Characterization of esterases in a Brazilian population of Zaprionus indianus (Diptera : Drosophilidae)

The aim of this study was to characterize esterases in Zaprionus indianus, a drosophilid recently introduced into Brazil. A further aim was study the variation of activity of esterases in the presence of inhibitors and their expression according to sex, sexual activity and age of individual flies. Polymorphisms were detected in two esterase loci (Est-2 and Est-3) and monomorphisms in four others (Est-1, Est-4, Est-5 and Est-6). Biochemical tests using alpha- and beta-naphthyl acetate and the inhibitors malathion, eserine sulphate and PMSF allowed us to classify EST-2 and EST-5 as beta-esterases, both carboxyl-esterases, and EST-1, EST-3, EST-4 and EST-6 as alpha-esterases. EST-1 and EST-3 were classified as carboxyl-esterases and EST-4 and EST-6 as cholinesterases. EST-5 activity was more pronounced in males and EST-2 was restricted to them or to recently copulated females. EST-4, rarely detected, was not characterized. Based on their biochemical characteristics possible roles for these enzymes are suggested.

Onset of sexual maturity and relative growth of the freshwater crab Trichodactylus fluviatilis (Trichodactyloidea) in south-eastern Brazil

The main purpose of this study was to provide an estimate for the onset of morphological sexual maturity in the freshwater crab Trichodactylus fluviatilis, from a population located in south-eastern Brazil, based on the relative growth relationships, as well as the characterization of ''handedness''. Monthly collections were carried out at night, from January 2006 to January 2007, in a small water body. In the laboratory, the crabs were sexed; the following body structures were measured and the estimate of onset of sexual maturity was given by the logistic function, as y a/(1\+becx), for 50% of the population to reach the adult phase (CW50carapace width). Right propodus length and abdomen width were the best-fit relationships to represent the onset of sexual maturity for males and females, respectively. Based on the logistic function, the onset of sexual maturity was estimated to occur at around 18mm CW for both sexes. The relative growth recorded for this species is consistent with the model for brachyuran crabs (Hartnoll RG. 1982, Growth. In: Bliss, DE, editor. The biology of Crustacea: embryology, morphology and genetics. New York: Academic Press. p. 111-196), except for the abdomen growth, which is likely to be an adaptive condition related to protection of the newly hatched young. © 2013 Taylor & Francis.

Variação sexual, ontogenética e ambiental do veneno de Crotalus durissus terrificus da região de Botucatu - São Paulo: caracterização enzimática, bioquímica e farmacológica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo da filogenia dos anofelinos brasileiros do subgênero Nyssorhynchus e padronização da amplificação dos genes do receptor do peptídeo sexual (SPR) e do peptídeo sexual (SP)

Pós-graduação em Ciências Biológicas (Genética) - IBB

A novel form of CYP3A from rat brain: cDNA cloning, protein characterization and gene regulation of cytochrome P450 3A9

One full length cDNA clone, designated 3aH15, was isolated from a rat brain cDNA library using a fragment of CYP3A2 cDNA as a probe. 3aH15 encoded a protein composed of 503 amino acid residues. The deduced amino acid sequence of 3aH15 was 92% identical to mouse Cyp3a-13 and had a 68.4% to 76.5% homology with the other reported rat CYP3A sequences. Clone 3aH15 was thus named CYP3A9 by Cytochrome P450 Nomenclature Committee. CYP3A9 seems to the major CYP3A isozyme expressed in rat brain. Sexual dimorphism of the expression of CYP3A9 was shown for the first time in rat brain as well as in rat liver. CYP3A9 appears to be female specific in rat liver based on the standards proposed by Kato and Yamazoe who defined sex specific expression of P450s as being a 10-fold or higher expression level in one sex compared with the other. CYP3A9 gene expression was inducible by estrogen treatment both in male and in female rats. Male rats treated with estrogen had a similar expression level of CYP3A9 mRNA both in the liver and brain. Ovariectomy of adult female rats drastically reduced the mRNA level of CYP3A9 which could be fully restored by estrogen replacement. On the other hand, only a two-fold induction of CYP3A9 expression by dexamethasone was observed in male liver and no significant induction of CYP3A9 mRNA was observed in female liver or in the brains. These results suggest that estrogen may play an important role in the female specific expression of the CYP3A9 gene and that CYP3A9 gene expression is regulated differently from other CYP3A isozymes. ^ P450 3A9 recombinant protein was expressed in E. coli using the pCWOri+ expression vector and the MALLLAVF amino terminal sequence modification. This construct gave a high level of expression (130 nmol P450 3A9/liter culture) and the recombinant protein of the modified P450 3A9 was purified to electrophoretic homogeneity (10.1 nmol P450/mg protein) from solubilized fractions using two chromatographic steps. The purified P450 3A9 protein was active towards the metabolism of many clinically important drugs such as imipramine, erythromycin, benzphetamine, ethylmorphine, chlorzoxazone, cyclosporine, rapamycin, etc. in a reconstituted system containing lipid and rat NADPH-P450 reductase. Although P450 3A9 was active towards the catabolism of testosterone, androstenedione, dehydroepiandrosterone (DHEA) and 17β-estradiol, P450 3A9 preferentially catalyzes the metabolism of progesterone to form four different hydroxylated products. Optimal reconstitution conditions for P450 3A9 activities required a lipid mixture and GSH. The possible mechanisms of the stimulatory effects of GSH on P450 3A9 activities are discussed. Sexually dimorphic expression of P450 3A9 in the brain and its involvement in many neuroactive drugs as well as neurosteroids suggest the possible role of P450 3A9 in some mental disorders and brain functions. ^

Characterization of the NOP -1 opsin photoreceptor in the filamentous fungus Neurospora crassa

Light absorption is an important process for energy production and sensory perception in many organisms. In the filamentous fungus, Neurospora crassa, blue-light is an important regulator of both asexual and sexual development, but the identity of the blue-light receptor is unknown. The work presented in this dissertation initiated the characterization of the putative N. crassa opsin photoreceptor, NOP-1. Opsins were thought to exist only in the archaea and mammals until the discovery of nop-1. All opsins have the same conserved structure of seven transmembrane helical domains with a lysine residue in the seventh helix specific for forming a Schiff-base linkage with retinal. The predicted NOP-1 protein sequence is equally similar to archaeal rhodopsins and a newly identified fungal opsin-related protein group (ORPs). ORPs maintain the seven transmembrane helical structure of opsins, but lack the conserved lysine residue for binding retinal. An ORP gene, orp-1 was identified in N. crassa and this work includes the cloning and sequence analysis of this gene. Characterization of NOP-1 function in N. crassa development began with the construction of a Δnop-1 deletion mutant. Extensive phenotypic analysis of Δnop-1 mutants revealed only subtle defects during development primarily under environmental conditions that induce a stress response. NOP-1 was overexpressed in the heterologous system Pichia pastoris, and it was demonstrated that NOP-1 protein bound all-trans retinal to form a green-light absorbing pigment (λmax = 534 nm) with a photochemical reaction cycle similar to archaeal sensory rhodopsins. nop-1 gene expression was monitored during N. crassa development. nop-1 transcript is highly expressed during asexual sporulation (conidiation) and transcript levels are abundant in the later stages of conidial development. nop-1 expression is not regulated by blue-light or elevated temperatures. Potential functions for NOP-1 were discovered through the transcriptional analysis of conidiation-associated genes in Δnop-1 mutants. NOP-1 exhibits antagonistic transcriptional regulation of conidiation-associated genes late in conidial development, by enhancing the carotenogenic gene, al-2 and repressing the conidiation-specific genes, con-10 and con-13. ^

Cloning and characterization of a specific coactivator, ARA70, for the androgen receptor in human prostate cells.

The androgen receptor (AR) is a member of the steroid receptor superfamily that plays an important role in male sexual differentiation and prostate cell proliferation. Mutations or abnormal expression of AR in prostate cancer can play a key role in the process that changes prostate cancer from androgen-dependent to an androgen-independent stage. Using a yeast two-hybrid system, we were able to isolate a ligand-dependent AR-associated protein (ARA70), which functions as an activator to enhance AR transcriptional activity 10-fold in the presence of 10(-10) M dihydrotestosterone or 10(-9) M testosterone, but not 10(-6) M hydroxyflutamide in human prostate cancer DU145 cells. Our data further indicated that ARA70 Will only slightly induce the transcriptional activity of other steroid receptors such as estrogen receptor, glucocorticoid receptor, and progesterone receptor in DU145 cells. Together, these data suggest that AR may need a specific coactivator(s) such as ARA70 for optimal androgen activity.

Relational and reproductive trajectories leading to adolescent pregnancy in Portugal: a national and regional characterization

INTRODUCTION: The current study aimed to describe the relational and reproductive trajectories leading to adolescent pregnancy in Portugal, and to explore whether there were differences in this process according to adolescents' place of residence. MATERIAL AND METHODS: Data were collected between 2008 and 2013 in 42 public health services using a self-report questionnaire developed by the researchers. The sample consisted of a nationally representative group of pregnant adolescents (n = 459). RESULTS: Regardless of having had one (59.91%) or multiple sexual partners (40.09%), the majority of adolescents became pregnant in a romantic relationship, using contraception at the time of the conception and knowing the contraceptive failure which led to pregnancy (39.22%). In some regions other trajectories were highly prevalent, reflecting options such as planning the pregnancy (Alentejo Region/ Azores Islands), not using contraception (Centro Region/Madeira Islands) or using it incorrectly, without identifying the contraceptive failure (Madeira Islands). On average, romantic relationships were longer than 19 months and adolescents' partners were older than themselves (> 4 years) and no longer in school (75.16%); these results were particularly significant when the pregnancy was planned. DISCUSSION: The knowledge gained in this study shows that prevention efforts must be targeted according to the adolescents' needs in each region and should include high-risk male groups. CONCLUSION: Our results may enable more efficient health policies to prevent adolescent pregnancy in different country regions and support educators and health care providers on sexual education and family planning efforts.

Relational and reproductive trajectories leading to adolescent pregnancy in Portugal: a national and regional characterization

INTRODUCTION: The current study aimed to describe the relational and reproductive trajectories leading to adolescent pregnancy in Portugal, and to explore whether there were differences in this process according to adolescents' place of residence. MATERIAL AND METHODS: Data were collected between 2008 and 2013 in 42 public health services using a self-report questionnaire developed by the researchers. The sample consisted of a nationally representative group of pregnant adolescents (n = 459). RESULTS: Regardless of having had one (59.91%) or multiple sexual partners (40.09%), the majority of adolescents became pregnant in a romantic relationship, using contraception at the time of the conception and knowing the contraceptive failure which led to pregnancy (39.22%). In some regions other trajectories were highly prevalent, reflecting options such as planning the pregnancy (Alentejo Region/ Azores Islands), not using contraception (Centro Region/Madeira Islands) or using it incorrectly, without identifying the contraceptive failure (Madeira Islands). On average, romantic relationships were longer than 19 months and adolescents' partners were older than themselves (> 4 years) and no longer in school (75.16%); these results were particularly significant when the pregnancy was planned. DISCUSSION: The knowledge gained in this study shows that prevention efforts must be targeted according to the adolescents' needs in each region and should include high-risk male groups. CONCLUSION: Our results may enable more efficient health policies to prevent adolescent pregnancy in different country regions and support educators and health care providers on sexual education and family planning efforts.

Synthesis and characterization of K2Ca5(SO4)6· H2O, the equivalent of görgeyite, a rare evaporite mineral

Görgeyite, K2Ca5(SO4)6··H2O, is a very rare monoclinic double salt found in evaporites related to the slightly more common mineral syngenite. At 1 atmosphere with increasing external temperature from 25 to 150 °C, the following succession of minerals was formed: first gypsum and K2O, followed at 100 °C by görgeyite. Changes in concentration at 150 °C due to evaporation resulted in the formation of syngenite and finally arcanite. Under hydrothermal conditions, the succession is syngenite at 50 °C, followed by görgyeite at 100 and 150 °C. Increasing the synthesis time at 100 °C and 1 atmosphere showed that initially gypsum was formed, later being replaced by görgeyite. Finally görgeyite was replaced by syngenite, indicating that görgeyite is a metastable phase under these conditions. Under hydrothermal conditions, syngenite plus a small amount of gypsum was formed, after two days being replaced by görgeyite. No further changes were observed with increasing time. Pure görgeyite showed elongated crystals approximately 500 to 1000 µ m in length. The infrared and Raman spectra are mainly showing the vibrational modes of the sulfate groups and the crystal water (structural water). Water is characterized by OH-stretching modes at 3526 and 3577 cm–1 , OH-bending modes at 1615 and 1647 cm–1 , and an OH-libration mode at 876 cm–1 . The sulfate 1 mode is weak in the infrared but showed strong bands at 1005 and 1013 cm–1 in the Raman spectrum. The 2 mode also showed strong bands in the Raman spectrum at 433, 440, 457, and 480 cm–1 . The 3 mode is characterized by a complex set of bands in both infrared and Raman spectra around 1150 cm–1 , whereas 4 is found at 650 cm–1.