Karyotypic evolution trends in Rhamdia quelen (Siluriformes, Heptapteridae) with considerations about the origin and differentiation of its supernumerary chromosomes

Among catfish species of the genus Rhamdia reported for the Brazilian territory, R. quelen is the most widespread, being found in nearly all hydrographic basins of Brazil. Nowadays, R. quelen is a synonym for at least 47 other species in this genus, its taxonomic status still being controversial. The available cytogenetic reports show a wide variation in the karyotypic macrostructure, with the frequent presence of supernumerary chromosomes. The remarkable cytogenetic variability associated with taxonomic issues in this species indicates that R. quelen is actually a species complex. In order to carry out a wide comparative cytogenetic study in R. quelen from southern and southeastern Brazil and examine a species complex, we analyzed the chromosomes of 14 populations from the main hydrographic basins of these two regions. Using classic and molecular cytogenetic techniques, we found seven distinct karyotypic formulae, all bearing 2n = 58 chromosomes. Supernumerary chromosomes were present in most of the populations; their number, size and C-banding pattern allowed us to differentiate populations with similar karyotypic compositions. We examined patterns of chromosomal evolution as well as the probable mechanisms involved in the origin and morphological differentiation of their supernumerary chromosomes.

Intracellular Ca(2+) Regulation During Neuronal Differentiation of Murine Embryonal Carcinoma and Mesenchymal Stem Cells

Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) play a central role in neuronal differentiation. However, Ca(2+) signaling in this process remains poorly understood and it is unknown whether embryonic and adult stem cells share the same signaling pathways. To clarify this issue, neuronal differentiation was analyzed in two cell lines: embryonic P19 carcinoma stem cells (CSCs) and adult murine bone-marrow mesenchymal stem cells (MSC). We studied Ca(2+) release from the endoplasmic reticulum via intracellular ryanodine-sensitive (RyR) and IP(3)-sensitive (IP(3)R) receptors. We observed that caffeine, a RyR agonist, induced a [Ca(2+)](i) response that increased throughout neuronal differentiation. We also demonstrated a functional coupling between RyRs and L-but not with N-, P-, or Q-type Ca(v)1 Ca(2+) channels, both in embryonal CSC and adult MSC. We also found that agonists of L-type channels and of RyRs increase neurogenesis and neuronal differentiation, while antagonists of these channels have the opposite effect. Thus, our data demonstrate that in both cell lines RyRs control internal Ca(2+) release following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels. This study shows that both in embryonal CSC and adult MSC [Ca(2+)](i) is controlled by a common pathway, indicating that coupling of L-type Ca(2+) channels and RyRs may be a conserved mechanism necessary for neuronal differentiation.

MAMMALIAN TARGET OF RAPAMYCIN COMPLEX 1 IS INVOLVED IN DIFFERENTIATION OF REGENERATING MYOFIBERS IN VIVO

This work was undertaken to provide further insight into the role of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle regeneration, focusing on myofiber size recovery. Rats were treated or not with rapamycin, an mTORC1 inhibitor. Soleus muscles were then subjected to cryolesion and analyzed 1, 10, and 21 days later. A decrease in soleus myofiber cross-section area on post-cryolesion days 10 and 21 was accentuated by rapamycin, which was also effective in reducing protein synthesis in these freeze-injured muscles. The incidence of proliferating satellite cells during regeneration was unaltered by rapamycin, although immunolabeling for neonatal myosin heavy chain (MHC) was weaker in cryolesion+rapamycin muscles than in cryolesion-only muscles. In addition, the decline in tetanic contraction of freeze-injured muscles was accentuated by rapamycin. This study indicates that mTORC1 plays a key role in the recovery of muscle mass and the differentiation of regenerating myofibers, independently of necrosis and satellite cell proliferation mechanisms. Muscle Nerve 42: 778-787,2010

Nickel sorption capacity of ground xylem of Quercus ilex trees and effects of selected ligands present in the xylem sap

In this work the influence of four different ligands present in the xylem sap of Quercus ilex (histidine, citric, oxalic and aspartic acids) on Ni(II) adsorption by xylem was investigated. Grinded xylem was trapped in acrylic columns and solutions of Ni(II), in the absence and presence of the four ligands prepared in KNO(3) 0-1 mol L(-1) at pH 5.5, were percolated through the column. Aliquots of solutions were recovered in the column end for Ni determination by graphite furnace atomic absorption spectrometry (GFAAS). The experimental. data to describe Ni sorption by xylem in both the presence and absence of ligands was better explained by the Freundlich isotherm model. The decreasing affinity order of ligands for Ni was: oxalic acid > citric acid > histidine > aspartic acid. On the other hand, the Ni(II) adsorption by xylem increased following the inverse sequence of ligands. Potentiometric titrations of acidic groups were carried out to elucidate the sorption site groups available in Q. ilex xylem. The potentiometric titration has shown three sorption sites: pK(a) 2.6 (57.7% of the sorption sites), related to monobasic aliphatic carboxylic acids or nitrogen aromatic bases, pK(a) 8.1 (9.6%) and pK(a) 9.9 (32.7%), related to phenolic groups. (C) 2008 Elsevier GmbH. All rights reserved.

Molecular detection and differentiation of Brazilian and African isolates of the entomopathogen Neozygites tanajoae (Entomophthorales: Neozygitaceae) with PCR using specific primers

Neozygites tanajoae is an entomopathogenic fungus which has been used for biocontrol of the cassava green mite (Mononychellus tanajoa, CGM) in Africa. Establishment and dispersal of Brazilian isolates which have been introduced into some African countries in recent years to improve CGM control was followed with specific PCR assays. Two primer pairs, NEOSSU_F/NEOSSU_R and 8DDC_F/8DDC_R, were used to differentiate isolates collected from several locations in Brazil and from three countries in Africa, Benin, Ghana and Tanzania. The first primer pair enabled the species-specific detection of Neozygites tanajoae, while the second differentiated the Brazilian isolates from those of other geographical origin. PCR assays were designed for detection of fungal DNA in the matrix of dead infested mites since N. tanajoae is difficult to isolate and culture on selective artificial media. Our results show that all isolates (Brazilian and African) that sporulated on mummified mites were amplified with the first primer pair confirming their Neozygites tanajoae identity. The second pair amplified DNA from all the Brazilian isolates, but did not amplify any DNA samples from the African isolates. None of the two primers showed amplification neither from any of the non-sporulating mite extracts nor from the dead uninfected mites used as negative controls. We confirmed that the two primer pairs tested are suitable for the detection and differential identification of N. tanajoae isolates from Brazil and Africa and that they are useful to monitor the establishment and spread of the Brazilian isolates of N. tanajoae introduced into Benin or into other African countries for improvement of CGM biocontrol.

A new polymorphism in the Growth and Differentiation Factor 9 (GDF9) gene is associated with increased ovulation rate and prolificacy in homozygous sheep

P>Brazilian Santa Ines (SI) sheep are very well-adapted to the tropical conditions of Brazil and are an important source of animal protein. A high rate of twin births was reported in some SI flocks. Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) are the first two genes expressed by the oocyte to be associated with an increased ovulation rate in sheep. All GDF9 and BMP15 variants characterized, until now, present the same phenotype: the heterozygote ewes have an increased ovulation rate and the mutated homozygotes are sterile. In this study, we have found a new allele of GDF9, named FecGE (Embrapa), which leads to a substitution of a phenylalanine with a cysteine in a conservative position of the mature peptide. Homozygote ewes presenting the FecGE allele have shown an increase in their ovulation rate (82%) and prolificacy (58%). This new phenotype can be very useful in better understanding the genetic control of follicular development; the mechanisms involved in the control of ovulation rate in mammals; and for the improvement of sheep production.

Plasmodium vivax recombinant vaccine candidate AMA-1 plays an important role in adaptive immune response eliciting differentiation of dendritic cells

The Apical Membrane Antigen-1 (AMA-1) is a well-characterized and functionally important merozoite protein and is currently considered a major candidate antigen for a malaria vaccine. Previously, we showed that AMA-1 has an influence on cellular immune responses of malaria-naive subjects, resulting in an alternative activation of monocyte-derived dendritic cells and induction of a pro-inflammatory response by stimulated PBMCs. Although there is evidence, from human and animal malaria model systems that cell-mediated immunity may contribute to both protection and pathogenesis, the knowledge on cellular immune responses in vivax malaria and the factors that may regulate this immunity are poorly understood. In the current work, we describe the maturation of monocyte-derived dendritic cells of P. vivax naturally infected individuals and the effect of P. vivax vaccine candidate Pv-AMA-1 on the immune responses of the same donors. We show that malaria-infected subjects present modulation of DC maturation, demonstrated by a significant decrease in expression of antigen-presenting molecules (CD1a, HLA-ABC and HLA-DR), accessory molecules (CD40, CD80 and CD86) and Fc gamma RI (CD64) receptor (P <= 0.05). Furthermore, Pv-AMA-1 elicits an upregulation of CD1a and HLA-DR molecules on the surface of monocyte-derived dendritic cells (P=0.0356 and P=0.0196, respectively), and it is presented by AMA-1-stimulated DCs. A significant pro-inflammatory response elicited by Pv-AMA-1-pulsed PBMCs is also demonstrated, as determined by significant production of TNF-alpha, IL-12p40 and IFN-gamma (P <= 0.05). Our results suggest that Pv-AMA-1 may partially revert DC down-modulation observed in infected subjects, and exert an important role in the initiation of pro-inflammatory immunity that might contribute substantially to protection. (c) 2009 Elsevier Ltd. All rights reserved.

Involvement of an Alternative Oxidase in Oxidative Stress and Mycelium-to-Yeast Differentiation in Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a thermodimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), which is the most prevalent systemic mycosis in Latin America. Differentiation from the mycelial to the yeast form (M-to-Y) is an essential step for the establishment of PCM. We evaluated the involvement of mitochondria and intracellular oxidative stress in M-to-Y differentiation. M-to-Y transition was delayed by the inhibition of mitochondrial complexes III and IV or alternative oxidase (AOX) and was blocked by the association of AOX with complex III or IV inhibitors. The expression of P. brasiliensis aox (Pbaox) was developmentally regulated through M-to-Y differentiation, wherein the highest levels were achieved in the first 24 h and during the yeast exponential growth phase; Pbaox was upregulated by oxidative stress. Pbaox was cloned, and its heterologous expression conferred cyanide-resistant respiration in Saccharomyces cerevisiae and Escherichia coli and reduced oxidative stress in S. cerevisiae cells. These results reinforce the role of PbAOX in intracellular redox balancing and demonstrate its involvement, as well as that of other components of the mitochondrial respiratory chain complexes, in the early stages of the M-to-Y differentiation of P. brasiliensis.

Population subdivision in marine environments: the contributions of biogeography, geographic distance, and discontinuous habitat to genetic differentiation in a blennioid

The relative importance of factors that may promote genetic differentiation in marine organisms is largely unknown. Here, contributions to population structure from biogeography, habitat distribution, and isolation by distance were investigated in Axoclinus nigricaudus, a small subtidal rock reef fish, throughout its range in the Gulf of California. A 408 basepair fragment of the mitochondrial control region was sequenced from 105 individuals. Variation was significantly partitioned between many pairs of populations. Phylogenetic analyses, hierarchical analyses of variance, and general linear models substantiated a major break between two putative biogeographic regions. This genetic discontinuity coincides with an abrupt change in ecological characteristics (including temperature and salinity) but does not coincide with known oceanographic circulation patterns. Geographic distance and the nature of habitat separating populations (continuous habitat along a shoreline, discontinuous habitat along a shoreline, and open water) also contributed to population structure in general linear model analyses. To verify that local populations are genetically stable over time, one population was resampled on four occasions over eighteen months; it showed no evidence of a temporal component to diversity. These results indicate that having a planktonic life stage does not preclude geographically partitioned genetic variation over relatively small geographic distances in marine environments. Moreover, levels of genetic differentiation among populations of Axoclinus nigricaudus cannot be explained by a single factor, but are due to the combined influences of a biogeographic boundary, habitat, and geographic distance.

Proliferation in monocyte-derived dendritic cell cultures is caused by progenitor cells capable of myeloid differentiation

Dendritic cells (DC) can be generated by culture of adherent peripheral blood (PB) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). There is controversy as to whether these DC arise from proliferating precursors or simply from differentiation of monocytes. DC were generated from myeloid-enriched PB non-T cells or sorted monocytes. DC generated from either population functioned as potent antigen-presenting cells. Uptake of [H-3]-thymidine was observed in DC cultured from myeloid-enriched non-T cells. Addition of lipopolysaccharide or tumor necrosis factor-alpha led to maturation of the DC, but did not inhibit proliferation. Ki67(+) cells were observed in cytospins of these DC, and by double staining were CD3(-)CD19(-)CD11c(-)CD40(-) and myeloperoxidase(+), suggesting that they were myeloid progenitor cells. Analysis of the starting population by flow cytometry demonstrated small numbers of CD34(+)CD33(-)CD14(-) progenitor cells, and numerous granulocyte-macrophage colony-forming units were generated in standard assays. Thus, production of DC in vitro from adherent PB cells also enriches for progenitor cells that are capable of proliferation after exposure to GM-CSF. Of clinical importance, the yield of DC derived in the presence of GM-CSF and IL-4 cannot be expanded beyond the number of starting monocytes. (C) 1998 by The American Society of Hematology.

Proliferation in monocyte-derived dendritic cell cultures is due to cells capable of myeloid differentiation

Dendritic cells (DC) can be generated by culture of adherent peripheral blood (PB) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). There is controversy as to whether these DC arise from proliferating precursors or simply from differentiation of monocytes. DC were generated from myeloid-enriched PB non-T cells or sorted monocytes. DC generated from either population functioned as potent antigen-presenting cells. Uptake of [H-3]-thymidine was observed in DC cultured from myeloid-enriched non-T cells. Addition of lipopolysaccharide or tumor necrosis factor-alpha led to maturation of the DC, but did not inhibit proliferation. Ki67(+) cells were observed in cytospins of these DC, and by double staining were CD3(-)CD19(-)CD11c(-)CD40(-) and myeloperoxidase(+), suggesting that they were myeloid progenitor cells. Analysis of the starting population by flow cytometry demonstrated small numbers of CD34(+)CD33(-)CD14(-) progenitor cells, and numerous granulocyte-macrophage colony-forming units were generated in standard assays. Thus, production of DC in vitro from adherent PB cells also enriches for progenitor cells that are capable of proliferation after exposure to GM-CSF. Of clinical importance, the yield of DC derived in the presence of GM-CSF and IL-4 cannot be expanded beyond the number of starting monocytes. (C) 1998 by The American Society of Hematology.

Differentiation of the mononuclear phagocyte system during mouse embryogenesis: The role of transcription factor PU.1

During mouse embryogenesis, macrophage-like cells arise first in the yolk sac and are produced subsequently in the liver. The onset of liver hematopoiesis is associated with the transition from primitive to definitive erythrocyte production. This report addresses the hypothesis that a similar transition in phenotype occurs in myelopoiesis. We have used whole mount in situ hybridization to detect macrophage-specific genes expressed during mouse development. The mouse c-fms mRNA, encoding the receptor for macrophage colony-stimulating factor (CSF-1), was expressed on phagocytic cells in the yolk sac and throughout the embryo before the onset of liver hematopoiesis, Similar cells were detected using the mannose receptor, the complement receptor (CR3), or the Microphthalmia transcription factor (MITF) as mRNA markers. By contrast, other markers including the F4/80 antigen, the macrophage scavenger receptor, the S-100 proteins, S100A8 and S100A9, and the secretory product lysozyme appeared later in development and appeared restricted to only a subset of c-fms-positive cells. Two-color immunolabeling on disaggregated cells confirmed that CR3 and c-fms proteins are expressed on the same cells. Among the genes appearing later in development was the macrophage-restricted transcription factor, PU.1, which has been shown to be required for normal adult myelopoiesis. Mice with null mutations in PU.1 had normal numbers of c-fms-positive phagocytes at 11.5dpc. PU.1(-/-) embryonic stem cells were able to give rise to macrophagelike cells after cultivation in vitro. The results support previous evidence that yolk sac-derived fetal phagocytes are functionally distinct from those arising in the liver and develop via a different pathway. (C) 1999 by The American Society of Hematology.

Phylogeographic differentiation in the mitochondrial control region in the koala, Phascolarctos cinereus (Goldfuss 1817)

The koala, Phascolarctos cinereus, is a geographically widespread species endemic to Australia, with three currently recognized subspecies: P.c. adustus, P.c. cinereus, and P.c. victor. Intraspecific variation in the mitochondrial DNA (mtDNA) control region was examined in over 200 animals from 16 representative populations throughout the species' range. Eighteen different haplotypes were defined in the approximate to 860 bp mtDNA control region as determined by heteroduplex analysis/temperature gradient gel electrophoresis (HDA/TGGE). Any single population typically possessed only one or two haplotypes yielding an average within-population haplotypic diversity of 0.180 +/- 0.003, and nucleotide diversity of 0.16%. Overall, mtDNA control region sequence diversity between populations averaged 0.67%, and ranged from 0% to 1.56%. Nucleotide divergence between populations averaged 0.51%, and ranged from 0% to 1.53%. Neighbour-joining methods revealed limited phylogenetic distinction between geographically distant populations of koalas, and tentative support for a single evolutionarily significant unit (ESU). This is consistent with previous suggestions that the morphological differences formalized by subspecific taxonomy may be interpreted as clinal variation. Significant differentiation in mtDNA-haplotype frequencies between localities suggested that little gene now currently exists among populations. When combined with microsatellite analysis, which has revealed substantial differentiation among koala populations, we conclude that the appropriate short-term management unit (MU) for koalas is the local population.

Differentiation of soil properties related to the spatial association of wheat roots and soil macropores

Under certain soil conditions, e.g. hardsetting clay B-horizons of South-Eastern Australia, wheat plants do not perform as well as would be expected given measurements of bulk soil attributes. In such soils, measurement indicates that a large proportion (80%) of roots are preferentially located in the soil within 1 mm of macropores. This paper addresses the question of whether there are biological and soil chemical effects concomitant with this observed spatial relationship. The properties of soil manually dissected from the 1-3 mm wide region surrounding macropores, the macropore sheath, were compared to those that are measured in a conventional manner on the bulk soil. Field specimens of two different soil materials were dissected to examine biological differentiation. To ascertain whether the macropore sheath soil differs from rhizosphere soil, wheat was grown in structured and repacked cores under laboratory conditions. The macropore sheath soil contained more microbial biomass per unit mass than both the bulk soil and the rhizosphere. The bacterial population in the macropore sheath was able to utilise a wider range of carbon substrates and to a greater extent than the bacterial population in the corresponding bulk soil. These differences between the macropore sheath and bulk soil were almost non-existent in the repacked cores. Evidence for larger numbers of propagules of the broad host range fungus Pythium in the macropore sheath soil were also obtained.