Polimorfismo da beta-lactoglobulina não afeta as características físico-químicas e a estabilidade do leite bovino

O objetivo deste trabalho foi avaliar o efeito do polimorfismo genético da beta-lactoglobulina, da raça e da sazonalidade sobre as características físico-químicas e estabilidade do leite bovino. Foram selecionados 5 rebanhos da raça Holandesa e 6 da Girolando. Amostras de leite e sangue foram coletadas de 660 vacas Holandesas e 293 Girolandos, num total de 953 amostras, obtidas em duas coletas na estação seca e duas na estação chuvosa. As amostras de leite foram submetidas à análise de acidez titulável, pH, crioscopia, e ao teste de estabilidade ao etanol (70, 76, 80 e 84ºGL). As amostras de sangue foram submetidas à reação em cadeia de polimerase, para determinação do polimorfismo da beta-lactoglobulina em ambas as raças estudadas. Não houve efeito do polimorfismo da beta-lactoglobulina sobre as características físico-químicas do leite. Observou-se efeito de raça (Holandesa e Girolando, respectivamente) sobre a acidez titulável (16,16 e 17,07°D), e da sazonalidade (estações chuvosa e seca, respectivamente) sobre a crioscopia (-0,5411 e -0,5376°H). Nas condições do estudo, observou-se efeito de raça e sazonalidade sobre a estabilidade do leite, com maior instabilidade do leite de Girolando, durante a estação seca. Não houve efeito do polimorfismo da beta-lactoglobulina sobre essa característica.

In leishmaniasis due to Leishmania guyanensis infection, distinct intralesional interleukin-10 and Foxp3 mRNA expression are associated with unresponsiveness to treatment.

The presence of intralesional natural regulatory T cells, characterized by the expression of Foxp3 mRNA, was analyzed in patients with localized leishmaniasis due to Leishmania guyanensis infection that was unresponsive to treatment with pentamidine isethionate. Foxp3 mRNA levels were associated with unresponsiveness to treatment among patients with a lesion duration of 1 month, but this association was not observed among patients with a lesion duration of <1 month. In conclusion, high intralesional expression of Foxp3 might be an indicator of poor response to treatment, depending on the duration of lesions.

Modulation du métabolisme du glucose astrocytaire par les cytokines pro-inflammatoires et le peptide beta-amyloïde. A la recherche d'une composante gliale aux processus neurodégénératifs

Résumé large public: Une altération localisée du métabolisme du glucose, le substrat énergétique préférentiellement utilisé dans le cerveau, est un trait caractéristique précoce de la maladie d'Alzheimer (MA). Il est maintenant largement admis que le beta-amyloïde, la neuroinflammation et le stress oxydatif participent au développement de la MA. Cependant les mécanismes cellulaires de la pathogenèse restent à identifier. Le métabolisme cérébral a ceci de remarquable qu'il repose sur la coopération entre deux types cellulaires, ainsi les astrocytes et les neurones constituent une unité métabolique. Les astrocytes sont notamment responsables de fournir aux neurones des substrats énergétiques, ainsi que des précurseurs du glutathion pour la défense contre le stress oxydatif. Ces fonctions astrocytaires sont essentielles au bon fonctionnement et à la survie neuronale; par conséquent, une altération de ces fonctions astrocytaires pourrait participer au développement de certaines maladies cérébrales. Le but de ce travail est, dans un premier temps, d'explorer les effets de médiateurs de la neuroinflammation (les cytokines pro-inflammatoires) et du peptide beta-amyloïde sur le métabolisme des astrocytes corticaux, en se focalisant sur les éléments en lien avec le métabolisme énergétique et le stress oxydatif. Puis, dans un second temps, de caractériser les conséquences pour les neurones des modifications du métabolisme astrocytaire induites par ces substances. Les résultats obtenus ici montrent que les cytokines pro-inflammatoires et le beta-amyloïde induisent une profonde altération du métabolisme astrocytaire, selon deux profils distincts. Les cytokines pro-inflammatoires, particulièrement en combinaison, agissent comme « découpleurs » du métabolisme énergétique du glucose, en diminuant l'apport potentiel de substrats énergétiques aux neurones. En plus de son effet propre, le peptide beta-amyloïde potentialise les effets des cytokines pro-inflammatoires. Or, dans le cerveau de patients atteints de la MA, les astrocytes sont exposés simultanément à ces deux types de substances. Les deux types de substances ont un effet ambivalent en termes de stress oxydatif. Ils induisent à la fois une augmentation de la libération de glutathion (potentiellement protecteur pour les neurones voisins) et la production d'espèces réactives de l'oxygène (potentiellement toxiques). Etant donné l'importance de la coopération entre astrocytes et neurones, ces modulations du métabolisme astrocytaire pourraient donc avoir un retentissement majeur sur les cellules environnantes, et en particulier sur la fonction et la survie neuronale. Résumé Les astrocytes et les neurones constituent une unité métabolique. Les astrocytes sont notamment responsables de fournir aux neurones des substrats énergétiques, tels que le lactate, ainsi que des précurseurs du glutathion pour la défense contre le stress oxydatif. Une altération localisée du métabolisme du glucose, le substrat énergétique préférentiellement utilisé dans le cerveau, est un trait caractéristique, précoce, de la maladie d'Alzheimer (MA). Il est maintenant largement admis que le beta-amyloïde, la neuroinflammation et le stress oxydatif participent au développement de la MA. Cependant, les mécanismes cellulaires de la pathogenèse restent à identifier. Le but de ce travail est d'explorer les effets des cytokines pro-inflammatoires (Il-1 ß et TNFα) et du beta-amyloïde (Aß) sur le métabolisme du glucose des astrocytes corticaux en culture primaire ainsi que de caractériser les conséquences, pour la viabilité des neurones voisins, des modifications du métabolisme astrocytaire induites par ces substances. Les résultats obtenus montrent que les cytokines pro-inflammatoires et le beta-amyloïde induisent une profonde altération du métabolisme astrocytaire, selon deux profils distincts. Les cytokines pro-inflammatoires, particulièrement en combinaison, agissent comme « découpleurs » du métabolisme glycolytique astrocytaire. Après 48 heures, le traitement avec TNFα et Il-lß cause une augmentation de la capture de glucose et de son métabolisme dans la voie des pentoses phosphates et dans le cycle de Krebs. A l'inverse, il cause une diminution de la libération de lactate et des stocks cellulaires de glycogène. En combinaison avec les cytokines tel qu'in vivo dans les cerveaux de patients atteints de MA, le peptide betaamyloïde potentialise les effets décrits ci-dessus. Isolément, le Aß cause une augmentation coordonnée de la capture de glucose et de toutes les voies de son métabolisme (libération de lactate, glycogenèse, voie des pentoses phosphate et cycle de Krebs). Les traitements altèrent peu les taux de glutathion intracellulaires, par contre ils augmentent massivement la libération de glutathion dans le milieu extracellulaire. A l'inverse, les deux types de traitements augmentent la production intracellulaire d'espèces réactives de l'oxygène (ROS). De plus, les cytokines pro-inflammatoires en combinaison augmentent massivement la production des ROS dans l'espace extracellulaire. Afin de caractériser l'impact de ces altérations métaboliques sur la viabilité des neurones environnants, un modèle de co-culture et des milieux conditionnés astrocytaires ont été utilisés. Les résultats montrent qu'en l'absence d'une source exogène d'antioxydants, la présence d'astrocytes favorise la viabilité neuronale ainsi que leur défense contre le stress oxydatif. Cette propriété n'est cependant pas modulée par les différents traitements. D'autre part, la présence d'astrocytes, et non de milieu conditionné, protège les neurones contre l'excitotoxicité due au glutamate. Les astrocytes prétraités (aussi bien avec le beta-amyloïde qu'avec les cytokines pro-inflammatoires) perdent cette propriété. Cet élément suggère que la perturbation du métabolisme astrocytaire causé par les cytokines pro-inflammatoires ou le beta-amyloïde pourrait participer à l'atteinte de la viabilité neuronale associée à certaines pathologies neurodégénératives.

Exendin-4 protects beta-cells from interleukin-1 beta-induced apoptosis by interfering with the c-Jun NH2-terminal kinase pathway.

OBJECTIVE: The pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) generates pancreatic beta-cells apoptosis mainly through activation of the c-Jun NH(2)-terminal kinase (JNK) pathway. This study was designed to investigate whether the long-acting agonist of the hormone glucagon-like peptide 1 (GLP-1) receptor exendin-4 (ex-4), which mediates protective effects against cytokine-induced beta-cell apoptosis, could interfere with the JNK pathway. RESEARCH DESIGN AND METHODS: Isolated human, rat, and mouse islets and the rat insulin-secreting INS-1E cells were incubated with ex-4 in the presence or absence of IL-1 beta. JNK activity was assessed by solid-phase JNK kinase assay and quantification of c-Jun expression. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Ex-4 inhibited induction of the JNK pathway elicited by IL-1 beta. This effect was mimicked with the use of cAMP-raising agents isobutylmethylxanthine and forskolin and required activation of the protein kinase A. Inhibition of the JNK pathway by ex-4 or IBMX and forskolin was concomitant with a rise in the levels of islet-brain 1 (IB1), a potent blocker of the stress-induced JNK pathway. In fact, ex-4 as well as IBMX and forskolin induced expression of IB1 at the promoter level through cAMP response element binding transcription factor 1. Suppression of IB1 levels with the use of RNA interference strategy impaired the protective effects of ex-4 against apoptosis induced by IL-1 beta. CONCLUSIONS: The data establish the requirement of IB1 in the protective action of ex-4 against apoptosis elicited by IL-1 beta and highlight the GLP-1 mimetics as new potent inhibitors of the JNK signaling induced by cytokines.

Epithelium-mesenchyme interactions control the activity of peroxisome proliferator-activated receptor beta/delta during hair follicle development.

Hair follicle morphogenesis depends on a delicate balance between cell proliferation and apoptosis, which involves epithelium-mesenchyme interactions. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and Akt1 are highly expressed in follicular keratinocytes throughout hair follicle development. Interestingly, PPARbeta/delta- and Akt1-deficient mice exhibit similar retardation of postnatal hair follicle morphogenesis, particularly at the hair peg stage, revealing a new important function for both factors in the growth of early hair follicles. We demonstrate that a time-regulated activation of the PPARbeta/delta protein in follicular keratinocytes involves the up-regulation of the cyclooxygenase 2 enzyme by a mesenchymal paracrine factor, the hepatocyte growth factor. Subsequent PPARbeta/delta-mediated temporal activation of the antiapoptotic Akt1 pathway in vivo protects keratinocytes from hair pegs against apoptosis, which is required for normal hair follicle development. Together, these results demonstrate that epithelium-mesenchyme interactions in the skin regulate the activity of PPARbeta/delta during hair follicle development via the control of ligand production and provide important new insights into the molecular biology of hair growth.

Genetic variation and environmental effects on beta-conglycinin and glycinin content in Brazilian soybean cultivars

The objective of this work was to determine genetic and environmental effects on beta-conglycinin and glycinin content in Brazilian soybean cultivars. The concentrations of these protein fractions were analyzed by scanning densitometry after electrophoresis, in 90 Brazilian soybean cultivars sown in Ponta Grossa, PR, in 2001. The effects of the sowing location were determined in the cultivar MG/BR 46 (Conquista), sown in 16 locations of Goiás and Minas Gerais states (Central Brazil), and in the cultivar IAS 5, sown in 12 locations of Paraná and São Paulo states (Southern Brazil), in 2002 soybean season. A significant variability for beta-conglycinin (7S) and glycinin (11S) protein fractions ratio was observed among the 90 Brazilian soybean cultivars. 'MS/BRS 169' (Bacuri) and 'BR-8' (Pelotas) presented the highest and the lowest 11S/7S ratios (2.76 and 1.17, respectively). Beta-conglycinin protein fractions presented more variability than glycinin protein fractions. Grouping test classified 7S proteins in seven groups, 11S proteins in four groups, and protein fraction ratios (11S/7S) in nine groups. Significant effect of sowing locations was also observed on protein fractions contents. There is a good possibility of breeding for individual protein fractions, and their subunits, without affecting protein content.

Antagonistic regulation of a proline-rich transcription factor by transforming growth factor beta and tumor necrosis factor alpha.

Transforming growth factor beta (TGF-beta) and tumor necrosis factor alpha (TNF-alpha) often exhibit antagonistic actions on the regulation of various activities such as immune responses, cell growth, and gene expression. However, the molecular mechanisms involved in the mutually opposing effects of TGF-beta and TNF-alpha are unknown. Here, we report that binding sites for the transcription factor CTF/NF-I mediate antagonistic TGF-beta and TNF-alpha transcriptional regulation in NIH3T3 fibroblasts. TGF-beta induces the proline-rich transactivation domain of specific CTF/NF-I family members, such as CTF-1, whereas TNF-alpha represses both the uninduced as well as the TGF-beta-induced CTF-1 transcriptional activity. CTF-1 is thus the first transcription factor reported to be repressed by TNF-alpha. The previously identified TGF-beta-responsive domain in the proline-rich transcriptional activation sequence of CTF-1 mediates both transcriptional induction and repression by the two growth factors. Analysis of potential signal transduction intermediates does not support a role for known mediators of TNF-alpha action, such as arachidonic acid, in CTF-1 regulation. However, overexpression of oncogenic forms of the small GTPase Ras or of the Raf-1 kinase represses CTF-1 transcriptional activity, as does TNF-alpha. Furthermore, TNF-alpha is unable to repress CTF-1 activity in NIH3T3 cells overexpressing ras or raf, suggesting that TNF-alpha regulates CTF-1 by a Ras-Raf kinase-dependent pathway. Mutagenesis studies demonstrated that the CTF-1 TGF-beta-responsive domain is not the primary target of regulatory phosphorylations. Interestingly, however, the domain mediating TGF-beta and TNF-alpha antagonistic regulation overlapped precisely the previously identified histone H3 interaction domain of CTF-1. These results identify CTF-1 as a molecular target of mutually antagonistic TGF-beta and TNF-alpha regulation, and they further suggest a molecular mechanism for the opposing effects of these growth factors on gene expression.

Dual inhibitors of beta-amyloid aggregation and acetylcholinesterase as multi-target anti-Alzheimer drug candidates

Notwithstanding the functional role that the aggregates of some amyloidogenic proteins can play in different organisms, protein aggregation plays a pivotal role in the pathogenesis of a large number of human diseases. One of such diseases is Alzheimer"s disease (AD), where the overproduction and aggregation of the β-amyloid peptide (Aβ) are regarded as early critical factors. Another protein that seems to occupy a prominent position within the complex pathological network of AD is the enzyme acetylcholinesterase (AChE), with classical and non-classical activities involved at the late (cholinergic deficit) and early (Aβ aggregation) phases of the disease. Dual inhibitors of Aβ aggregation and AChE are thus emerging as promising multi-target agents with potential to efficiently modify the natural course of AD. In the initial phases of the drug discovery process of such compounds, in vitro evaluation of the inhibition of Aβ aggregation is rather troublesome, as it is very sensitive to experimental assay conditions, and requires expensive synthetic Aβ peptides, which makes cost-prohibitive the screening of large compound libraries. Herein, we review recently developed multi-target anti-Alzheimer compounds that exhibit both Aβ aggregation and AChE inhibitory activities, and, in some cases also additional valuable activities such as BACE-1 inhibition or antioxidant properties. We also discuss the development of simplified in vivo methods for the rapid, simple, reliable, unexpensive, and high-throughput amenable screening of Aβ aggregation inhibitors that rely on the overexpression of Aβ42 alone or fused with reporter proteins in Escherichia coli.

FXYD Proteins Reverse Inhibition of the Na+-K+ Pump Mediated by Glutathionylation of Its {beta}1 Subunit.

The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

The CD8 beta polypeptide is required for the recognition of an altered peptide ligand as an agonist.

T cell activation is triggered by the specific recognition of cognate peptides presented by MHC molecules. Altered peptide ligands are analogs of cognate peptides which have a high affinity for MHC molecules. Some of them induce complete T cell responses, i.e. they act as agonists, whereas others behave as partial agonists or even as antagonists. Here, we analyzed both early (intracellular Ca2+ mobilization), and late (interleukin-2 production) signal transduction events induced by a cognate peptide or a corresponding altered peptide ligand using T cell hybridomas expressing or not the CD8 alpha and beta chains. With a video imaging system, we showed that the intracellular Ca2+ response to an altered peptide ligand induces the appearance of a characteristic sustained intracellular Ca2+ concentration gradient which can be detected shortly after T cell interaction with antigen-presenting cells. We also provide evidence that the same altered peptide ligand can be seen either as an agonist or a partial agonist, depending on the presence of CD8beta in the CD8 co-receptor dimers expressed at the T cell surface.

Beta-catenin Status in P?aediatric Medulloblastomas: correlation of immunohistochemical expression with mutational status, genetic profiles, and clinical characteristics.

Medulloblastoma is the most frequent malignant paediatric brain tumour. The activation of the Wnt/beta-catenin pathway occurs in 10-15% of medulloblastomas and has been recently described as a marker for favourable patient outcome. We report a series of 72 paediatric medulloblastomas evaluated for beta-catenin protein expression, CTNNB1 mutations, and comparative genomic hybridization. Gene expression profiles were also available in a subset of 40 cases. Immunostaining of beta-catenin showed extensive nuclear staining (>50% of the tumour cells) in six cases and focal nuclear staining (<10% of cells) in three cases. The other cases either exhibited a signal strictly limited to the cytoplasm (58 cases) or were negative (five cases). CTNNB1 mutations were detected in all beta-catenin extensively nucleopositive cases. The expression profiles of these cases documented strong activation of the Wnt/beta-catenin pathway. Remarkably, five out of these six tumours showed a complete loss of chromosome 6. In contrast, cases with focal nuclear beta-catenin staining, as well as tumours with negative or cytoplasmic staining, never demonstrated CTNNB1 mutation, Wnt/beta-catenin pathway activation or chromosome 6 loss. Patients with extensive nuclear staining were significantly older at diagnosis and were in continuous complete remission after a mean follow-up of 75.7 months (range 27.5-121.2 months) from diagnosis. All three patients with focal nuclear staining of beta-catenin died within 36 months from diagnosis. Altogether, these data confirm and extend previous observations that CTNNB1-mutated tumours represent a distinct molecular subgroup of medulloblastomas with favourable outcome, indicating that therapy de-escalation should be considered. International consensus on the definition criteria of this distinct medulloblastoma subgroup should be achieved.

Post-switching beta synchronization reveals concomitant sensory reafferences and active inhibition processes

It is known that post-movement beta synchronization (PMBS) is involved both in active inhibition and in sensory reafferences processes. The aim of this study was examine the temporal and spatial dynamics of the PMBS involved during multi-limb coordination task. We investigated post-switching beta synchronization (assigned PMBS) using time-frequency and source estimations analyzes. Participants (n = 17) initiated an auditory-paced bimanual tapping. After a 1500 ms preparatory period, an imperative stimulus required to either selectively stop the left while maintaining the right unimanual tapping (Switch condition: SWIT) or to continue the bimanual tapping (Continue condition: CONT). PMBS significantly increased in SWIT compared to CONT with maximal difference within right central region in broad-band 14âeuro"30 Hz and within left central region in restricted-band 22âeuro"26 Hz. Source estimations localized these effects within right pre-frontal cortex and left parietal cortex, respectively. A negative correlation showed that participants with a low percentage of errors in SWIT had a large PMBS amplitude within right parietal and frontal cortices. This study shows for the first time simultaneous PMBS with distinct functions in different brain regions and frequency ranges. The left parietal PMBS restricted to 22âeuro"26 Hz could reflect the sensory reafferences of the right hand tapping disrupted by the switching. In contrast, the right pre-frontal PMBS in a broad-band 14âeuro"30 Hz is likely reflecting the active inhibition of the left hand stopped. Finally, correlations between behavioral performance and the magnitude of the PMBS suggest that beta oscillations can be viewed as a marker of successful active inhibition.

Finite mixture analysis of beauty-contest data using generalised beta distributions

This paper introduces a mixture model based on the beta distribution, without preestablishedmeans and variances, to analyze a large set of Beauty-Contest data obtainedfrom diverse groups of experiments (Bosch-Domenech et al. 2002). This model gives a bettert of the experimental data, and more precision to the hypothesis that a large proportionof individuals follow a common pattern of reasoning, described as iterated best reply (degenerate),than mixture models based on the normal distribution. The analysis shows thatthe means of the distributions across the groups of experiments are pretty stable, while theproportions of choices at dierent levels of reasoning vary across groups.

Regulation of the transforming growth factor beta-responsive transcription factor CTF-1 by calcineurin and calcium/calmodulin-dependent protein kinase IV.

Transforming growth factor beta (TGF-beta) is a pluripotent peptide hormone that regulates various cellular activities, including growth, differentiation, and extracellular matrix protein gene expression. We previously showed that TGF-beta induces the transcriptional activation domain (TAD) of CTF-1, the prototypic member of the CTF/NF-I family of transcription factors. This induction correlates with the proposed role of CTF/NF-I binding sites in collagen gene induction by TGF-beta. However, the mechanisms of TGF-beta signal transduction remain poorly understood. Here, we analyzed the role of free calcium signaling in the induction of CTF-1 transcriptional activity by TGF-beta. We found that TGF-beta stimulates calcium influx and mediates an increase of the cytoplasmic calcium concentration in NIH3T3 cells. TGF-beta induction of CTF-1 is inhibited in cells pretreated with thapsigargin, which depletes the endoplasmic reticulum calcium stores, thus further arguing for the potential relevance of calcium mobilization in TGF-beta action. Consistent with this possibility, expression of a constitutively active form of the calcium/calmodulin-dependent phosphatase calcineurin or of the calcium/calmodulin-dependent kinase IV (DeltaCaMKIV) specifically induces the CTF-1 TAD and the endogenous mouse CTF/NF-I proteins. Both calcineurin- and DeltaCaMKIV-mediated induction require the previously identified TGF-beta-responsive domain of CTF-1. The immunosuppressants cyclosporin A and FK506 abolish calcineurin-mediated induction of CTF-1 activity. However, TGF-beta still induces the CTF-1 TAD in cells treated with these compounds or in cells overexpressing both calcineurin and DeltaCaMKIV, suggesting that other calcium-sensitive enzymes might mediate TGF-beta action. These results identify CTF/NF-I as a novel calcium signaling pathway-responsive transcription factor and further suggest multiple molecular mechanisms for the induction of CTF/NF-I transcriptional activity by growth factors.