RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes

RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.     

Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes

Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.     

The transmembrane domain of podoplanin is required for its association with lipid rafts and the induction of epithelial-mesenchymal transition

Podoplanin is a transmembrane glycoprotein that is upregulated in cancer and was reported to induce an epithelial-mesenchymal transition (EMT) in MDCK cells. The promotion of EMT was dependent on podoplanin binding to ERM (ezrin, radixin, moesin) proteins through its cytoplasmic (CT) domain, which led to RhoA-associated kinase (ROCK)-dependent ERM phosphorylation. Using detergent-resistant membrane (DRM) assays, as well as transmembrane (TM) interactions and ganglioside GM1 binding, we present evidence supporting the localization of podoplanin in raft platforms important for cell signalling. Podoplanin mutant constructs harbouring a heterologous TM region or lacking the CT tail were unable to associate with DRMs, stimulate ERM phosphorylation and promote EMT or cell migration. Similar effects were observed upon disruption of a GXXXG motif within the TM domain, which is involved in podoplanin self-assembly. In contrast, deletion of the extracellular (EC) domain did not affect podoplanin DRM association. Together, these data suggest that both the CT and TM domains are required for podoplanin localization in raft platforms, and that this association appears to be necessary for podoplanin-mediated EMT and cell migration.     

The comparative analysis of homologous fresh frozen bone and autogenous bone graft,associated or not with autogenous bone marrow,in rabbit calvaria: a clinical and histomorphometric study

The aim of this study was to evaluate the potential of fresh frozen homologous and autogenous grafts, associated or not with autogenous bone marrow, to form bone. Sixty titanium cylinders were used, and were fixed to the skulls of 30 rabbits. These cylinders were filled with (A) autogenous bone (AM) autogenous bone associated with the bone marrow (H) fresh frozen homologous bone (HM) fresh frozen homologous bone associated with the bone marrow (M) pure autogenous bone marrow and (C) blood clot. The animals were sacrificed after 02 and 03 months. After clinical evaluation, the samples were stained with hematoxylin, eosin and Mallory Trichrome dyes for optical microscopy analysis and histomorphometric analysis. Experimental groups that received mineralized materials (A, AM, H, HM) showed the best bone formation results, presenting no statistical difference between them (P > 0.05). Groups that did not receive mineralized materials (M and C) showed the worst results (P < 0.05), but the M group showed better results than the C group. Most of the autogenous and homologous bone particles were resorbed and there was a larger amount of residual particles in the homologous graft (H, HM) when compared with the autogenous graft (A, AM; P < 0.05). These findings suggest that fresh frozen homologous grafts produced similar amounts of new bone when compared with the autogenous grafts. However, the amount of residual bone particles was larger in the homogenous groups, which may indicate a slower remodeling process. The homologous fresh frozen bone seems to be a good osteoconductive material. The use of only autogenous bone marrow showed better results when compared to the bood clot. However, this research indicates that association with mineralized materials is required.     

Genetic parameters for body weight, carcass chemical composition and yield in a broiler-layer cross developed for QTL mapping

The objective of this study was to estimate genetic and phenotypic correlations of body weight at 6 weeks of age (BW6), as well as final carcass yield, and moisture, protein, fat and ash contents, using data from 3,422 F2 chickens originated from reciprocal cross between a broiler and a layer line. Variance components were estimated by the REML method, using animal models for evaluating random additive genetic and fixed contemporary group (sex, hatch and genetic group) effects. The heritability estimates (h(2)) for BW6, carcass yield and percentage of carcass moisture were 0.31 ± 0.07, 0.20 ± 0.05 and 0.33 ± 0.07, respectively. The h(2) for the percentages of protein, fat and ash on a dry matter basis were 0.48 ± 0.09, 0.55 ± 0.10 and 0.36 ± 0.08, respectively. BW6 had a positive genetic correlation with fat percentage in the carcass, but a negative one with protein and ash contents. Carcass yield, thus, appears to have only low genetic association with carcass composition traits. The genetic correlations observed between traits, measured on a dry matter basis, indicated that selection for carcass protein content may favor higher ash content and a lower percentage of carcass fat.     

The karyotype of Adenomera diptyx (Boettger 1885) (Anura, Leptodactylidae) from northeastern Argentina

In this work we analyzed the karyotype of five populations of Adenomera diptyx from Argentina after conventional staining, Ag-NOR and C-banding. All specimens presented 2n = 26 and FN = 34. The karyotype was formed by three submetacentric, one metacentric and nine telocentric pairs. Silver staining revealed that the NOR was located on a secondary constriction in pair 7. C- banding evidenced constitutive heterochromatin at the pericentromeric region of all chromosomes. The karyotype of A. diptyx was similar to that of A. hylaedactyla (2n = 26, FN = 34) and different from that of A. andreae (2n = 26, FN = 40) in the fundamental number and secondary constriction position. It also differed from the karyotypes of A. marmorata (2n = 24, FN = 34 and 36) and of A. aff. bokermanni (2n = 23, FN = 34) in diploid number. Until a comprehensive cytogenetic analysis of all the species of the genus is performed, their chromosome evolution will remain poorly understood.     

Allelic frequencies and statistical data obtained from 12 codis STR loci in an admixed population of the Brazilian Amazon

The allelic frequencies of 12 short tandem repeat loci were obtained from a sample of 307 unrelated individuals living in Macapá, a city in the northern Amazon region, Brazil. These loci are the most commonly used in forensics and paternity testing. Based on the allele frequency obtained for the population of Macapá, we estimated an interethnic admixture for the three parental groups (European, Native American and African) of, respectively, 46%, 35% and 19%. Comparing these allele frequencies with those of other Brazilian populations and of the Iberian Peninsula population, no significant distances were observed. The interpopulation genetic distances (F(ST) coefficients) to the present database ranged from F(ST) = 0.0016 between Macapá and Belém to F(ST) = 0.0036 between Macapá and the Iberian Peninsula.     

A new paradigm for the understanding of obesity: the role of stem cells

Obesity is a pandemic disorder that can be defined as a chronic excess of adipose tissue that increases the risk of suffering chronic diseases such as, diabetes, arterial hypertension, stroke and some forms of cancer. We now know that adipose tissue, aside from being an energy store, is also an important endocrine and metabolic organ. Recently, new mechanisms that control obesity have been identified, such as the equilibrium between white and brown adipose tissue, the localization of adipose mass (visceral or ventral), and the presence of adipose and mesenchymal stem cells. In this review, we describe the implication of these stem cell types in the normal physiology and dysfunction of adipose tissue. These stem cells provide a potential target for modulating the response of the body to obesity and diabetes, as well as a potential tool for regenerative medicine.