-se o delineamento inteiramente casualizado em arranjo fatorial 5 × 2, com cinco níveis de lisina (0,841; 0,876; 0,997; 1,022 e 1,030%) e dois de zinco (43 e 243 ppm). No primeiro ensaio, utilizaram-se 900 aves com peso médio inicial de 957,4 g distribuídas em parcelas experimentais de 30 aves e três repetições e, no segundo ensaio, 180 frangos com peso médio inicial de 866,22 g, divididos em parcelas de três aves e seis repetições. As características avaliadas foram desempenho, rendimento de cortes e composição corporal (1º ensaio), balanço de nitrogênio e digestibilidade aparente das dietas (2º ensaio). Houve interação entre os níveis de lisina e zinco para o ganho de peso, a conversão alimentar, a matéria seca ingerida e o balanço energético. Os rendimentos de peito, coxa e sobrecoxa tiveram aumentos lineares em resposta ao acréscimo no nível de lisina digestível na dieta. O melhor desempenho foi obtido com o nível de 0,997% de lisina digestível (ou 1,134% lisina total) e de 43 ppm de zinco. Para maior rendimento dos cortes, o nível de lisina digestível recomendável deve ser no mínimo 1,002% (ou 1,139% de lisina total), independentemente dos níveis de zinco quelato. A maior inclusão de zinco em dietas para frangos de corte dos 22 aos 42 dias de idade não melhora a utilização da lisina na dieta e aumenta a deposição de gordura corporal.

Interleukin-22 forms dimers that are recognized by two interleukin-22R1 receptor chains

Interleukin-22 (IL-22) is a class 2 cytokine whose primary structure is similar to that of interleukin 10 (IL-10) and interferon-gamma (IFN-gamma). IL-22 induction during acute phase immune response indicates its involvement in mechanisms of inflammation. Structurally different from IL-10 and a number of other members of IL-10 family, which form intertwined inseparable V-shaped dimers of two identical polypeptide chains, a single polypeptide chain of IL-22 folds on itself in a relatively globular structure. Here we present evidence, based on native gel electrophoresis, glutaraldehyde cross-linking, dynamic light scattering, and small angle x-ray scattering experiments, that human IL-22 forms dimers and tetramers in solution under protein concentrations assessable by these experiments. Unexpectedly, low-resolution molecular shape of IL-22 dimers is strikingly similar to that of IL-10 and other intertwined cytokine dimeric forms. Furthermore, we determine an ab initio molecular shape of the IL-22/IL-22R1 complex which reveals the V-shaped IL-22 dimer interacting with two cognate IL-22R1 molecules. Based on this collective evidence, we argue that dimerization might be a common mechanism of all class 2 cytokines for the molecular recognition with their respective membrane receptor. We also speculate that the IL-22 tetramer formation could represent a way to store the cytokine in nonactive form at high concentrations that could be readily converted into functionally active monomers and dimers upon interaction with the cognate cellular receptors.

Survival and Neuronal Differentiation of Mesenchymal Stem Cells Transplanted into the Rodent Brain Are Dependent upon Microenvironment

Introduction: The successful integration of stem cells in adult brain has become a central issue in modern neuroscience. In this study we sought to test the hypothesis that survival and neurodifferentiation of mesenchymal stem cells (MSCs) may be dependent upon microenvironmental conditions according to the site of implant in the brain. Methods: MSCs were isolated from adult rats and labeled with enhanced-green fluorescent protein (eGFP) lentivirus. A cell suspension was implanted stereotactically into the brain of 50 young rats, into one neurogenic area (hippocampus), and into another nonneurogenic area (striatum). Animals were sacrificed 6 or 12 weeks after surgery, and brains were stained for mature neuronal markers. Cells coexpressing NeuN (neuronal specific nuclear protein) and GFP (green fluorescent protein) were counted stereologically at both targets. Results: The isolated cell population was able to generate neurons positive for microtubule-associated protein 2 (MAP2), neuronal-specific nuclear protein (NeuN), and neurofilament 200 (NF200) in vitro. Electrophysiology confirmed expression of voltage-gated ionic channels. Once implanted into the hippocampus, cells survived for up to 12 weeks, migrated away from the graft, and gave rise to mature neurons able to synthesize neurotransmitters. By contrast, massive cell degeneration was seen in the striatum, with no significant migration. Induction of neuronal differentiation with increased cyclic adenosine monophosphate in the culture medium before implantation favored differentiation in vivo. Conclusions: Our data demonstrated that survival and differentiation of MSCs is strongly dependent upon a permissive microenvironment. Identification of the pro-neurogenic factors present in the hippocampus could subsequently allow for the integration of stem cells into nonpermissive areas of the central nervous system.

Expression and cellular localization of microRNA-29b and RAX, an activator of the RNA-dependent protein kinase (PKR), in the retina of streptozotocin-induced diabetic rats

Purpose: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats. Methods: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis. Results: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b. Conclusions: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.

Effects of perinatal protein deprivation and recovery on esophageal myenteric plexus

AIM: To evaluate effects of pre- and postnatal protein deprivation and postnatal recovery on the myenteric plexus of the rat esophagus. METHODS: Three groups of young Wistar rats (aged 42 d) were studied: normal-fed (N42), protein-deprived (D42), and protein-recovered (R42). The myenteric neurons of their esophagi were evaluated by histochemical reactions for nicotinamide adenine dinucleotide (NADH), nitrergic neurons (NADPH)-diaphorase and acetylcholinesterase (AChE), immunohistochemical reaction for vasoactive intestinal polypeptide (VIP), and ultrastructural analysis by transmission electron microscopy. RESULTS: The cytoplasms of large and medium neurons from the N42 and R42 groups were intensely reactive for NADH. Only a few large neurons from the D42 group exhibited this aspect. NADPH detected in the D42 group exhibited low reactivity. The AChE reactivity was diffuse in neurons from the D42 and R42 groups. The density of large and small varicosities detected by immunohistochemical staining of VIP was low in ganglia from the D42 group. In many neurons from the D42 group, the double membrane of the nuclear envelope and the perinuclear cisterna were not detectable. NADH and NADPH histochemistry revealed no group differences in the profile of nerve cell perikarya (ranging from 200 to 400 mu m(2)). CONCLUSION: Protein deprivation causes a delay in neuronal maturation but postnatal recovery can almost completely restore the normal morphology of myenteric neurons. (C) 2010 Baishideng. All rights reserved.

Crystallization, data collection and data processing of maltose-binding protein (MalE) from the phytopathogen Xanthomonas axonopodis pv. citri

Maltose-binding protein is the periplasmic component of the ABC transporter responsible for the uptake of maltose/maltodextrins. The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 Kusing the hanging-drop vapour-diffusion method. The crystal belonged to the primitive hexagonal space group P6(1)22, with unit-cell parameters a = 123.59, b = 123.59, c = 304.20 angstrom, and contained two molecules in the asymetric unit. It diffracted to 2.24 angstrom resolution.

Crystallization and preliminary X-ray diffraction analysis of human IL-22 bound to its soluble decoy receptor IL-22BP

Interleukin-22 (IL-22) is a pleiotropic cytokine that is involved in inflammatory responses. Human IL-22 was incubated with its soluble decoy receptor IL-22BP (IL-22 binding protein) and the IL-22 -IL-22BP complex was crystallized in hanging drops using the vapour-diffusion method. Suitable crystals were obtained from polyethylene glycol solutions and diffraction data were collected to 2.75 angstrom resolution. The crystal belonged to the tetragonal space group P41, with unit-cell parameters a = b = 67.9, c = 172.5 angstrom, and contained two IL-22-IL- 22BP complexes per asymmetric unit.

Schistosoma mansoni Tegument Protein Sm29 Is Able to Induce a Th1-Type of Immune Response and Protection against Parasite Infection

Background: Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate. Methods and Findings: We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p<0.05). Protective immunity in mice was associated with high titers of specific anti-Sm29 IgG1 and IgG2a and elevated production of IFN-gamma, TNF-alpha and IL-12, a typical Th1 response. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to worms from control mice revealed a significant (q<0.01) down-regulation of 495 genes and up-regulation of only 22 genes. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals, suggesting that under immune attack schistosomes reduce the expression of critical surface proteins. Conclusion: This study demonstrates that Sm29 surface protein is a new vaccine candidate against schistosomiasis and suggests that Sm29 vaccination associated with other protective critical surface antigens is the next logical strategy for improving protection.

Growth performance and body composition of pacu Piaractus mesopotamicus (Holmberg 1887) in response to dietary protein and energy levels

Improper dietary protein and energy levels and their ratio will lead to increased fish production cost. This work evaluated effects of dietary protein : energy ratio on growth and body composition of pacu, Piaractus mesopotamicus. Fingerling pacu (15.5 +/- 0.4 g) were fed twice a day for 10 weeks until apparent satiation with diets containing 220, 260, 300, 340 or 380 g kg-1 crude protein (CP) and 10.9, 11.7, 12.6, 13.4 or 14.2 MJ kg-1 digestible energy (DE) in a totally randomized experimental design, 5 x 5 factorial scheme (n = 3). Weight gain, specific growth rate increased and feed conversion ratio (FCR) decreased significantly (P < 0.05) when CP increased from 220 to 271, 268 and 281 g kg-1 respectively. Pacu was able to adjust feed consumption in a wide range of dietary DE concentration. Fish fed 260 CP diets showed best (P < 0.05) protein efficiency ratio and FCR with 11.7-12.6 MJ kg-1; but for the 380 CP-diets group, significant differences were observed only at 14.2 MJ kg-1 dietary energy level, suggesting that pacu favours protein as energy source. DE was the chief influence on whole body chemical composition. Minimum dietary protein requirement of pacu is 270 g kg-1, with an optimum CP : DE of 22.2 g MJ-1.