Long-term dexamethasone therapy may induce peripheral insulin resistance (IR), which in turn elicits increased beta-cell function and proliferation. However, whether such adaptive compensations occur during short-term treatment with dexamethasone is unclear. Here, we compared morphofunctional parameters in endocrine pancreas after short- and long-term dexamethasone administration. Groups of rats received daily i. p. injection of 1 mg/kg b. w. dexamethasone for 1 (DEX-1), 3 (DEX-3), or 5 consecutive days (DEX-5), whilst control rats were saline-treated (CTL). Despite the absence of apparent IR in DEX-1 rats, this group exhibited increased circulating insulin levels and glucose-stimulated insulin secretion (GSIS), compared to the CTL group (p<0.05). Evident IR as well as marked hyperinsulinemia and GSIS, as judged by the static and dynamic insulin secretion values, were observed in DEX-3 and DEX-5 rats (p<0.05). GSIS in islets cultured with 1 μM dexamethasone was lower compared to the control (p<0.05). Marked increases in beta-cell proliferation were observed in DEX-3 and DEX-5 rats, compared to CTL and DEX-1 rats (p<0.05). The alterations observed in DEX-3 rats were more pronounced in DEX-5 rats, which also exhibited a higher content of islet Cdk4 and Cd2 proteins, compared to the CTL group (p<0.05). We conclude that short-term dexamethasone treatment (DEX-1) induces an increase in beta-cell function that does not require the presence of discernible IR. As the treatment continues, the IR develops rapidly, and increased insulin secretion as well as beta-cell hyperplasia is demanded for the appropriate maintenance of glucose homeostasis.

Although glucocorticoids are widely used as antiinflammatory agents in clinical therapies, they may cause serious side effects that include insulin resistance and hyperinsulinemia. To study the potential functional adaptations of the islet of Langerhans to in vivo glucocorticoid treatment, adult Wistar rats received dexamethasone (DEX) for 5 consecutive days, whereas controls (CTL) received only saline. The analysis of insulin release in freshly isolated islets showed an enhanced secretion in response to glucose in DEX-treated rats. The study of Ca(2+) signals by fluorescence microscopy also demonstrated a higher response to glucose in islets from DEX-treated animals. However, no differences in Ca(2+) signals were found between both groups with tolbutamide or KCl, indicating that the alterations were probably related to metabolism. Thus, mitochondrial function was explored by monitoring oxidation of nicotinamide dinucleotide phosphate autofluorescence and mitochondrial membrane potential. Both parameters revealed a higher response to glucose in islets from DEX-treated rats. The mRNA and protein content of glucose transporter-2, glucokinase, and pyruvate kinase was similar in both groups, indicating that changes in these proteins were probably not involved in the increased mitochondrial function. Additionally, we explored the status of Ca(2+)-dependent signaling kinases. Unlike calmodulin kinase II, we found an augmented phosphorylation level of protein kinase C alpha as well as an increased response of the phospholipase C/inositol 1,4,5-triphosphate pathway in DEX-treated rats. Finally, an increased number of docked secretory granules were observed in the beta-cells of DEX animals using transmission electron microscopy. Thus, these results demonstrate that islets from glucocorticoid-treated rats develop several adaptations that lead to an enhanced stimulus-secretion coupling and secretory capacity.

Azospirillum brasilense is an important plant-growth promoting bacterium (PGPB) that requires several critical steps for root colonization, including biofilm and exopolysaccharide (EPS) synthesis and cell motility. In several bacteria these mechanisms are mediated by quorum sensing (QS) systems that regulate the expression of specific genes mediated by the autoinducers N-acyl-homoserine lactones (AHLs). We investigated QS mechanisms in strains Ab-V5 and Ab-V6 of A. brasilense, which are broadly used in commercial inoculants in Brazil. Neither of these strains carries a luxI gene, but there are several luxR solos that might perceive AHL molecules. By adding external AHLs we verified that biofilm and EPS production and cell motility (swimming and swarming) were regulated via QS in Ab-V5, but not in Ab-V6. Differences were observed not only between strains, but also in the specificity of LuxR-type receptors to AHL molecules. However, Ab-V6 was outstanding in indole acetic acid (IAA) synthesis and this molecule might mimic AHL signals. We also applied the quorum quenching (QQ) strategy, obtaining transconjugants of Ab-V5 and Ab-V6 carrying a plasmid with acyl-homoserine lactonase. When maize (Zea mays L.) was inoculated with the wild-type and transconjugant strains, plant growth was decreased with the transconjugant of Ab-V5-confirming the importance of an AHL-mediated QS system-but did not affect plant growth promotion by Ab-V6.

Endurance training has been shown to increase pancreatic β-cell function and mass. However, whether exercise modulates β-cell growth and survival pathways signaling is not completely understood. This study investigated the effects of exercise on growth and apoptotic markers levels in rat pancreatic islets. Male Wistar rats were randomly assigned to 8-wk endurance training or to a sedentary control group. After that, pancreatic islets were isolated; gene expression and the total content and phosphorylation of several proteins related to growth and apoptotic pathways as well as the main antioxidant enzymes were determined by real-time polymerase chain reaction and Western blot analysis, respectively. Reactive oxygen species (ROS) production was measured by fluorescence. Endurance training increased the time to reach fatigue by 50%. Endurance training resulted in increased protein phosphorylation content of AKT (75%), AKT substrate (AS160; 100%), mTOR (60%), p70s6k (90%), and ERK1/2 (50%), compared with islets from control group. Catalase protein content was 50% higher, whereas ROS production was 49 and 77% lower in islets from trained rats under basal and stimulating glucose conditions, respectively. Bcl-2 mRNA and protein levels increased by 46 and 100%, respectively. Bax and cleaved caspase-3 protein contents were reduced by 25 and 50% in islets from trained rats, respectively. In conclusion, these results demonstrate that endurance training favors the β-cell growth and survival by activating AKT and ERK1/2 pathways, enhancing antioxidant capacity, and reducing ROS production and apoptotic proteins content.

Summary:We conducted a phase I trial to determine the dose and schedule of paclitaxel, when given together with filgrastim, which would optimally promote mobilization of stem cells with tolerable toxicity. Dose escalation began at 275 mg/m 2 3 h infusion. Dose-limiting neuropathy was observed at the 300 mg/m 2 dose level. A second dose escalation was conducted utilizing 24 h infusion schedules, beginning at 225 mg/m 2 . Dose escalation was continued by 25 mg/m 2 increments to 300 mg/m 2 , at which dose neuropathy was again dose-limiting. The recommended dose and schedule of paclitaxel for the purpose of mobilization of stem cells, when given together with filgrastim, are 275 mg/m 2 as a 24 h infusion. The median stem cell yield after this dose of paclitaxel was 6.6 ؋ 10 6 CD34 ؉ cells/kg/apheresis (range 3.6 ؋ 10 6 -7.7 ؋ 10 6 ). Keywords: breast neoplasm; mobilization; stem cells; paclitaxel Peripheral blood progenitor cells are now preferred to bone marrow for hematopoietic reconstitution after high-dose chemotherapy because they are associated with shorter periods of cytopenia and fewer acute complications. Rapid hematopoietic recovery following stem cell infusion renders multicycle high-dose chemotherapy feasible, which has led to improvements in complete response rates in the treatment of metastatic breast cancer. 1 The risk of reintroducing malignant cells may be reduced with the use of peripheral blood stem cells, compared with bone marrow. 2 The optimal means of mobilizing peripheral blood stem cells is not known. Adequate numbers of stem cells, estimated by CD34 ϩ cell determination, can be obtained by apheresis after administration of colony-stimulating factors or during rebound leukocytosis after moderately or severely myelosuppressive chemotherapy. CD34 ϩ cells collected after the use of filgrastim are significantly less likely to undergo apoptosis than are CD34 ϩ cells in the peripheral blood in the unstimulated state. 3 Non-randomized comparisons suggest that hematopoietic recovery will be shortened after infusion of peripheral blood progenitor cells harvested during filgrastim-supported recovery from chemotherapy. 4,5 There is an inverse relationship between yield and prior treatment with radiotherapy and chemotherapy, and this is the most important predictive factor for yield. Among patients with relapsed breast cancer, prior adjuvant therapy and radiotherapy may impair mobilization. In addition, the recent trend towards greater use of double or multicycle high-dose therapy, and use of purging systems which may reduce cell number, have created a need for higher stem cell yields. Use of chemotherapy to aid in the mobilization of stem cells had been reported by Brugger et al 6 to mobilize epithelial cells in a time-dependent manner, but subsequent investigators have found no significant difference in contamination of progenitor cells mobilized by filgrastim alone compared with an historical group mobilized with high-dose cyclophosphamide followed by GM-CSF. 7 Paclitaxel contributes to mobilization ...

Treatment of metastatic melanoma still remains a challenge, since in advanced stage it is refractory to conventional treatments. Most patients with melanoma have either B-RAF or N-RAS mutations, and these oncogenes lead to activation of the RAS-RAF-MEK-ERK and AKT signal pathway, keeping active the proliferation and survival pathways in the cell. Therefore, the identification of small molecules that block metastatic cell proliferation and induce cell death is needed. Violacein, a pigment produced by Chromobacterium violaceum found in Amazon River, has been used by our group as a biotool for scrutinizing signaling pathways associated with proliferation, survival, aggressiveness, and resistance of cancer cells. In the present study, we demonstrate that violacein diminished the viability of RAS- and RAF-mutated melanoma cells (IC value ∼500 nM), and more important, this effect was not abolished after treatment medium removal. Furthermore, violacein was able to reduce significantly the invasion capacity of metastatic melanoma cells in 3D culture. In the molecular context, we have shown for the first time that violacein causes a strong drop on histone deacetylase 6 expression, a proliferating activator, in melanoma cells. Besides, an inhibition of AXL and AKT was detected. All these molecular events propitiate an inhibition of autophagy, and consequently, melanoma cell death by apoptosis.

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