Norepinephrine acts as D1-dopaminergic agonist in the embryonic avian retina: late expression of beta1-adrenergic receptor shifts norepinephrine specificity in the adult tissue

Dopamine is the main catecholamine found in the chick retina whereas norepinephrine is only found in trace amounts. We compared the effectiveness of dopamine and norepinephrine in promoting cyclic AMP accumulation in retinas at embryonic day 13 (E13) and from post-hatched chicken (P15). Dopamine (EC(50)=10microM) and norepinephrine (EC(50)=30microM), but not the beta(1)-adrenergic agonist isoproterenol, stimulated over seven-fold the production of cyclic AMP in E13 retina. The cyclic AMP accumulation induced by both catecholamines in embryonic tissue was entirely blocked by 2microM SCH23390, a D(1) receptor antagonist, but not by alprenolol (beta-adrenoceptor antagonist). In P15 retinas, 100microM isoproterenol stimulated five-fold the accumulation of cAMP. This effect was blocked by propanolol (10microM), but not by 2microM SCH23390. Embryonic and adult retina display beta(1) adrenergic receptor mRNA as detected by RT-PCR, but the beta(1) adrenergic receptor protein was detected only in post-hatched tissue. We conclude that norepinephrine cross-reacts with D(1) dopaminergic receptor with affinity similar to that of dopamine in the embryonic retina. In the mature retina, however, D(1) receptors become restricted to activation by dopamine. Moreover, as opposed to the embryonic tissue, norepinephrine seems to stimulate cAMP accumulation via beta(1)-like adrenergic receptors in the mature tissue.     

Phylogenetic analysis and a time tree for a large drosophilid data set (Diptera: Drosophilidae)

Drosophila is the genus responsible for the birth of experimental genetics, but the taxonomy of drosophilids is difficult because of the overwhelming diversity of the group. In this study, we assembled sequences for 358 species (14 genera, eight subgenera, 57 species groups, and 65 subgroups) to generate a maximum‐likelihood topology and a Bayesian timescale. In addition to sampling an unprecedented diversity of Drosophila lineages, our analyses incorporated a geographical perspective because of the high levels of endemism. In our topology, Drosophila funebris (Fabricius, 1787) (the type species of Drosophila) is tightly clustered with the pinicola subgroup in a North American clade within subgenus Drosophila. The type species of other drosophilid genera fall within the Drosophila radiation, presenting interesting prospects for the phylogenetic taxonomy of the group. Our timescale suggests that a few drosophilid lineages survived the Cretaceous–Palaeogene (K‐Pg) extinction. The drosophilid diversification began during the Palaeocene in Eurasia, but peaked during the Miocene, an epoch of drastic climatic changes. The most recent common ancestor of the clades corresponding to subgenera Sophophora and Drosophila lived approximately 56 Mya. Additionally, Hawaiian drosophilids diverged from an East Asian lineage approximately 26 Mya, which is similar to the age of the oldest emerging atoll in the Hawaiian–Emperor Chain. Interestingly, the time estimates for major geographical splits (New World versus Asia and Africa versus Asia) were highly similar for independent lineages. These results suggest that vicariance played a significant role in the radiation of fruit flies. © 2013 The Linnean Society of London     

Chromosome evolution and phylogeny in Ronderosia (Orthoptera,Acrididae, Melanoplinae): clues of survivors to the challenge of sympatry?

In an attempt to unveil the origin of neo‐sex chromosomes in Ronderosia Cigliano grasshoppers, we performed a combined phylogenetic analysis based on morphological (external morphology and male genitalia) and molecular data (COI, COII, 16S and ITS2) to explore the chromosome evolution within the genus. We also analysed the distributional patterns of the various Ronderosia species and considered the possible role of chromosome rearrangements (CRs) in speciation processes within the genus in the light of ‘suppressed‐recombination’ models. We mapped the states of three chromosomal characters on the combined tree topology. The combined evidence supported Ronderosia as a monophyletic group. The cytogenetic analyses of the genus demonstrated the importance of rearranged karyotypes with single, complex and multiples neo‐sex chromosome determination systems in all species. The chromosome character optimisation suggests X‐autosome centric fusion as the mechanism responsible for neo‐sex chromosome formation in most Ronderosia species, except in R. dubia and R. bergii. Similar autosomes were involved in fusions with the ancestral X chromosome in Ronderosia, supporting previous hypotheses on the unique origin of X‐autosome fusion for the sex chromosome in the genus. As a source of chromosome variation, autosome‐autosome centric fusion played a secondary role in Ronderosia compared with other Dichroplini. Given the homogeneity in the morphological features, the sympatric distribution of closely related species and the intrinsic property of centric fusion as suppressors of the crossing over, we suggest that CRs may have played a key role during the speciation process within Ronderosia.     

Evolutionary History of the PER3 Variable Number of Tandem Repeats (VNTR): Idiosyncratic Aspect of Primate Molecular Circadian Clock

The PER3 gene is one of the clock genes, which function in the core mammalian molecular circadian system. A variable number of tandem repeats (VNTR) locus in the 18th exon of this gene has been strongly associated to circadian rhythm phenotypes and sleep organization in humans, but it has not been identified in other mammals except primates. To better understand the evolution and the placement of the PER3 VNTR in a phylogenetical context, the present study enlarges the investigation about the presence and the structure of this variable region in a large sample of primate species and other mammals. The analysis of the results has revealed that the PER3 VNTR occurs exclusively in simiiforme primates and that the number of copies of the primitive unit ranges from 2 to 11 across different primate species. Two transposable elements surrounding the 18th exon of PER3 were found in primates with published genome sequences, including the tarsiiforme Tarsius syrichta, which lacks the VNTR. These results suggest that this VNTR may have evolved in a common ancestor of the simiiforme branch and that the evolutionary copy number differentiation of this VNTR may be associated with primate simiiformes sleep and circadian phenotype patterns.     

High density micromass cultures of embryonic limb bud mesenchymal cells: An in vitro model of endochondral skeletal development

Summary To study the mechanisms regulating endochondral skeletal development, we examined the characteristics of long-term, high density micromass cultures of embryonic chicken limb bud mesenchymal cells. By culture Day 3, these cells underwent distinct chondrogenesis, evidenced by cellular condensation to form large nodules exhibiting cartilage-like morphology and extracellular matrix. By Day 14, extensive cellular hypertrophy was seen in the core of the nodules, accompanied by increased alkaline phosphatase activity, and the limitation of cellular proliferation to the periphery of the nodules and to internodular areas. By Day 14, matrix calcification was detected by alizarin red staining, and calcium incorporation increased as a function of culture time up to 2 to 3 wk and then decreased. X-ray probe elemental analysis detected the presence of hydroxyapatite. Analogous to growth cartilage developing in vivo, these cultures also exhibited time-dependent apoptosis, on the basis of DNA fragmentation detected in situ by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), ultrastructural nuclear morphology, and the appearance of internucleosomal DNA degradation. These findings showed that cellular differentiation, maturation, hypertrophy, calcification, and apoptosis occurred sequentially in the embryonic limb mesenchyme micromass cultures and indicate their utility as a convenient in vitro model to investigate the regulatory mechanisms of endochondral ossification.     

Dengue and Zika virus infection patterns vary among Aedes aegypti field populations from Belo Horizonte,a Brazilian endemic city

Dengue virus (DENV) and Zika virus (ZIKV) belong to the same viral family, the Flaviviridae. They cause recurring threats to the public health systems of tropical countries such as Brazil. The primary Brazilian vector of both viruses is the mosquito Aedes aegypti. After the mosquito ingests a blood meal from an infected person, the viruses infect and replicate in the midgut, disseminate to secondary tissues and reach the salivary gland (SG), where they are ready to be transmitted to a vertebrate host. It is thought that the intrinsic discrepancies among mosquitoes could affect their ability to deal with viral infections. This study confirms that the DENV and ZIKV infection patterns of nine Ae. aegypti field populations found in geographically separate health districts of an endemic Brazilian city vary. We analyzed the infection rate, disseminated infection, vector competence, and viral load through quantitative PCR. Mosquitoes were challenged using the membrane-feeding assay technique and were tested seven and fourteen days post-infection (early and late infection phases, respectively). The infection responses varied among the Ae. aegypti populations for both flaviviruses in the two infection phases. There was no similarity between DENV and ZIKV vector competencies or viral loads. According to the results of our study, the risk of viral transmission overtime after infection either increases or remains unaltered in ZIKV infected vectors. However, the risk may increase, decrease, or remain unaltered in DENV-infected vectors depending on the mosquito population. For both flaviviruses, the viral load persisted in the body even until the late infection phase. In contrast to DENV, the ZIKV accumulated in the SG over time in all the mosquito populations. These findings are novel and may help direct the development of control strategies to fight dengue and Zika outbreaks in endemic regions, and provide a warning about the importance of understanding mosquito responses to arboviral infections.     

Cell death evaluation in benzo[a]pyrene-transformed human breast epithelial cells after microcell-mediated transfer of chromosomes 11 and 17

The incidence of apoptosis and nuclear instability, including the incidence of catastrophic death, were investigated in benzo[a]pyrene (BP)-transformed human breast epithelial cells (BP1-E cell line) after microcell-mediated transfer of chromosomes 11 and 17. Since the introduction of normal chromosomes 11 and 17 into tumorigenic human breast BP1-E cells reverts some of these cells' characteristics (especially those affected by microsatellite instabilities and loss of heterozygosity) to those of parental non-transformed MCF-10F cells, it was expected that the cell death rates would also be affected by this treatment. The transfer of the mentioned chromosomes, especially chromosome 17, to tumorigenic BP1-E cells increased the apoptotic ratios and decreased the nuclear instability ratios, thus showing that the microsatellite instability and loss of heterozygosity induced by BP in these chromosomes of MCF-10F cells affect the control of cell death mechanisms.     

Dimethyl Sulfoxide and Ebselen Prevent Convulsions Induced by 5-Aminolevulinic Acid

We investigated whether intrastriatal (i.s.) administration of 5-aminolevulinic acid (ALA) induces oxidative damage and whether behavioral alterations induced by i.s. administration of ALA could be affected by antioxidants. Unilateral injection of ALA (6 micromol/striatum) increased (approximately 30%) thiobarbituric acid-reactive substances (TBARS), but did not affect striatal content of total thiol groups. ALA-induced body asymmetry was not prevented by pretreatment with ascorbic acid (100 mg/kg, s.c.), dimethyl sulfoxide (DMSO, 0.5 microl/striatum, i.s.) or ebselen (10 nmol/striatum, i.s.). ALA-induced convulsions were not prevented by ascorbic acid, but were partially prevented by DMSO and completely prevented by ebselen. Ebselen completely prevented the increase of striatal TBARS induced by ALA. Results obtained suggest the involvement of reactive species in ALA-induced convulsions and may be of value in understanding the physiopathology of neurological dysfunctions associated to ALA overload.     

Probiotic Properties of Lactic Acid Bacteria Isolated from Water-Buffalo Mozzarella Cheese

This study evaluated the probiotic properties (stability at different pH values and bile salt concentration, auto-aggregation and co-aggregation, survival in the presence of antibiotics and commercial drugs, study of β-galactosidase production, evaluation of the presence of genes encoding MapA and Mub adhesion proteins and EF-Tu elongation factor, and the presence of genes encoding virulence factor) of four LAB strains (Lactobacillus casei SJRP35, Leuconostoc citreum SJRP44, Lactobacillus delbrueckii subsp. bulgaricus SJRP57 and Leuconostoc mesenteroides subsp. mesenteroides SJRP58) which produced antimicrobial substances (antimicrobial peptides). The strains survived the simulated GIT modeled in MRS broth, whole and skim milk. In addition, auto-aggregation and the cell surface hydrophobicity of all strains were high, and various degrees of co-aggregation were observed with indicator strains. All strains presented low resistance to several antibiotics and survived in the presence of commercial drugs. Only the strain SJRP44 did not produce the β-galactosidase enzyme. Moreover, the strain SJRP57 did not show the presence of any genes encoding virulence factors; however, the strain SJRP35 presented vancomycin resistance and adhesion of collagen genes, the strain SJRP44 harbored the ornithine decarboxylase gene and the strain SJRP58 generated positive results for aggregation substance and histidine decarboxylase genes. In conclusion, the strain SJRP57 was considered the best candidate as probiotic cultures for further in vivo studies and functional food products development.     

Cytogenetic divergence in two sympatric fish species of the genus Astyanax Baird and Girard, 1854 (Characiformes,Characidae) from northeastern Brazil

The fish genus Astyanax is widespread throughout the Neotropical region and is one of the most species-rich genera of the Characiformes. Cytogenetic studies of Astyanax have revealed marked intra- and interspecific diversity, with the identification of various species complexes. In this report, we describe the karyotypic structure of two sympatric species of Astyanax (Astyanax sp. and Astyanax aff. fasciatus) from the Middle Contas River basin in the northeastern Brazilian state of Bahia. Both species had 2n = 48 but differed in their karyotypic formulae. Small heterochromatic blocks and multiple nucleolar organizer regions (NORs) were identified in both species. Terminal CMA₃⁺/DAPI⁻ signals were observed in Astyanax sp. and A. aff. fasciatus, mostly coincident with NORs. These results show that chromosomal markers can be used to identify species in this fish complex. These markers can provide useful information for evolutionary studies and investigations on the mechanisms of chromosomal diversity in Astyanax.