Rapid immunological diagnosis of active tuberculosis, and differentiation from asymptomatic infection

info:eu-repo/semantics/nonPublished

Immediate early gene expression in dog thyrocytes in response to growth, proliferation, and differentiation stimuli.

In dog thyroid cells, insulin or IGF-1 induces cell growth and is required for the mitogenic action of TSH through cyclic AMP, of EGF, and of phorbol esters. HGF per se stimulates cell proliferation and is thus the only full mitogenic agent. TSH and cAMP enhance, whereas EGF phorbol esters and HGF repress differentiation expression. In this study, we have investigated for each factor and regulatory cascade of the intermediate step of immediate early gene induction, that is, c-myc, c-jun, jun D, jun B, c-fos, fos B, fra-1, fra-2, and egr1; fra-1 and fra-2 expressions were very low. TSH or forskolin increased the levels of c-myc, jun B, jun D, c-fos, and fos B while decreasing those of c-jun and egr1. Phorbol myristate ester stimulated the expression of all the genes. EGF and HGF stimulated the expression of all the genes except jun D and for EGF fos B. All these effects were obtained in the presence and in the absence of insulin, which shows that insulin is not necessary for the effects of the mitogens on immediate early gene expression. The definition of the repertoire of early immediate genes inductible by the various growth cascades provides a framework for the analysis of gene expression in tumors. (1) Insulin was able to induce all the protooncogenes investigated except fos B. This suggests that fos B could be the factor missing for insulin to induce mitogenesis. (2) No characteristic pattern of immediate early gene expression has been observed for insulin, which induces cell hypertrophy and is permissive for the action of the other growth factors. These effects are therefore not accounted for by a specific immediate early gene expression. On the other hand, insulin clearly enhances the effects of TSH, phorbol ester, and EGF on c-myc, junB, and c-fos expression. This suggests that the effect of insulin on mitogenesis might result from quantitative differences in the transcription complexes formed. (3) c-myc, c-fos, and jun B mRNA induction by all stimulating agents, whether inducing cell hypertrophy, or growth and dedifferentiation, or growth and differentiation, suggests that, although these expressions are not sufficient, they may be necessary for the various growth responses of thyroid cells. (4) The inhibition of c-jun and egr1 mRNA expression, and the marked induction of jun D mRNA appear to be specific features of the TSH cAMP pathway. They might be related to its differentiating action. (5) fos B, which is induced by TSH, forskolin, phorbol ester, and HGF but not by insulin, could be involved in the mitogenic action of the former factors.

Small Proline Rich Protein-2 Expression and Regulation in the Caco-2 model of Intestinal Epithelial Differentiation along the Crypt-Villus Axis

Small proline-rich protein-2 (SPRR2) functions as a determinant of flexibility and permeability in the mature cornified envelope of the skin. SPRR2 is strongly upregulated by the commensal flora and may mediate signaling to differentiated epithelia of the small intestine and colon. Yet, SPRR2 function in the GI tract is largely unexplored. Using the Caco-2 model of intestinal epithelial differentiation along the crypt-villus axis, we hypothesized that SPRR2 would be preferentially expressed in post-confluent differentiated Caco-2 cells and examined SPRR2 regulation by the protein kinase A pathway (PKA) and short chain fatty acids (SCFAs). Differentiation-dependent SPRR2 expression was examined in cytoskeletal-, membrane-, and nuclear-enriched fractions by immunoblotting and confocal immunofluorescence. We studied the effect of SCFAs, known inducers of differentiation, on SPRR2 expression in pre-confluent undifferentiated Caco-2 cells and explored potential mechanisms involved in this induction using MAP kinase inhibitors. SPRR2 expression was also compared between HIEC crypt cells and 16 to 20 week primary fetal villus cells as well as in different segments in mouse small intestine and colon. We determined if SPRR2 is increased by gram negative bacteria such as S. typhimurium. SPRR2 expression increased in a differentiation-dependent manner in Caco-2 cells and was present in human fetal epithelial villus cells but absent in HIEC crypt cells. Differentiation-induced SPRR2 was down-regulated by 8-Br-cAMP as well as by forskolin/IBMX co-treatment. SPRR2 was predominantly cytoplasmic and did not accumulate in Triton X-100-insoluble cytoskeletal fractions. SPRR2 was present in the membrane- and nuclear-enriched fractions and demonstrated co-localization with F-actin at the apical actin ring. No induction was seen with the specific HDAC inhibitor trichostatin A, while SCFAs and the HDAC inhibitor SBHA all induced SPRR2. SCFA responses were inhibited by MAP kinase inhibitors SB203580 and U0126, thus suggesting that the SCFA effect may be mediated by orphan G-protein receptors GPR41 and GPR43. S. typhimurium induced SPRR2 in undifferentiated cells. We conclude that SPRR2 protein expression is associated with differentiated epithelia and is regulated by PKA signaling and by by-products of the bowel flora. This is the first report to establish an in vitro model to study the physiology and regulation of SPRR2.

Reproductive isolation and genetic differentiation of ferox trout from sympatric brown trout in Loch Aweand Loch Laggan, Scotland

Molecular marker studies reported here, involving allozymes, mitochondrial DNA and microsatellites, demonstrate that ferox brown trout Salmo trutta in Lochs Awe and Laggan, Scotland, are reproductively isolated and genetically distinct from co-occurring brown trout. Ferox were shown to spawn primarily, and possibly solely, in a single large river in each lake system making them particularly vulnerable to environmental changes. Although a low level of introgression seems to have occurred with sympatric brown trout, possibly as a result of human-induced habitat alterations and stocking, ferox trout in these two lakes meet the requirements for classification as a distinct biological, phylogenetic and morphological species. It is proposed that the scientific name Salmo ferox Jardine, 1835, as already applied to Lough Melvin (Ireland) ferox, should be extended to Awe and Laggan ferox.

An investigation into the role of Bcl-2 in neuroendocrine differentiation

INTRODUCTION: In addition to its role in apoptosis suppression, Bcl-2 has been reported to be co-expressed with neuroendocrine markers in several tissues, leading to speculation that this oncoprotein may promote neuroendocrine differentiation. AIM: This study investigated whether Bcl-2 modulated neuroendocrine biopeptide expression. METHODS: Levels of chromogranin A, neurone specific enolase, protein gene peptide 9.5, pancreatic polypeptide, and the chromogranin-derived peptides, intervening peptide and vasostatin-1 were examined by immunocytochemistry in rat phaeochromocytoma (PC12) cell lines genetically engineered to over-express Bcl-2 and their mock-transfected controls. Intensity of fluorescence was graded using a semi-quantitative scale from (-) indicating negative expression to (+++) indicating intense positivity. RESULTS: Mann-Whitney U analysis indicated that no significant differences in expression existed between control and Bcl2 over-expressing cell lines for any of the six peptides examined. CONCLUSIONS: The results of this study do not support the hypothesis that Bcl-2 promotes the acquisition of a neuroendocrine phenotype.