The induction of NOS2 expression by the hybrid cecropin A-melittin antibiotic peptide CA(1-8)M(1-18) in the monocytic line RAW 264.7 is triggered by a temporary and reversible plasma membrane permeation

There is an increasing awareness of immune cell modulation by antimicrobial peptides. While this process often requires specific receptors for the peptides involved, several reports point out to a receptor-independent process. The cecropin A-melittin hybrid peptide CA(1-8)M(1-18) (KWKLFKKIGIGAVLKVLTTGLPALIS-amide) modifies gene expression in the macrophage line RAW 264.7 in the absence of any previous macrophage priming, suggesting a membrane permeation process. To further analyze the initial steps of this mechanism, we have studied the interaction of the peptide with these cells. Below 2 microM, CA(1-8)M(1-18) causes a concentration-dependent membrane depolarization partially reversible with time. At 2 microM, the accumulation of the SYTOX green vital dye is one half of that achieved with 0.05% Triton X-100. The binding level, as assessed by fluorescein-labeled CA(1-8)M(1-18), varies from 7.7+/-1.2 to 37.4+/-3.9 x 10(6) molecules/cell over a 0.5-4.0 microM concentration range. Electrophysiological experiments with 0.5 microM CA(1-8)M(1-18), a concentration that triggers maximal NOS2 expression and minimal toxicity, show a reversible current induction in the RAW 264.7 plasma membrane that is maintained as far as peptide is present. This activation of the macrophage involves the production of nitric oxide, a metabolite lethal for many pathogens that results from unspecific membrane permeation by antimicrobial peptides, and represents a new mode of action that may open new therapeutic possibilities for these compounds against intracellular pathogens.     

Modeling human mitochondrial diseases in flies

Human mitochondrial diseases are associated with a wide range of clinical symptoms, and those that result from mutations in mitochondrial DNA affect at least 1 in 8500 individuals. The development of animal models that reproduce the variety of symptoms associated with this group of complex human disorders is a major focus of current research. Drosophila represents an attractive model, in large part because of its short life cycle, the availability of a number of powerful techniques to alter gene structure and regulation, and the presence of orthologs of many human disease genes. We describe here Drosophila models of mitochondrial DNA depletion, deafness, encephalopathy, Freidreich's ataxia, and diseases due to mitochondrial DNA mutations. We also describe several genetic approaches for gene manipulation in flies, including the recently developed method of targeted mutagenesis by recombinational knock-in.     

Effect of stress on hepatic 11beta-hydroxysteroid dehydrogenase activity and its influence on carbohydrate metabolism

Stress activates the synthesis and secretion of catecholamines and adrenal glucocorticoids, increasing their circulating levels. In vivo, hepatic 11beta-hydroxysteroid dehydrogenase 1 (HSD1) stimulates the shift of 11-dehydrocorticosterone to corticosterone, enhancing active glucocorticoids at tissue level. We studied the effect of 3 types of stress, 1 induced by bucogastric overload with 200 mmol/L HCl causing metabolic acidosis (HCl), the second induced by bucogastric overload with 0.45% NaCl (NaCl), and the third induced by simulated overload (cannula), on the kinetics of hepatic HSD1 of rats and their influence on the activity of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase, glycemia, and glycogen deposition. Compared with unstressed controls, all types of stress significantly increased HSD1 activity (146% cannula, 130% NaCl, and 253% HCl), phosphoenolpyruvate carboxykinase activity (51% cannula, 48% NaCl, and 86% HCl), and glycemia (29% cannula, 30% NaCl, and 41% HCl), but decreased hepatic glycogen (68% cannula, 68% NaCl, and 78% HCl). Owing to these results, we suggest the following events occur when stress is induced: an increase in hepatic HSD1 activity, augmented active glucocorticoid levels, increased gluconeogenesis, and glycemia. Also involved are the multiple events indirectly related to glucocorticoids, which lead to the depletion of hepatic glycogen deposits, thereby contributing to increased glycemia. This new approach shows that stress increments the activity of hepatic HSD1 and suggests that this enzyme could be involved in the development of the Metabolic Syndrome.     

Oxidative damage in the livers of senescence-accelerated mice: a gender-related response

The prevalence of liver diseases emphasizes the need of animal models to research on the mechanism of disease pathogenesis. Furthermore, most of the liver pathologies have the oxidative stress as an important component. The senescence-accelerated mouse strain SAMP8 was proposed as a valuable animal model for the study of liver diseases. To gain a better understanding of the mechanisms underlying degenerative processes in SAMP8 mice livers, we studied the oxidative-induced damage in 5-month-old SAMP8 mice and SAMR1, senescence-accelerated-resistant mice. We found profound differences in the antioxidant response to aging between sexes, with males displaying lowest levels of main antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) in SAMP8; whereas females had no difference in their activities, except for GR, when compared with their SAMR1 controls. The results obtained show the binomial SOD/CAT as an important factor for counteracting reactive oxygen species-dependent damage. There were not pathological differences at the morphological level between both strains, although the decay in protection against free radicals had an immediate response by increasing lipid and protein oxidative damage in SAMP8 mice liver. At 5 months, both male and female SAMP8 mice confront the oxidative stress challenge to different extents. Indeed, proteins seem to be the most vulnerable biomolecule in SAMP8 male mice.     

Preparation and biological evaluation of some 1,2-O-isopropylidene-D-hexofuranose esters

The synthesis and biological evaluation of some new glycose esters bearing the 1,2-O-isopropylidene-d-hexofuranose functionality and belonging to the 3-O-acyl-d-allose and 6-O-acyl-d-glucose series are reported. When the results concerning cell growth inhibition are compared, it appears that the 6-O-acyl-d-glucose derivatives are more active than the 3-O-acyl-d-allose compounds. Within both 6-O-acyl-d-glucose and 3-O-acyl-d-allose derivatives, butyric esters displayed the highest inhibitory effects. Inhibition of cell growth is not associated with high induction levels of erythroid differentiation, despite the fact that pivaloates induce erythroid differentiation to an extent similar to that exhibited by previously reported molecules [Bioorg. Med. Chem. Lett.1999, 9, 3153-3158].     

Nested collagen matrices: a new model to study migration of human fibroblast populations in three dimensions

Fibroblast-3D collagen matrix culture provides a model system to analyze cell physiology under conditions that more closely resemble tissue than conventional 2D cell culture. Previous work has focused primarily on remodeling and contraction of collagen matrices by fibroblasts, and there has been little research on migration of cell populations within the matrix. Here, we introduce a nested collagen matrix model to analyze migration of fibroblasts in 3D collagen matrices. Nested collagen matrices were prepared by embedding contracted cell-containing matrices (also called dermal equivalents) inside cell-free matrices; migration occurred from the former to the latter. Control experiments with human dermal fragments in place of dermal equivalents confirmed the reliability of the model. Human fibroblast migration in nested collagen matrices occurred after a lag phase of 8-16 h, and cells migrating out of the inner matrices were bipolar with leading dendritic extensions. Migration was myosin II, Rho kinase and metalloproteinase-dependent but did not require plasma fibronectin. Platelet-derived growth factor but not lysophosphatidic acid or serum stimulated cell migration, although all three of these physiological agonists promote matrix remodeling and contraction. The nested collagen matrix model is a relatively easy, rapid and quantitative method to measure migration of cell populations. Our studies using this model demonstrate important differences between regulation of fibroblast migration and remodeling in collagen matrices.     

The p53-induced Wig-1 protein binds double-stranded RNAs with structural characteristics of siRNAs and miRNAs

Wig-1 is a p53-induced zinc finger protein. Here we show that human Wig-1 binds long (>or=23 bp) dsRNAs with 5'-overhangs. The first zinc finger domain is necessary but not sufficient for this dsRNA-binding in vitro. Wig-1 also binds dsRNA in living cells via zinc fingers 1 and 2. Both zinc fingers 1 and 2 are important for Wig-1-mediated growth suppression. Moreover, Wig-1 binds 21 bp dsRNAs with 3'-protruding ends. These findings demonstrate that human Wig-1 can bind different types of dsRNAs, including dsRNAs resembling small interfering RNAs (siRNAs) and microRNAs (miRNAs), and indicate that dsRNA binding has a role in Wig-1-mediated regulation of cell growth.     

Banding pattern of A and B chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae), with comments on B chromosomes evolution

B chromosomes in Prochilodus lineatus, a migratory neotropical fish, were analyzed in a comparative study among populations from the Dourada lagoon (State of Paraná, Brazil) and from Mogi-Guaçu river (State of São Paulo, Brazil). The data on C-banding and fluorescent in situ hybridization with a satellite DNA probe (SATH1), indicate that the small metacentric B chromosome might correspond to an isochromosome. On the other hand, both populations presented a distinct set of B chromosomes, differentiated either by their number and by the presence of variant B types in the population from Mogi-Guaçu river. The present results indicate that the B chromosomes of P. lineatus should have an ancient origin, and have undergone a differential evolutionary pathway among distinct populations.