Cytoarchitectureal changes in hippocampal subregions of the NZB/W F1 mouse model of lupus.

Over 50% of clinical patients affected by the systemic lupus erythematosus disease display impaired neurological cognitive functions and psychiatric disorders, a form called neuropsychiatric systemic lupus erythematosus. Hippocampus is one of the brain structures most sensitive to the cognitive deficits and psychiatric disorders related to neuropsychiatric lupus. The purpose of this study was to compare, layer by layer, neuron morphology in lupus mice model NZB/W F1 versus Wild Type mice. By a morphometric of cells identified on Nissl-stained sections, we evaluated structural alterations between NZB/W F1 and Wild Type mice in seven hippocampal subregions: Molecular dentate gyrus, Granular dentate gyrus, Polymorph dentate gyrus, Oriens layer, Pyramidal layer, Radiatum layer and Lacunosum molecular layer. By principal component analysis we distinguished healthy Wild Type from NZB/W F1 mice. In NZB/W F1 mice hippocampal cytoarchitecture, the neuronal cells resulted larger in size and more regular than those of Wild Type. In NZB/W F1, neurons were usually denser than in WT. The Pyramidal layer neurons were much denser in Wild Type than in NZB/W F1. Application of principal component analysis, allowed to distinguish NZB/W F1 lupus mice from healthy, showing as NZBW subjects presented a scattered distribution and intrasubject variability. Our results show a hypertrophy of the NZB/W F1 hippocampal neurons associated with an increase in perikaryal size within the CA1, CA2, CA3 region and the DG. These results help advance our understanding on hippocampal organization and structure in the NZB/W F1 lupus model, suggesting the hypothesis that the different subregions could be differentially affected in neuropsychiatric systemic lupus erythematosus disease. Leveraging an in-depth analysis of the morphology of neural cells in the hippocampal subregions and applying dimensionality reduction using PCA, we propose an efficient methodology to distinguish pathological NZBW mice from WT mice."

Multi-aspect testing and ranking inference to quantify dimorphism in the cytoarchitecture of cerebellum of male, female and intersex individuals: a model applied to bovine brains.

The dimorphism among male, female and freemartin intersex bovines, focusing on the vermal lobules VIII and IX, was analyzed using a novel data analytics approach to quantify morphometric differences in the cytoarchitecture of digitalized sections of the cerebellum. This methodology consists of multivariate and multi-aspect testing for cytoarchitecture-ranking, based on neuronal cell complexity among populations defined by factors, such as sex, age or pathology. In this context, we computed a set of shape descriptors of the neural cell morphology, categorized them into three domains named size, regularity and density, respectively. The output and results of our methodology are multivariate in nature, allowing an in-depth analysis of the cytoarchitectonic organization and morphology of cells. Interestingly, the Purkinje neurons and the underlying granule cells revealed the same morphological pattern: female possessed larger, denser and more irregular neurons than males. In the Freemartin, Purkinje neurons showed an intermediate setting between males and females, while the granule cells were the largest, most regular and dense. This methodology could be a powerful instrument to carry out morphometric analysis providing robust bases for objective tissue screening, especially in the field of neurodegenerative pathologies.

The laminar organization of the motor cortex in monodactylous mammals: a comparative assessment based on horse, chimpanzee, and macaque.

The architecture of the neocortex classically consists of six layers, based on cytological criteria and on the layout of intra/interlaminar connections. Yet, the comparison of cortical cytoarchitectonic features across different species proves overwhelmingly difficult, due to the lack of a reliable model to analyze the connection patterns of neuronal ensembles forming the different layers. We first defined a set of suitable morphometric cell features, obtained in digitized Nissl-stained sections of the motor cortex of the horse, chimpanzee, and crab-eating macaque. We then modeled them using a quite general non-parametric data representation model, showing that the assessment of neuronal cell complexity (i.e., how a given cell differs from its neighbors) can be performed using a suitable measure of statistical dispersion such as the mean absolute deviation-mean absolute deviation (MAD). Along with the non-parametric combination and permutation methodology, application of MAD allowed not only to estimate, but also to compare and rank the motor cortical complexity across different species. As to the instances presented in this paper, we show that the pyramidal layers of the motor cortex of the horse are far more irregular than those of primates. This feature could be related to the different organizations of the motor system in monodactylous mammals.

Gene expression profiles of estrogen receptors α and ß in the fetal bovine hypothalamus and immunohistochemical characterization during development.

Steroid hormones intervene in the structural and functional regulation of neuronal processes during development and thus determine brain differentiation. The effects of estrogens are mediated by two transcription factors, namely estrogen receptor α (ER-α) and estrogen receptor ß (ER-ß), that regulate the expression of target genes through their binding to specific DNA target sequences. We describe the mRNA expression of ER-α and ER-ß in the hypothalamus of developing male and female bovines as revealed by quantitative real-time polymerase chain reaction, and the distribution of the two ERs in hypothalamic sections of all fetal stages as shown by immunohistochemistry. The expression profiles of the mRNAs of both ERs are mutually correlated throughout the gestation period, and their levels increase significantly in the last stages of gestation. No sexual differences in the mRNA expression of either ER-α or ER-ß have been found in our fetal specimens. The use of specific antisera against ER-α and ER-ß has allowed us to characterize and confirm the distribution of these receptors in the hypothalami of all fetal stages considered. Our results offer detailed information concerning the distribution of ER-α and ER-ß in the developing bovine hypothalamus and provide additional insights into the processes involved in the hypothalamic development of a mammal with a long gestation and a highly gyrencephalic brain.

Expression profile of the pore-forming subunits α1A and α1D in the foetal bovine hypothalamus: a mammal with a long gestation.

This study describes the expression of the voltage operated calcium channels (VOCCs) subunits α1A (typical of the P/Q family) and α1D (of the L family) in the bovine hypothalamus. The expression of both P/Q and L families has been characterized in the brain of adult mammals. However, their distribution and expression during foetal neuronal differentiation have not yet been determined. The expression profile of the α1A and α1D pore-forming subunits was investigated during four embryonic stages in bovine foetuses. Our data suggest that the expressions of α1A and α1D are correlated during development, with an increase only in males that peaks on the last period of gestation. Bovine male hypothalami showed significantly higher α1A and α1D expression values in comparison to female ones during the whole developmental period. In the females, the expression profiles of both genes were constant during all the developmental time. Immunohistochemical studies confirmed the presence of the α1A and α1D protein subunits in foetal hypothalamic neurones starting from the third foetal stage. Our data provide new information on the hypothalamic expression of α1A and α1D subunits during development in a mammal with a long gestation period and a large and convoluted brain.

Characterization of an established endothelial cell line from primary cultures of fetal sheep hypothalamus.

Immortalized cell lines from fetal brain are an experimental model for studying the in vitro molecular pathways regulating neural cell differentiation and the development of neural networks. The procedures are described to obtain an established cell line from the 90-day old fetal sheep hypothalamus. Viral oncogene LT-SV40 transformation was used to isolate a stable cell line (ENOS-01) that was characterized immunocytochemically. Immortalized cells can be classified as an endothelial cell line of hypothalamic microvasculature. Furthermore, mRNA expression and immunocytochemical of estrogen receptors α and ß were also evaluated. Since it is known that cerebral vessels are directly targeted by sex steroids, our established cell line represents an alternative system to study estradiol/receptor interactions during brain development. Our in vitro model can provide a tool to investigate the complex relationships among the cell types forming the blood-brain barrier, which is known to be involved in the pathogenesis of sheep transmissible neurological diseases.

Expression of aromatase P450(AROM) in the human fetal and early postnatal cerebral cortex.

Aromatase (P450(AROM)), the enzyme responsible for the conversion of testosterone (T) into 17-ß estradiol (E(2)), plays a crucial role in the sexual differentiation of specific hypothalamic nuclei. Moreover, recent findings indicate that local E(2) synthesis has an impact on other brain areas including hippocampus, temporal cortex and cerebellum, and may thus influence also cognitive functions. Numerous studies have described the expression and the distribution of P450(AROM) throughout ontogenesis and postnatal development of the central nervous system in several mammals, but data referring to humans are scarce. In the adult human brain, P450(AROM) has been detected in the hypothalamus, limbic areas, and in the basal forebrain, and described in glial cells of the cerebral cortex and hippocampus. In this study we report the expression, distribution and cellular localization of P450(AROM) in the human fetal and early postnatal cerebral cortex. In our series of fetal brains of the second trimester, P450(AROM) expression appeared at gestational week (GW) 17 and resulted limited to groups of cells localized close to the growing neuroepithelium in the ventricular and subventricular zones. At GWs 20-24, scattered P450(AROM) immunoreactive (-ir) neural cells were identified in the intermediate plate and subplate, and in the parietal cortical plate. In perinatal and early postnatal individuals the quantity of P450(AROM)-ir elements increased, and revealed the morphology typical of glial cells. Double labeling immunostaining with anti-GFAP and anti-P450(AROM) antisera, and subsequent confocal analysis, confirmed this observation. Our data show that the expression of P450(AROM) in the fetal cortex starts approx at the end of the fourth gestational month, but increases steadily only in the last trimester or in the early postnatal period. This temporal trend may suggest that P450(AROM) could act as a differentiation-promoting factor, based on timing of the steroid actions.

Expression and localization of aromatase P450AROM, estrogen receptor-α, and estrogen receptor-ß in the developing fetal bovine frontal cortex.

The enzyme aromatase (P450(AROM)) converts testosterone (T) into 17-ß estradiol (E(2)) and is crucial for the control of development of the central nervous system during ontogenesis. The effects of E(2) in various brain areas are mediated by the estrogen receptor alpha (ER-α) and the estrogen receptor beta (ER-ß). During fetal development, steroids are responsible for the sexual differentiation of the hypothalamus. Estrogens are also able to exert effects in other brain areas of the fetus including the frontal cortex, where they act through estrogen receptors (ERs) modulating cognitive function and affective behaviors. In this study we have determined the expression profiles of P450(AROM) and ERs in the fetal bovine frontal cortex by quantitative Real-Time PCR (qRT-PCR) throughout the prenatal development. The data show that the patterns of expression of both ERs are strongly correlated during pregnancy and increase in the last stage of gestation. On the contrary, the expression of P450(AROM) has no correlation with ERs expression and is not developmentally regulated. Moreover, we performed immunochemical studies showing that fetal neurons express P450(AROM) and the ERs. P450(AROM) is localized in the cytoplasm and only seldom present in the fine extensions of the cells; ER-α is detected predominantly in the soma whereas ER-ß is only present in the nucleus of a few cells. This study provides new data on the development of the frontal cortex in a long gestation mammal with a large convoluted brain.

Stimulation of Ca²+ signals in neurons by electrically coupled electrolyte-oxide-semiconductor capacitors.

Electrolyte-oxide-semiconductor capacitors (EOSCs) are a class of microtransducers for extracellular electrical stimulation that have been successfully employed to activate voltage-dependent sodium channels at the neuronal soma to generate action potentials in vitro. In the present work, we report on their use to control Ca²+ signalling in cultured mammalian cells, including neurons. Evidence is provided that EOSC stimulation with voltage waveforms in the microsecond or nanosecond range activates two distinct Ca²+ pathways, either by triggering Ca²+ entry through the plasma membrane or its release from intracellular stores. Ca²+ signals were activated in non-neuronal and neuronal cell lines, CHO-K1 and SH-SY5Y. On this basis, stimulation was tailored to rat and bovine neurons to mimic physiological somatic Ca²+ transients evoked by glutamate. Being minimally invasive and easy to use, the new method represents a versatile complement to standard electrophysiology and imaging techniques for the investigation of Ca²+ signalling in dissociated primary neurons and cell lines.

The upper respiratory tract of dolphins.

The functional anatomy of the respiratory system of dolphins has been scarcely studied. Specifically, the capacity of the system to resist pressure changes during diving has not been fully understood. Here we shortly describe the upper respiratory tract of dolphins based on three common species, the bottlenose dolphin Tursiops truncatus, the Risso's dolphin Grampus griseus, and the striped dolphin Stenella coeruleoalba. We emphasize the keymorphological features that represent evolutionary adaptations to life in the water, and, furthermore, also present a model of the tracheo-bronchial tree based on mechanical characterization and subsequent computational simulation of its biomechanical behaviour. Comparisons with the goat allowed us to determine how different structures may respond to diving-related pressure.

IgE-mediated allergy to corn: a 50 kDa protein, belonging to the Reduced Soluble Proteins, is a major allergen.

BACKGROUND: Although corn is often cited as an allergenic food, very few studies have been devoted to the identification of corn allergens and corn allergy has been rarely confirmed by double-blind, placebo-controlled food challenge (DBPCFC). Recently, Pastorello et al. (1) identified some salt-soluble IgE-binding proteins of corn flour as potential allergens. One of these, corresponding to corn Lipid Transfer Protein (LTP), appeared to be the major one. The aim of this study was to verify the clinical significance of the skin prick test (SPT) and CAP-FEIA CAP-System IgE fluozoenzyme immunosorbent assay (Pharmacia Diagnostic, Uppsala, Sweden) positivities to corn and to identify the presence of IgE-binding proteins in the corn flour salt-insoluble protein fractions (comprising up to 96% of the total protein) using sera of patients with DBPCFC-documented food allergy to corn. In addition the effect of cooking and proteolytic digestion on the corn allergens was investigated. METHODS: Sixteen subjects with SPT and CAP-FEIA positivities to corn flour were examined. Only six of them complained of suffering from urticaria and/or other symptoms after ingestion of corn-based foods. The patients were food challenged with cooked corn flour (polenta). IgE-binding proteins were detected by immunoblotting. The digestibility of the IgE-binding proteins was examined during a pepsin attack followed by a pancreatin digestion performed on a cooked corn flour sample. RESULTS: Oral challenge was positive only for six patients with symptoms after ingestion of corn. A 50 kDa protein, belonging to the corn Reduced Soluble Protein (RSP) fraction was recognized by the serum IgE of all the DBPCFC-positive subjects and resulted to be resistant to both heating and peptic/pancreatic digestion. SPT with the purified RSP fraction gave positive results for all of the DBPCFC-positive patients examined. CONCLUSIONS: SPT and CAP-FEIA positivities to corn flour had no clinical significance for most of the patients and food allergy to corn has to be proved by DBPCFC. A salt-unextractable protein of 50 kDa, belonging to the RSP fraction, represents a potential allergen in food hypersensitivity to corn because of its stability to cooking and digestion.

IgE binding to soluble and insoluble wheat flour proteins in atopic and non-atopic patients suffering from gastrointestinal symptoms after wheat ingestion.

BACKGROUND: The involvement of IgE-mediated hypersensitivity reactions in the genesis of gastrointestinal symptoms after ingestion of foods containing wheat has been rarely reported. OBJECTIVE: To detect IgE specifically binding to wheat proteins in the sera of atopic and non-atopic patients suffering from gastrointestinal symptoms after ingestion of wheat and to evaluate the reliability of skin prick test and CAP in the diagnosis of food allergy to wheat. METHODS: The sera of patients (10 atopic and 10 non-atopic) previously diagnosed as suffering from irritable bowel syndrome and complaining of symptoms after wheat ingestion were analysed by immunoblotting for IgE binding to water/salt-soluble and insoluble wheat flour proteins. RESULTS: All the atopic patients and only one of the non-atopic patients were positive to wheat CAP. For the patients tested, skin prick test was positive for all the atopic patients and for only one of the non-atopic patients. However, immunoblotting experiments showed the presence of specific IgE to wheat proteins in all the patients. Ten out of 11 of the wheat CAP-positive patients had IgE binding to a soluble 16-kDa band, but the same band was recognized, in a slighter way, by only two out of nine of the wheat CAP-negative patients. Moreover, although almost all of the patients were negative in CAP testing with gluten, 19 out of 20 recognized protein bands belonging to the prolamin fraction. CONCLUSIONS: For the atopic patients the positivity to skin prick test and CAP to wheat was in accordance with the immunoblotting results and a food allergy to wheat could be diagnosed. In these patients a major allergen was a 16-kDa band corresponding to members of the cereal alpha-amylase/trypsin inhibitors protein family, the major allergens involved in baker's asthma. In the non-atopic patients the positive immunoblotting results contrasted with the responses of the allergologic tests, indicating that the allergenic wheat protein preparations currently used are of limited value in detecting specific IgE to wheat and that the fraction of irritable bowel syndrome (IBS) patients with food allergy may be larger than believed.

Food allergy to wheat products: the effect of bread baking and in vitro digestion on wheat allergenic proteins. A study with bread dough, crumb, and crust.

The effect of baking and digestion on the allergenicity of wheat flour proteins has been studied. Pooled sera of patients suffering from food allergy to wheat products were tested for IgE binding to the proteins of the wheat dough and of the bread crumb and crust, before and after being in vitro digested. During in vitro digestion, the IgE binding protein components of the unheated dough tended to disappear, whereas a permanence of IgE recognition was evident for both the bread crumb and crust. This indicates that the baking process increases the resistance of the potential allergens of the wheat flour to proteolytic digestion, allowing them to reach the gastrointestinal tract, where they can elicit the immunological response. Therefore, the effects of baking must be carefully considered in studying food allergies to wheat products.

Modifications of wheat flour proteins during in vitro digestion of bread dough, crumb, and crust: an electrophoretic and immunological study.

The proteins of wheat flour have several biological activities that can affect human health and physiology when wheat-based foods are consumed. The modifications of bread crumb and crust proteins during an in vitro peptic/pancreatic digestion process were studied by electrophoresis and immunoblotting with polyclonal antibodies specific for single proteins or groups of homologous proteins of the wheat flour, and the results were compared to those obtained for an unheated dough sample. The results show that baking affects the extent of proteolysis and the immunological and physicochemical features of the digestion products in relation to the level of the heat treatment. Therefore, the results concerning the digestion of the unheated wheat flour or dough are not representative of what happens when baked products enter the human digestive tract.

In vitro assessment of acetic-acid-soluble proteins (glutenin) toxicity in celiac disease.

Acetic-acid-soluble storage proteins from gluten of the bread wheat cv. Sprint 3 were fractionated by adsorption chromatography on 2000 A controlled-pore glass (CPG) beads, and glutenin polymers with molecular mass higher than 10(7) Da and free from monomeric gliadins were recovered. The glutenin polymers were found to consist of high-molecular-weight (HMW) and low-molecular-weight (LMW) glutenin subunits. Peptic-tryptic (PT) digests of glutenins were examined for their agglutination activity on human myelogenous leukemia K 562(S) cells, agglutination being strongly correlated with toxicity for the celiac intestine. The peptide fraction at a concentration of 1 g/L of culture medium was able to agglutinate 30% of K 562(S) cells, suggesting a moderate toxic effect. This toxicity may be accounted for by homologies in amino acid sequences between glutenin subunits and alpha/beta- and gamma-gliadins.

Purification of wheat glutenin subunits by preparative acid and two-dimensional electrophoresis.

Reduced alkylated glutenin subunits from wheat flour were fractionated by preparative electrophoresis at acid pH. The high molecular weight glutenin subunits (HMW-GS) and some of the low molecular weight glutenin subunits (LMW-GS) were purified by this one-step procedure, whereas the remainder of the LMW-GS, comigrating in the acid system, were purified in a second step by electroendosmotic preparative electrophoresis in the presence of sodium dodecyl sulfate. The quantities of recovered protein were sufficient for biochemical characterization and/or antibody production.

Isolation of the catalytic subunit of a membrane-bound H(+)-pyrophosphatase from pea stem mitochondria.

The catalytic subunit of a membrane-bound pyrophosphatase was purified by electroendosmotic preparative electrophoresis from etiolated pea stem mitochondria. The enzyme was identified as a single peak relatively pure, because only a very limited number of polypeptides were detectable by SDS/PAGE of the active fractions. The pyrophosphatase was associated to a band with a molecular mass of 35 kDa, showing a specific activity of 0.7 mumol Pi . mg-1 protein . min-1 (37 degrees C, pH 8.0) and an apparent Km value of 200 microM. The hydrolytic activity required Mg2+, was inhibited by imidodiphosphate (HNO6P2Na4), Ca2+, F- and was stimulated by phospholipids. Cardiolipin, phophatidylcholine and phosphatidylethanolamine had the maximal activating effect. The isolated protein is very similar to the catalytic subunit of pyrophosphatases isolated from rat liver (beta-subunit) and Saccharomyces cerevisiae mitochondria.

Production and genetic characterization of near-isogenic lines in the bread-wheat cultivar Alpe.

Two biotypes of the bread-wheat cultivar Alpe were shown to possess contrasting alleles at each of the glutenin (Glu-B1, Glu-D1, Glu-B3 and Glu-D3) and gliadin (Gli-B1 and Gli-D1) loci on chromosomes 1B and 1D. Fourteen near-isogenic lines (NILs) were produced by crossing these biotypes and used to determine the genetic control of both low-molecular-weight (LMW) glutenin subunits and gliadins by means of one-dimensional or two-dimensional electrophoresis. Genes coding for the B, C and D groups of EMW subunits were found to be inherited in clusters tightly linked with those controlling gliadins. Southern-blot analysis of total genomic DNAs hybridized to a γ-gliadin-specific cDNA clone revealed that seven NILs lack both the Gli-D1 and Glu-D3 loci on chromosome 1D. Segregation data indicated that these "null" alleles are normally inherited. Comparison of the "null" NILs with those possessing allele b at the Glu-D3 locus showed one B subunit, seven C subunits and two D subunits, as fractionated by two-dimensional A-PAGExSDS-PAGE, to be encoded by this allele. Alleles b and k at Glu-B3 were found to code for two C subunits plus eight and six B subunits respectively, whereas alleles b and k at Gli-B1 each controlled the synthesis of two ß-gliadins, one γ and two ω-gliadins. The novel Gli-B5 locus coding for two ω-gliadins was shown to recombine with the Gli-B1 locus on chromosome 1B. The two-dimensional map of glutenin subunits showed α-gliadins encoded at the Gli-A2 locus on chromosome 6A. The use of Alpe NILs in the study of the individual and combined effects of glutenin subunits on dough properties is discussed.