Dietary nucleotide supplementation as an alternative to in-feed antibiotics in weaned piglets.

Nucleotides are important to cell growth and division and are crucial to the rapid proliferation of such cells as the intestinal mucosa and immune cells. Accordingly, the nucleotide requirements of animals are high during periods of rapid growth and periods of stress like post-weaning period. Thus, nucleotide supplementation may be a possible alternative to in-feed antibiotics as growth promoter in this phase. The study aimed to evaluate dietary nucleotide supplementation as an alternative to in-feed antibiotics on performance and gut health of weaned piglets. Ninety-six 21-day-old piglets, weighing 7.44 ±â€¯0.65 kg, were allocated into 1 of 3 treatments (8 pens per treatment; 4 pigs per pen) in a 14-day trial. Dietary treatments consisted of control: corn-soybean meal-based diet; nucleotides: control +2 g/kg of a nutritional additive with purified nucleotides; and antibiotic: control +0.8 g/kg of antibiotic growth promoter based on colistin and tylosin. Performance variables and fecal score were not affected (P > 0.05) by supplementing nucleotide or antibiotic. Nucleotides treatment had similar effect to antibiotic and superior to control (P < 0.05) on enhancing duodenum villus height, jejunum crypt depth, and reduction of Paneth cellular area. Duodenum and ileum of animals supplemented with nucleotides or antibiotics had higher (P < 0.05) number of proliferating cells than did those of control animals, whereas the jejunum of animals that received antibiotic diets presented more (P < 0.05) proliferating cells than either the nucleotides or control animals. Jejunum of nucleotide-treated piglets showed a greater number of apoptotic cells than those fed antibiotic or control diets (P < 0.05). Nucleotides and antibiotic treatments decreased the B lymphocyte counts in duodenum and ileum (P < 0.05) but increased in the jejunum (P < 0.05), when compared to the control treatment. Relative abundance of mitogen-activated protein kinases-6, haptoglobin, and tumor necrosis factor-α mRNA was not influenced (P > 0.05) by treatments. In the ileal, antibiotic supplementation reduced total bacteria quantification compared to nucleotide supplementation or the control (P < 0.05), whereas nucleotides supplementation increased enterobacteria proliferation compared to the antibiotic or control diets (P < 0.05). However, nucleotides and antibiotic reduced (P < 0.05) colon total bacteria quantification when compared to control. These results suggest that the nucleotides source used to weaned piglets improved gut health by modulating the local immune response and modulating intestinal mucosa development, and, therefore, nucleotides may be an alternative to antibiotics as growth promoters.

The oral cavity in leprosy: what clinicians need to know.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, a bacillus that has a tropism for skin and peripheral nerves. Leprosy treatment is based on a multidrug therapy established by the World Health Organization in 1982 and, despite its widespread use, Brazil ranks second worldwide in numbers of cases. Oral involvement in leprosy has been poorly described in the literature, and few studies have shown that although the bacillus is found in mucosa, specific leprosy lesions are rare and affect patients with advanced stages of the disease. This review aimed to assess the literature on oral manifestations in leprosy and the aspects involving oral cavity in leprosy pathogenesis.

Determinação de parâmetros farmacognósticos para as folhas de Erythroxylum suberosum A. St.-Hilaire (Erythroxylaceae) coletadas no município de Goiânia, GO. / Determination of pharmacognostic parameters for the leaves of Erythroxylum suberosum A. St.-Hilaire (Erythroxylaceae) collected in the city of Goiania, GO

RESUMO As plantas medicinais representam um dos principais recursos terapêuticos utilizado pelo homem para a cura e prevenção de doenças, no entanto, para que o tratamento seja seguro e eficaz é necessário conhecer seus componentes químicos. Dessa forma, o intuito deste trabalho foi determinar os parâmetros farmacognosticos para o estabelecimento do controle de qualidade da droga vegetal composta pelas folhas de Erythroxylum suberosum A. St.-Hil., pertencentes à família Erythroxylaceae ocorrente no Cerrado. Para tanto, foram coletadas folhas adultas nas proximidades da Universidade Federal de Goiás, Goiânia-GO. Foram determinados o teor de umidade por meio de balança com irradiação de raios infravermelho e o teor de cinzas totais. Na prospecção fitoquímica foram pesquisadas as classes de metabólitos secundários e realizou-se o doseamento de fenóis totais, flavonoides e taninos totais. Os resultados dos testes de pureza realizados, teor de umidade e teor de cinzas totais, estão de acordo com os limites estabelecidos pelas especificações farmacopeicas. As análises fitoquímicas evidenciaram a presença de flavonoides, taninos, cumarinas, saponinas e resinas. No doseamento de fenóis totais, taninos e flavonoides presentes nas folhas de E. suberosum A. St.-Hil. obteve-se respectivamente 17,97%, 6,31%, 3,87%. Estes resultados confirmam os dados da literatura quanto à presença destes compostos em Erythroxylaceae, pois de acordo com nas folhas de E. tortuosum obteve-se valores de 10%, 8,4% e 0,064% de Fenóis, Taninos e flavonoides, respectivamente e nas folhas de E. deciduum foram encontrados 12,04% de fenóis totais, 0,87% de taninos e 1,37% de flavonoides. Os resultados encontrados no presente trabalho também se tornam relevantes quando comparadas as quantidades desses metabólitos com espécies clássicas na biossíntese de compostos fenólicos como o teor de taninos de Stryphnodendron adstringens (Mart.) Coville e em Eugenia uniflora L. foi de 29,9% e 2,96%, respectivamente. Teores de flavonóides que variaram entre 0,65% a 0,79% na Calendula officinalis L. e Ginkgo bilobaL. 0,59%, 0,75% e 0,79%. Sendo assim, a quantidade considerável de compostos fenólicos obtida em E. suberosum A. St.-Hil. sugere que a espécie possua um importante potencial terapêutico e quem sabe potencial antioxidante, a ser explorado em estudos posteriores.

ABSTRACT Medicinal plants represent a major therapeutic resources used by man for the cure and prevention of diseases, however, that the treatment be safe and effective it is necessary to know their chemical components. Thus, the aim of this work was to determine the pharmacognostic parameters for the establishment of the plant drug quality control made by the leaves of Erythroxylum suberosum A. St.-Hil., belonging to the family Erythroxylaceae occurring in the Cerrado. To this end, adult leaves were collected near the Federal University of Goiás, Goiânia-GO. It was determined the moisture content through scale radiating infrared rays and the content of total ash. The phytochemical screening of secondary metabolites classes were surveyed and performed the determination of total phenols, flavonoids and total tannins. The results of the purity tests, moisture content and total ash content, are in accordance with the limits established by the pharmacopeic specifications. The phytochemical analysis revealed the presence of flavonoids, tannins, coumarins, saponins and resins. The determination of total phenols, tannins and flavonoids quantities present in the leaves of E. suberosum A. St.-Hil. were obtained respectively as 17.97%, 6.31%, 3.87%. These results confirm the literature data for the presence of these compounds in Erythroxylaceae, since according to the leaves of E. tortuosum yielded values of 10%, 8.4% and 0.064% of phenols, tannins and flavonoids, respectively. On the E. deciduum leaves were found 12.04% of total phenols, tannins 0.87% and 1.37% of flavonoids. The results of this work also become relevant when comparing the amounts of these metabolites with classic species in the biosynthesis of phenolic compounds such as tannins content in barbatimão and pitanga was 29.9% and 2.96%, respectively. Flavonoid contents ranging from 0.65% to 0.79% in calendula and ginkgo 0.59%, 0.75% and 0.79%. Thus, considerable amount of phenolic compounds obtained in E. suberosum. A. St. Hil. It suggests that the species has an important therapeutic potential and who knows antioxidant potential to be explored in further studies.

A novel positive feedback loop mediated by the docking protein Gab1 and phosphatidylinositol 3-kinase in epidermal growth factor receptor signaling.

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4, 5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR.

Activation of the JNK pathway is essential for transformation by the Met oncogene.

The Met/Hepatocyte Growth Factor (HGF) receptor tyrosine kinase is oncogenically activated through a rearrangement that creates a hybrid gene Tpr-Met. The resultant chimeric p65(Tpr-Met) protein is constitutively phosphorylated on tyrosine residues in vivo and associates with a number of SH2-containing signaling molecules including the p85 subunit of PI-3 kinase and the Grb2 adaptor protein, which couples receptor tyrosine kinases to the Ras signaling pathway. Mutation of the binding site for Grb2 impairs the ability of Tpr-Met oncoprotein to transform fibroblasts, suggesting that the activation of the Ras/MAP kinase signaling pathway through Grb2 may be essential for cellular transformation. To test this hypothesis dominant-negative mutants of Grb2 with deletions of the SH3 domains were introduced into Tpr-Met transformed fibroblasts. Cells overexpressing the mutants were found to be morphologically reverted and exhibited reduced growth in soft agar. Surprisingly, the Grb2 mutants blocked activation of the JNK/SAPK but not MAP kinase activity induced by the Tpr-Met oncoprotein. Additionally, cells expressing dominant-negative Grb2 mutants had reduced PI-3-kinase activity and dominant-negative mutants of Rac1 blocked both Tpr-Met-induced transformation and activation of JNK. These experiments reveal a novel link between Met and the JNK pathway, which is essential for transformation by this oncogene.

Reduced activation of RAF-1 and MAP kinase by a fibroblast growth factor receptor mutant deficient in stimulation of phosphatidylinositol hydrolysis.

Signaling via the fibroblast growth factor receptor 1 (FGFR1, flg) was analyzed in Ba/F3 hematopoietic cells expressing either wild-type or a mutant FGF receptor (Y766F) unable to activate phospholipase C-gamma (PLC-gamma) and stimulate phosphatidylinositol (PI) hydrolysis. Stimulation of cells expressing wild-type or mutant FGFR with acidic FGF (aFGF) caused similar activation of Ras. However, an approximately 3-fold reduced activation of Raf-1 and MAP kinase was observed in aFGF-stimulated cells expressing mutant receptors as compared to cells expressing wild-type FGF receptors. Comparison of phosphopeptide maps of Raf-1 immunoprecipitated from the two cell types activated by either aFGF or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate) suggests that Raf-1 is phosphorylated by both Ras-dependent and PLC-gamma-dependent mechanisms. In spite of the differential effect on Raf-1 and MAP kinase activation, aFGF stimulated similar proliferation of cells expressing wild-type or mutant receptors indicating that Ras-dependent activation of Raf-1 and MAP kinase is sufficient for transduction of FGFR mitogenic signals. Ras may also activate signal transduction pathways that are complementary or parallel to the MAP kinase pathway to stimulate cell proliferation.

Efficient cell transformation by the Tpr-Met oncoprotein is dependent upon tyrosine 489 in the carboxy-terminus.

The receptor for hepatocyte growth factor/scatter factor (HGF/SF) was originally identified as an oncogene, Tpr-Met, which consists of the cytoplasmic tyrosine kinase domain of the HGF/SF receptor (Met) fused down-stream of sequences encoded by the tpr gene. As a consequence of this rearrangement the Tpr-Met fusion oncoprotein is localized to the cytoplasm and is a constitutively activated kinase. To identify signalling pathways important for Tpr-Met-mediated cell transformation we have generated tyrosine to phenylalanine mutants of Tpr-Met that are compromised in their ability to transform Fischer rat 3T3 (Fr3T3) cells in culture. We show that a single tyrosine residue in the carboxy terminus of Tpr-Met (residue 489) is essential for efficient transformation of Fr3T3 cells by this oncoprotein. Mutation of tyrosine 489 to phenylalanine does not affect the exogenous kinase activity of the Tpr-Met oncoprotein toward casein, but it impairs the ability of the mutant protein to bind to and activate phosphatidylinositol 3 kinase in vivo and completely abolishes the in vivo association with the Grb2 adaptor protein as well as the association and/or phosphorylation of an unknown protein of 110 kDa. These data are consistent with a single tyrosine residue in the Tpr-Met oncoprotein being essential for the activation of several signalling pathways which lead to the transformation of Fr3T3 fibroblasts.

Autophosphorylation modulates the kinase activity and oncogenic potential of the Met receptor tyrosine kinase.

The met oncogene is activated by a genomic rearrangement that generates a hybrid protein containing tpr sequences at its amino terminus fused directly to the met (hepatocyte growth factor/scatter factor) receptor tyrosine kinase domain. The resultant p65Tpr-Met hybrid protein possesses tyrosine kinase activity and is constitutively phosphorylated on tyrosine in vivo. A substitution of the conserved lysine in the catalytic domain abolished kinase activity and the transforming potential of Tpr-Met demonstrating the requirement for kinase activity for transformation. To study the role of autophosphorylation at particular tyrosine residues on the transforming activity of Tpr-Met, the major autophosphorylation sites were identified by two dimensional phosphopeptide mapping. Two tyrosine residues in the catalytic domain, tyrosine 365 and tyrosine 366, were found to be the major autophosphorylation sites both in vitro and in vivo. Mutation of these sites singly or together modulates the biochemical and biological properties of Tpr-Met. Compared with the wild-type Tpr-Met, mutants have a reduced in vitro kinase activity when measured by levels of autophosphorylation and by their ability to phosphorylate an exogenous substrate and have little or no transforming activity. These results indicate that autophosphorylation at critical tyrosine residues is required for activation of the kinase activity of Tpr-Met and for cell transformation by this oncogene.

Oncogenic activation of tyrosine kinases.

Tyrosine kinases comprise the largest group of oncoproteins, a fact that underscores the importance of reversible tyrosine phosphorylation in the regulation of essential cellular functions. Oncogenic activation of tyrosine kinases results in the constitutive activation of what is normally a conditionally regulated enzyme activity. Studies of tyrosine kinase oncoproteins, and a comparison with their corresponding proto-oncogene products, have identified important functional and regulatory domains within these proteins, positive and negative regulators of their enzyme activities and signalling cascades that control cell growth and differentiation.

Dimerization mediated through a leucine zipper activates the oncogenic potential of the met receptor tyrosine kinase.

Oncogenic activation of the met (hepatocyte growth factor/scatter factor) receptor tyrosine kinase involves a genomic rearrangement that generates a hybrid protein containing tpr-encoded sequences at its amino terminus fused directly to the met-encoded receptor kinase domain. Deletion of Tpr sequences abolishes the transforming ability of this protein, implicating this region in oncogenic activation. We demonstrate, by site-directed mutagenesis and coimmunoprecipitation experiments, that a leucine zipper motif within Tpr mediates dimerization of the tpr-met product and is essential for the transforming activity of the met oncogene. By analogy with ligand-stimulated activation of receptor tyrosine kinases, we propose that constitutive dimerization mediated by a leucine zipper motif within Tpr is responsible for oncogenic activation of the Met kinase. The possibility that this mechanism of activation represents a paradigm for a class of receptor tyrosine kinase oncogenes activated by DNA rearrangement is discussed.

Isoforms of the met receptor tyrosine kinase.

Hepatocyte growth factor/scatter factor (HGF-SF), a multifunctional cytokine, is the ligand for the met receptor tyrosine kinase. Multiple met mRNAs of 8, 7, 5, 3 and 1.6-kb in size have been identified in human cell lines and tissue. To investigate the biological function of these various isoforms we have isolated cDNA clones corresponding to some of the differentially spliced met mRNAs. Characterization of these cDNAs suggests that by alternative splicing and possibly by use of distinct transcription initiation sites the met HGF-SF receptor is expressed in various isoforms. We have demonstrated that there are two met 8-kb mRNAs that differ through alternative splicing of a 54-bp exon that maintains the open reading frame such that these proteins differ by only 18 aa in their extracellular domain. The -54-bp form corresponds to the most abundant 8-kb met RNA and encodes the p190 met alpha beta heterodimer. In contrast the +54-bp mRNA encodes a protein of 170 kd that is not cleaved yet is expressed at the cell surface and has in vitro kinase activity. The 7-kb mRNA differs by alternative splicing such that it encodes a protein with a distinct amino terminus. Unlike these met RNAs, the 1.6-kb mRNA has new 5' and 3' sequences and encodes a protein that shares homology with the extracellular domain of the met RTK but has a unique carboxy terminus. Thus multiple met RNAs encode proteins that differ in both the extracellular ligand binding domain and within the cytoplasmic domain suggesting that these different met isoforms may have distinct biological activities.

Alternative splicing generates isoforms of the met receptor tyrosine kinase which undergo differential processing.

The met proto-oncogene is a member of the family of tyrosine kinase growth factor receptors. We describe the isolation and characterization of a cDNA clone (pOK) for the met receptor from a gastric carcinoma cell line. This clone differs from the published cDNA clone by the absence of 54 bp predicted to encode 18 amino acids in the extracellular domain. The pOK cDNA corresponds to the most abundant met RNA species of 8 kb expressed in human cell lines and tissue, and we show that there are in fact two 8-kb met receptor tyrosine kinase (RTK) isoforms that are generated by alternative splicing. This newly described met isoform when transiently expressed in COS cells encodes a protein of 190 kDa which corresponds in size to the p190 met alpha beta heterodimer expressed in human cell lines. Furthermore, we show that the 190-kDa product of pOK consists of the 140-kDa met beta subunit associated with the 50-kDa met alpha subunit. This finding suggests that both the alpha and beta met chains are encoded by this construct and confirms the hypothesis that a single chain precursor is cleaved to produce both subunits of met. In contrast, the previously characterized met isoform corresponds to a minor met RNA species and encodes a protein of 170 kDa that is not cleaved yet is processed in a manner that allows cell surface expression. Both met RTK isoforms are autophosphorylated in the in vitro kinase assay. These results suggest that different isoforms of the met RTK may have distinct biological activities.

Characterization of an expanded large granular lymphocyte subset lacking natural killer activity present in renal allograft recipients.

We have previously reported that, in long-term renal allograft recipients who receive chronic chemical immunosuppression and who are at risk for late chronic viral infections and virus associated tumors, the percentage of lymphocytes the phenotype of which is Leu-7+/Leu-11(-) (CD16) is markedly and significantly increased compared with that in normal controls. Since this population may lack natural killer (NK) activity and may explain the state of decreased host resistance, we carried out studies in 16 kidney transplant recipients on conventional immunosuppression and 10 age-matched normal controls to further define the phenotype, the morphology, and the NK cell activity of this particular subset. Using two-color flow cytometry analysis we found that the Leu-7+ cell subset comprises two essentially nonoverlapping subpopulations, depending on whether cells are coexpressing the NK cell marker Leu-11/CD16 (Leu-7+/Leu-11+ phenotype) or the pan-T cell marker Leu-4/CD3 (Leu-7+/Leu-4+ phenotype). We thus demonstrated that Leu-7+/Leu-11- cells do coexpress the Leu-4+/CD3 surface determinant. The percentage of Leu-7+/Leu-4+ (CD3) is significantly elevated in transplant recipients compared with that in normal controls (26 +/- 4% versus 8 +/- 2%, P less than 0.005). In contrast, the size of the Leu-7+/Leu-11+ cell subset is similar in both groups. Although in transplant recipients 70% of Leu-7+ cells coexpress Leu-4/CD3, only 43% do so in the control group. Cell sorter experiments isolated the Leu-7+/Leu-4+ cells and showed that morphologically these cells are typical large granular lymphocytes that cannot be distinguished from Leu-11+ NK cells. NK-sensitive K562 target cells showed no cytotoxicity. In contrast, Leu-7+/Leu-11+ cells exhibited high killing activity. Therefore, in long-term stable renal allograft recipients at increased risk of developing cancers and chronic viral infections, a subpopulation of non-NK large granular lymphocytes, the phenotype of which is Leu-7+/Leu-11-/Leu-4+, is abnormally expanded. This subset likely contributes to the diminished functional attributes of the chronic drug-induced immunodeficiency.