Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli

To investigate the mechanism by which beta-hydroxy-beta-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25-50 muM). HMB (50 muM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70(S6k)), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1-2 h) stimulation of phosphorylation of mTOR and p70(S6k). However, in the presence of HMB, phosphorylation of mTOR, p70(S6k), and 4E-BP1 was increased, and inactive 4E-BP1-eIF4E complex was reduced, whereas the active eIF4G.eIF4E complex was increased, suggesting continual stimulation of protein synthesis. HMB alone reduced phosphorylation of elongation factor 2, but this effect was not seen in the presence of PIF. PIF induced autophosphorylation of the double-strand RNA-dependent protein kinase (PKR), leading to phosphorylation of eIF2 on the alpha-subunit, which would inhibit protein synthesis. However, in the presence of HMB, phosphorylation of PKR and eIF2alpha was attenuated, and this was also observed in skeletal muscle of cachectic mice administered HMB (0.25 g/kg). These results suggest that HMB attenuates the depression of protein synthesis by PIF in myotubes through multiple mechanisms.

Attenuation of proteasome-induced proteolysis in skeletal muscle by beta-hydroxy-beta-methylbutyrate in cancer-induced muscle loss

Loss of skeletal muscle is an important determinant of survival in patients with cancer-induced weight loss. The effect of the leucine metabolite beta-hydroxy-beta-methylbutyrate (HMB) on the reduction of body weight loss and protein degradation in the MAC16 model of cancer-induced weight loss has been compared with that of eicosapentaenoic acid (EPA), a recognized inhibitor of protein degradation. HMB was found to attenuate the development of weight loss at a dose greater than 0.125 g/kg accompanied by a small reduction in tumor growth rate. When EPA was used at a suboptimal dose level (0.6 g/kg) the combination with HMB seemed to enhance the anticachectic effect. Both treatments caused an increase in the wet weight of soleus muscle and a reduction in protein degradation, although there did not seem to be a synergistic effect of the combination. Proteasome activity, determined by the "chymotrypsin-like" enzyme activity, was attenuated by both HMB and EPA. Protein expression of the 20S alpha or beta subunits was reduced by at least 50%, as were the ATPase subunits MSS1 and p42 of the 19S proteasome regulatory subunit. This was accompanied by a reduction in the expression of E2(14k) ubiquitin-conjugating enzyme. The combination of EPA and HMB was at least as effective or more effective than either treatment alone. Attenuation of proteasome expression was reflected as a reduction in protein degradation in gastrocnemius muscle of cachectic mice treated with HMB. In addition, HMB produced a significant stimulation of protein synthesis in skeletal muscle. These results suggest that HMB preserves lean body mass and attenuates protein degradation through down-regulation of the increased expression of key regulatory components of the ubiquitin-proteasome proteolytic pathway, together with stimulation of protein synthesis.

Understanding the role of transforming growth factor beta signalling and epigenomics in osteoarthritis

Osteoarthritis (OA) is the most common form of arthritis with a high socioeconomic burden, with an incompletely understood etiology. Evidence suggests a role for the transforming growth factor beta (TGF-ß) signalling pathway and epigenomics in OA. The aim of this thesis was to understand the involvement of the TGF-ß pathway in OA and to determine the DNA methylation patterns of OA-affected cartilage as compared to the OA-free cartilage. First, I found that a common SNP in the BMP2 gene, a ligand in the Bone morphogenetic protein (BMP) subunit of TGF-ß pathway, was associated with OA in the Newfoundland population. I also showed a genetic association between SMAD3 - a signal transducer in the TGF-ß subunit of the TGF-ß signalling pathway - and the total radiographic burden of OA. I further demonstrated that SMAD3 is over-expressed in OA cartilage, suggesting an over activation of the TGF-ß signalling in OA. Next, I examined the connection of these genes in the regulation of matrix metallopeptidase 13 (MMP13) - an enzyme known to destroy extracellular matrix in OA cartilage - in the context of the TGF-ß signalling. The analyses showed that TGF-ß, MMP13, and SMAD3 were overexpressed in OA cartilage, whereas the expression of BMP2 was significantly reduced. The expression of TGF-ß was positively correlated with that of SMAD3 and MMP13, suggesting that TGF-ß signalling is involved in up-regulation of MMP13. This regulation, however, appears not to be controlled by SMAD3 signals, possibly due to the involvement of collateral signalling, and to be suppressed by BMP regulation in healthy cartilage, whose levels were reduced in end-stage OA. In a genome-wide DNA methylation analysis, I reported CpG sites differentially methylated in OA and showed that the cartilage methylome has a potential to distinguish between OA-affected and non-OA cartilage. Functional clustering analysis of the genes harbouring differentially methylated loci revealed that they are enriched in the skeletal system morphogenesis pathway, which could be a potential candidate for further OA studies. Overall, the findings from the present thesis provide evidence that the TGF-ß signalling pathway is associated with the development of OA, and epigenomics might be involved as a potential mechanism in OA.

Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography

Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 angstrom resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same 'double-psi beta-barrel' architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA transcription and DNA replication within the same protein superfamily. This study documents further the complex evolutionary history of the DNA replication apparatus in different domains of life and proposes a classification of all extant DNAPs.

Impaired Compensatory Beta-cell Function And Growth In Response To High-fat Diet In Ldl Receptor Knockout Mice.

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr-/- mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr-/- mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr-/- mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr-/- mice showed no significant changes in beta-cell mass, but lower islet-duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr-/- mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion.

Macrophage Cell Activation With Acute Apical Abscess Contents Determined By Interleukin-1 Beta And Tumor Necrosis Factor Alpha Production.

This clinical study has investigated the antigenic activity of bacterial contents from exudates of acute apical abscesses (AAAs) and their paired root canal contents regarding the stimulation capacity by levels of interleukin (IL)-1 beta and tumor necrosis factor alpha (TNF-α) throughout the root canal treatment against macrophage cells. Paired samples of infected root canals and exudates of AAAs were collected from 10 subjects. Endodontic contents were sampled before (root canal sample [RCS] 1) and after chemomechanical preparation (RCS2) and after 30 days of intracanal medication with calcium hydroxide + chlorhexidine gel (Ca[OH]2 + CHX gel) (RCS3). Polymerase chain reaction (16S rDNA) was used for detection of the target bacteria, whereas limulus amebocyte lysate was used to measure endotoxin levels. Raw 264.7 macrophages were stimulated with AAA exudates from endodontic contents sampled in different moments of root canal treatment. Enzyme-linked immunosorbent assays were used to measure the levels of TNF-α and IL-1 beta. Parvimonas micra, Porphyromonas endodontalis, Dialister pneumosintes, and Prevotella nigrescens were the most frequently detected species. Higher levels of endotoxins were found in samples from periapical exudates at RCS1 (P < .005). In fact, samples collected from periapical exudates showed a higher stimulation capacity at RCS1 (P < .05). A positive correlation was found between endotoxins from exudates with IL-1 beta (r = 0.97) and TNF-α (r = 0.88) production (P < .01). The significant reduction of endotoxins and bacterial species achieved by chemomechanical procedures (RCS2) resulted in a lower capacity of root canal contents to stimulate the cells compared with that at RCS1 (P < .05). The use of Ca(OH)2 + CHX gel as an intracanal medication (RCS3) improved the removal of endotoxins and bacteria from infected root canals (P < .05) whose contents induced a lower stimulation capacity against macrophages cells at RCS1, RCS2, and RCS3 (P < .05). AAA exudates showed higher levels of endotoxins and showed a greater capacity of macrophage stimulation than the paired root canal samples. Moreover, the use of intracanal medication improved the removal of bacteria and endotoxins from infected root canals, which may have resulted in the reduction of the inflammatory potential of the root canal content.

Enhanced Glucose-induced Intracellular Signaling Promotes Insulin Hypersecretion: Pancreatic Beta-cell Functional Adaptations In A Model Of Genetic Obesity And Prediabetes.

Obesity is associated with insulin resistance and is known to be a risk factor for type-2 diabetes. In obese individuals, pancreatic beta-cells try to compensate for the increased insulin demand in order to maintain euglycemia. Most studies have reported that this adaptation is due to morphological changes. However, the involvement of beta-cell functional adaptations in this process needs to be clarified. For this purpose, we evaluated different key steps in the glucose-stimulated insulin secretion (GSIS) in intact islets from female ob/ob obese mice and lean controls. Obese mice showed increased body weight, insulin resistance, hyperinsulinemia, glucose intolerance and fed hyperglycemia. Islets from ob/ob mice exhibited increased glucose-induced mitochondrial activity, reflected by enhanced NAD(P)H production and mitochondrial membrane potential hyperpolarization. Perforated patch-clamp examination of beta-cells within intact islets revealed several alterations in the electrical activity such as increased firing frequency and higher sensitivity to low glucose concentrations. A higher intracellular Ca(2+) mobilization in response to glucose was also found in ob/ob islets. Additionally, they displayed a change in the oscillatory pattern and Ca(2+) signals at low glucose levels. Capacitance experiments in intact islets revealed increased exocytosis in individual ob/ob beta-cells. All these up-regulated processes led to increased GSIS. In contrast, we found a lack of beta-cell Ca(2+) signal coupling, which could be a manifestation of early defects that lead to beta-cell malfunction in the progression to diabetes. These findings indicate that beta-cell functional adaptations are an important process in the compensatory response to obesity.

Partial-hepatectomized (70%) Model Shows A Correlation Between Hepatocyte Growth Factor Levels And Beta-cell Mass.

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Caracterização do complexo de inclusão ropivacaína: beta-ciclodextrina

Characteriza of the inclusion complex ropivacaine: beta-cyclodextrin. Ropivacaine (RVC) is a widely used local anesthetic. The complexation of RVC with beta-cyclodextrin (beta-CD) is of great interest for the development of more efficient local anesthetic formulations. The present work focuses on the characterization of the RVC:beta-CD complex by nuclear magnetic resonance (NMR). The stoichiometry of the complex is 1:2 RVC:beta-CD. DOSY-NMR shows that the association constant is 55.5 M-1. Longitudinal relaxation time results show that RVC changes its mobility in the presence of beta-CD. This study is focused on the physicochemical characterization of inclusion complexes that are potentials options for pain treatment.

Indução assimétrica 1,5-Anti na adição de enolatos de boro de metilcetonas beta-oxigenadas a aldeídos

High levels of substrate-based 1,5-stereoinduction are obtained in the boron-mediated aldol reactions of beta-oxygenated methyl ketones with achiral and chiral aldehydes. Remote induction from the boron enolates gives the 1,5-anti adducts, with the enolate pi-facial selectivity critically dependent upon the nature of the beta-alkoxy protecting group. This 1,5-anti aldol methodology has been strategically employed in the total synthesis of several natural products. At present, the origin of the high level of 1,5-anti induction obtained with the boron enolates is unclear, although a model based on a hydrogen bonding between the alkoxy oxygen and the formyl hydrogen has been recently proposed.

Expression of SMAD proteins, TGF-beta/activin signaling mediators, in human thyroid tissues

OBJECTIVE: To investigate the expression of SMAD proteins in human thyroid tissues since the inactivation of TGF-β/activin signaling components is reported in several types of cancer. Phosphorylated SMAD 2 and SMAD3 (pSMAD2/3) associated with the SMAD4 induce the signal transduction generated by TGF-β and activin, while SMAD7 inhibits this intracellular signaling. Although TGF-β and activin exert antiproliferative roles in thyroid follicular cells, thyroid tumors express high levels of these proteins. MATERIALS AND METHODS: The protein expression of SMADs was evaluated in multinodular goiter, follicular adenoma, papillary and follicular carcinomas by immunohistochemistry. RESULTS: The expression of pSMAD2/3, SMAD4 and SMAD7 was observed in both benign and malignant thyroid tumors. Although pSMAD2/3, SMAD4 and SMAD7 exhibited high cytoplasmic staining in carcinomas, the nuclear staining of pSMAD2/3 was not different between benign and malignant lesions. CONCLUSIONS: The finding of SMADs expression in thyroid cells and the presence of pSMAD2/3 and SMAD4 proteins in the nucleus of tumor cells indicates propagation of TGF-β/activin signaling. However, the high expression of the inhibitory SMAD7, mostly in malignant tumors, could contribute to the attenuation of the SMADs antiproliferative signaling in thyroid carcinomas.

Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7

No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) producedbyenterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.

Concentrações de retinol e de beta-caroteno séricos e perfil nutricional de crianças em Teresina, Piauí, Brasil

OBJETIVO: Avaliar as concentrações séricas de retinol e beta-caroteno de pré-escolares em Teresina, Piauí, com caracterização do perfil antropométrico e do consumo alimentar. MATERIAL E MÉTODOS: Estudo transversal envolvendo 135 crianças em creche municipal, com avaliação do estado nutricional pelos métodos: bioquímico (concentração sérica de retinol e beta-caroteno), antropométrico (índices de peso para estatura - P/E e estatura para idade - E/I) e dietético (freqüência de consumo alimentar). RESULTADOS: Observou-se prevalência de deficiência de vitamina A (DVA) de 8,9% (IC95%: 4,7 - 15,0%) e existência de associação entre suplementação anterior e concentrações de retinol, com maior proporção de crianças com níveis normais de retinol entre as suplementadas (p = 0,025). As concentrações de retinol e de beta-caroteno mostraram-se correlacionadas, porém com força leve a moderada (p < 0,021). Os percentuais de crianças com baixo P/E e de baixa E/I foram de 1,9% (IC95%: 0,2 - 6,8%) e 9,7% (IC95%: 4,8 - 17,1%), respectivamente. Na avaliação dietética verificou-se baixo consumo de alimentos ricos em vitamina A. CONCLUSÕES: A elevada prevalência de DVA nas crianças, combinada com a alta percentagem de crianças com valores aceitáveis de retinol, os baixos valores medianos de beta-caroteno, a alta percentagem de déficit estatural e a inadequação do consumo de alimentos ricos em vitamina A, indicam a necessidade de se aprimorar as estratégias de educação em saúde e nutrição da população, incentivando o consumo de alimentos fontes de vitamina A, como medidas auto-sustentáveis importantes no combate ao problema. Além disso, deve ser considerado o incentivo à fortificação dos alimentos e ao fortalecimento de Programas de suplementação.

Rheology of decane/water and triglyceride/water emulsions stabilized by 'beta'-casein and sodium caseinate

Emulsões estabilizadas por 'beta'-caseína e sódio caseinato tiveram suas propriedades reológicas investigadas em função da natureza da fase oleosa, da força iônica e do pH. Fases oleosas de características estruturais distintas, a saber, decano e óleos vegetais de alto teor triglicerídico, foram ensaiadas. A emulsificação dos sistemas contendo decano foi significativamente mais efetiva do que aquela das amostras contendo triglicérides. Efeitos de pH e força iônica mostraram-se relativamente pouco importantes sobre a capacidade emulsificante da proteína. As propriedades reológicas foram marcadamente distintas em cada caso, com estruturas de caráter sólido (G' G") sendo produzidas com decano, diferentemente do que foi observado para amostras contendo triglicérides, nas quais a viscoelasticidade não foi nem mesmo aparente. A relevância de aspectos espaciais da estrutura da fase oleosa no desenvolvimento do caráter viscoelástico é discutida. Propõe-se que os fatores responsáveis pelo comportamento distinto observado residam possivelmente na interface gotícula/meio dispersante, inacessível por microscopia óptica, e guardam pouca relação com tamanho ou forma da gotícula.

Emergence of Klebsiella pneumoniae carrying the novel extended-spectrum 'beta'-lactamase gene variants 'bla IND. SHV-40', 'bla IND. TEM-116' and the class I integron-associated 'bla IND. GES-7' in Brazil

A clinical Klebsiella pneumoniae isolate carrying the extended-spectrum beta-lactamase gene variants bla(SHV-40), bla(TEM-116) and bla(GES-7) was recovered. Cefoxitin and ceftazidime activity was most affected by the presence of these genes and an additional resistance to trimethoprim-sulphamethoxazole was observed. The bla(GES-7) gene was found to be inserted into a class 1 integron. These results show the emergence of novel bla(TEM) and bla(SHV) genes in Brazil. Moreover, the presence of class 1 integrons suggests a great potential for dissemination of bla(GES) genes into diverse nosocomial pathogens. Indeed, the bla(GES-7) gene was originally discovered in Enterobacter cloacae in Greece and, to our knowledge, has not been reported elsewhere