Induction of globin mRNA expression by interleukin-3 in a stem cell factor-dependent SV-40 T-antigen-immortalized multipotent hematopoietic cell line

Erythropoiesis requires the stepwise action on immature progenitors of several growth factors, including stem cell factor (SCF), interleukin 3 (IL-3), and erythropoietin (Epo). Epo is required to sustain proliferation and survival of committed progenitors and might further modulate the level of expression of several erythroid genes, including globin genes. Here we report a new SCF-dependent immortalized mouse progenitor cell line (GATA-1 ts SCF) that can also grow in either Epo or IL-3 as the sole growth factor. When grown in SCF, these cells show an "open" chromatin structure of the beta-globin LCR, but do not significantly express globin. However, Epo or IL-3 induce globin expression and are required for its maintainance. This effect of IL-3 is unexpected as IL-3 was previously reported either to be unable to induce hemoglobinization, or even to antagonize it. This suggests that GATA-1 ts SCF cells may have progressed to a stage in which globin genes are already poised for expression and only require signal(s) that can be elicited by either Epo or IL-3. Through the use of inhibitors, we suggest that p38 may be one of the molecules modulating induction and maintenance of globin expression.     

Functional interaction between PARP-1 and PARP-2 in chromosome stability and embryonic development in mouse

The DNA damage-dependent poly(ADP-ribose) polymerases, PARP-1 and PARP-2, homo- and heterodimerize and are both involved in the base excision repair (BER) pathway. Here, we report that mice carrying a targeted disruption of the PARP-2 gene are sensitive to ionizing radiation. Following alkylating agent treatment, parp-2(-/-)-derived mouse embryonic fibroblasts exhibit increased post-replicative genomic instability, G(2)/M accumulation and chromosome mis-segregation accompanying kinetochore defects. Moreover, parp-1(-/-)parp-2(-/-) double mutant mice are not viable and die at the onset of gastrulation, demonstrating that the expression of both PARP-1 and PARP-2 and/or DNA-dependent poly(ADP-ribosyl) ation is essential during early embryogenesis. Interestingly, specific female embryonic lethality is observed in parp-1(+/-)parp-2(-/-) mutants at E9.5. Meta phase analyses of E8.5 embryonic fibroblasts highlight a specific instability of the X chromosome in those females, but not in males. Together, these results support the notion that PARP-1 and PARP-2 possess both overlapping and non-redundant functions in the maintenance of genomic stability.     

Insulin/IGF-I Signaling Pathways Enhances Tumor Cell Invasion through Bisecting GlcNAc N-glycans Modulation. An Interplay with E-Cadherin

Changes in glycosylation are considered a hallmark of cancer, and one of the key targets of glycosylation modifications is E-cadherin. We and others have previously demonstrated that E-cadherin has a role in the regulation of bisecting GlcNAc N-glycans expression, remaining to be determined the E-cadherin-dependent signaling pathway involved in this N-glycans expression regulation. In this study, we analysed the impact of E-cadherin expression in the activation profile of receptor tyrosine kinases such as insulin receptor (IR) and IGF-I receptor (IGF-IR). We demonstrated that exogenous E-cadherin expression inhibits IR, IGF-IR and ERK 1/2 phosphorylation. Stimulation with insulin and IGF-I in MDA-MD-435 cancer cells overexpressing E-cadherin induces a decrease of bisecting GlcNAc N-glycans that was accompanied with alterations on E-cadherin cellular localization. Concomitantly, IR/IGF-IR signaling activation induced a mesenchymal-like phenotype of cancer cells together with an increased tumor cell invasion capability. Altogether, these results demonstrate an interplay between E-cadherin and IR/IGF-IR signaling as major networking players in the regulation of bisecting N-glycans expression, with important effects in the modulation of epithelial characteristics and tumor cell invasion. Here we provide new insights into the role that Insulin/IGF-I signaling play during cancer progression through glycosylation modifications.     

Prediction of Antimicrobial Activity of Synthetic Peptides by a Decision Tree Model

Antimicrobial resistance is a persistent problem in the public health sphere. However, recent attempts to find effective substitutes to combat infections have been directed at identifying natural antimicrobial peptides in order to circumvent resistance to commercial antibiotics. This study describes the development of synthetic peptides with antimicrobial activity, created in silico by site-directed mutation modeling using wild-type peptides as scaffolds for these mutations. Fragments of antimicrobial peptides were used for modeling with molecular modeling computational tools. To analyze these peptides, a decision tree model, which indicated the action range of peptides on the types of microorganisms on which they can exercise biological activity, was created. The decision tree model was processed using physicochemistry properties from known antimicrobial peptides available at the Antimicrobial Peptide Database (APD). The two most promising peptides were synthesized, and antimicrobial assays showed inhibitory activity against Gram-positive and Gram-negative bacteria. Colossomin C and colossomin D were the most inhibitory peptides at 5 μg/ml against Staphylococcus aureus and Escherichia coli. The methods described in this work and the results obtained are useful for the identification and development of new compounds with antimicrobial activity through the use of computational tools.     

Fructose addition to a glucose supplement modifies perceived exertion during strength and endurance exercise

The addition of fructose (F) to a glucose (G) supplement may modify the metabolic response during exercise; however, its effect on perceived exertion (PE) and its influence on postprandial metabolism have not been jointly studied in different types of exercise. This study sought to assess the acute effects of F addition to a G supplement on PE and on the postprandial metabolic response during a single bout of either strength exercise (SE) or endurance exercise (EE). Twenty physically trained men ingested an oral dose of G or GF 15 minutes before starting a 30-minute session of SE (10 sets of 10 repetitions of half squat) or EE (cycling). The combination resulted in 4 randomized interventions in a crossover design in which all subjects performed all experimental conditions: G + SE, GF + SE, G + EE, and GF + SE. Perceived exertion, heart rate (HR), G, insulin, lactate, and urinary catecholamine levels were measured before exercise, during the exercise, and during acute recovery. Perceived exertion during exercise was lower for GF than for G during SE and EE (mean ± SD; 8.95 ± 0.62 vs. 9.26 ± 0.65, p < 0.05 and 7.47 ± 0.84 vs. 7.74 ± 0.93, p < 0.05, respectively). The glycemic peak in GF + SE was lower than in G + SE (p < 0.05), and there was a second peak during recovery (p < 0.05), whereas in EE, no difference in blood G levels was noted between G and GF supplements. Moreover, HR, urinary adrenalin, and noradrenalin were lower in GF than in G (p < 0.05), though only for EE. The results showed that PE is positively affected by GF supplementation for both SE and EE and thus may be a useful dietary strategy for helping to achieve higher training loads.     

Rac and phosphatidylinositol 3-kinase regulate the protein kinase B in Fc epsilon RI signaling in RBL 2H3 mast cells

FcepsilonRI signaling in rat basophilic leukemia cells depends on phosphatidylinositol 3-kinase (PI3-kinase) and the small GTPase Rac. Here, we studied the functional relationship among PI3-kinase, its effector protein kinase B (PKB), and Rac using inhibitors of PI3-kinase and toxins inhibiting Rac. Wortmannin, an inhibitor of PI3-kinase, blocked FcepsilonRI-mediated tyrosine phosphorylation of phospholipase Cgamma, inositol phosphate formation, calcium mobilization, and secretion of hexosaminidase. Similarly, Clostridium difficile toxin B, which inactivates all Rho GTPases including Rho, Rac and Cdc42, and Clostridium sordellii lethal toxin, which inhibits Rac (possibly Cdc42) but not Rho, blocked these responses. Stimulation of the FcepsilonRI receptor induced a rapid increase in the GTP-bound form of Rac. Whereas toxin B inhibited the Rac activation, PI3-kinase inhibitors (wortmannin and LY294002) had no effect on activation of Rac. In line with this, wortmannin had no effect on tyrosine phosphorylation of the guanine nucleotide exchange factor Vav. Wortmannin, toxin B, and lethal toxin inhibited phosphorylation of PKB on Ser(473). Similarly, translocation of the pleckstrin homology domain of PKB tagged with the green fluorescent protein to the membrane, which was induced by activation of the FcepsilonRI receptor, was blocked by inhibitors of PI3-kinase and Rac inactivation. Our results indicate that in rat basophilic leukemia cells Rac and PI3-kinase regulate PKB and suggest that Rac is functionally located upstream and/or parallel of PI3-kinase/PKB in FcepsilonRI signaling.     

Somatostatin inhibition of insulin release from freshly isolated and organ cultured rat islets of langerhans in vitro

1×10^?6M somatostatin causes a 37–44% inhibition of glucose induced insulin release from freshly isolated rat islets of Langerhans. A 81 to 95% inhibition is observed when the isolated islets are maintained in organ culture for 2 days prior to the somatostatin treatment. The dose curve of somatostatin on cultured islets shows an apparent K_I of 1.4×10^?9. The tetradecapeptide also causes a reversible inhibition of the stimulation of insulin release by 5 mM theophylline and 23 mM K⁺.     

Severe abnormalities in the oral mucosa induced by suprabasal expression of epidermal keratin K10 in transgenic mice

Previous studies have demonstrated that keratin K10 plays an important role in mediating cell signaling processes, since the ectopic expression of this keratin induces cell cycle arrest in proliferating cells in vitro and in vivo. However, apart from its well known function of providing epithelial cells with resilience to mechanical trauma, little is known about its possible roles in nondividing cells. To investigate what these might be, transgenic mice were generated in which the expression of K10 was driven by bovine K6beta gene control elements (bK6(beta)hK10). The transgenic mice displayed severe abnormalities in the tongue and palate but not in other K6-expressing cells such as those of the esophagus, nails, and hair follicles. The lesions in the tongue and palate included the cytolysis of epithelial suprabasal cells associated with an acute inflammatory response and lymphocyte infiltration. The alterations in the oral mucosa caused the death of transgenic pups soon after birth, probably because suckling was impaired. These anomalies, together with others found in the teeth, are reminiscent of the lesions observed in some patients with pachyonychia congenita, an inherited epithelial fragility associated with mutations in keratins K6 and K16. Although no epithelial fragility was observed in the bK6(beta)hK10 oral epithelia of the experimental mice, necrotic processes were seen. Collectively, these data show that the carefully regulated tissue- and differentiation-specific patterns displayed by the keratin genes have dramatic consequences on the biological behavior of epithelial cells and that changes in the specific composition of the keratin intermediate filament cytoskeleton can affect their physiology, in particular those of the oral mucosa.