Population structure and phylogenetic relationships in Brassica rapa L. subspecies by using isozyme markers / Estrutura populacional e relações filogenéticas em subespécies de Brassica rapa L. utilizando marcadores isoenzimáticos

Abstract The present study aimed to assess population structure and phylogenetic relationships of nine subspecies of Brassica rapa L. represented with thirty-five accessions cover a wide range of species distribution area using isozyme analysis in order to select more diverse accessions as supplementary resources that can be utilized for improvement of B. napus. Enzyme analysis resulted in detecting 14 putative polymorphic loci with 27 alleles. Mean allele frequency 0.04 (rare alleles) was observed in Cat4A and Cat4B in sub species Oleifera accession CR 2204/79 and in subspecies trilocularis accessions CR 2215/88 and CR 2244/88. The highest genetic diversity measures were observed in subspecies dichotoma, accession CR 1585/96 (the highest average of observed (H0) and expected heterozygosity (He), and number of alleles per locus (Ae)). These observations make this accession valuable genetic resource to be included in breeding programs for the improvement of oilseed B. napus. The average fixation index (F) is significantly higher than zero for the analysis accessions indicating a significant deficiency of heteozygosity. The divergence among subspecies indicated very great genetic differentiation (FST = 0.8972) which means that about 90% of genetic diversity is distributed among subspecies, while 10% of the diversity is distributed within subspecies. This coincides with low value of gene flow (Nm = 0.0287). B. rapa ssp. oleifera (turnip rape) and B. rapa ssp. trilocularis (sarson) were grouped under one cluster which coincides with the morphological classification.

Resumo O presente estudo teve como objetivo avaliar a estrutura populacional e as relações filogenéticas de nove subespécies de Brassica rapa L. representadas com 35 acessos, cobrindo uma ampla gama de áreas de distribuição de espécies usando análise isoenzimática, a fim de selecionar acessos mais diversos como recursos suplementares que podem ser utilizados para melhoria de B. napus. A análise enzimática resultou na detecção de 14 loci polimórficos putativos com 27 alelos. A frequência média de 0,04 alelo (alelos raros) foi observada em Cat4A e Cat4B, nas subespécies Oleifera CR 2204/79 e nas subespécies trilocularis CR 2215/88 e CR 2244/88. As maiores medidas de diversidade genética foram observadas na subespécie dicotômica CR 1585/96 (a média mais alta observada (H0) e heterozigosidade esperada (He) e número de alelos por locus (Ae). Essas observações tornam esse acesso um valioso recurso genético a ser incluído em programas de melhoramento de oleaginosas B. napus. O índice médio de fixação (F) é significativamente maior que 0 para os acessos à análise, indicando uma deficiência significativa de heterozigose. A divergência entre as subespécies indicou uma grande diferenciação genética (FST = 0,8972), o que significa que cerca de 90% da diversidade genética é distribuída entre as subespécies, enquanto 10% da diversidade é distribuída nas subespécies. Isso coincide com o baixo valor do fluxo gênico (Nm = 0,0287). B. rapa ssp. oleifera (nabo) e B. rapa ssp. trilocularis (sarson) foram agrupados conforme a classificação morfológica.

Study on the distribution of human alanine aminotransferase isoenzyme in human tissues / 生物工程学报

This study intends to obtain recombinant proteins of ALT1 and ALT2 isozymes by using genetic recombination technology. Monoclonal antibodies ALT1 and ALT2 with high specificity and high activity were prepared and screened (ALT1 monoclonal antibody has been successfully prepared and published). The localization, distribution and expression of ALT1 and ALT2 isozymes in human tissues were discussed. The ALT2 genes were amplified from human liver cancer cell (HepG2) by RT-PCR method. The mature ALT2 gene was subcloned into the pET32a-ALT2 prokaryotic expression vector. Its ligation product was transformed into BL21(DE3) competent cells, and transformed into competent cells to express ALT2 proteins induced by IPTG. The recombinant proteins of ALT2 were purified by nickel column (Ni⁺) affinity chromatography. Balb/c mice were immunized with recombinant proteins of ALT2. Positive serum mouse spleen cells and myeloma cells SP2/0 were selected for cell fusion. The positive cell lines were selected by indirect ELISA and subcloned by limited dilution method. Affinity chromatography was used to purify ALT2 antibodies. The expression and distribution of ALT2 in human normal tissues were detected by RT-PCR and Western blotting. Results show that the expression of ALT isoenzyme in tissues was almost the same at gene mRNA level and protein level. ALT1 is highly expressed in liver, kidney and skeletal muscle, and moderately expressed in gastrointestinal smooth muscle. ALT2 is highly expressed in fat, skeletal muscle and myocardium, and is poorly expressed in gastrointestinal smooth muscle. Immunohistochemical studies show that ALT1 is highly expressed in hepatocytes, renal medullary tubules and muscle fibers, ALT2 is highly expressed in adipocytes and myocardial cells, and ALT1 and ALT2 in gastrointestinal tissues are mainly expressed in mucosa of upper intestinal wall region. The results showed that the isoenzymes ALT1 and ALT2 were mainly expressed in the mucosa of the upper part of the intestinal wall. It is widely distributed in the tissues, providing theoretical basis for understanding the mechanism of ALT activity increase under different pathological conditions.

Case report: is low α-Gal enzyme activity sufficient to establish the diagnosis of Fabry disease? / Relato de caso: a diminuição da enzimaα-Gal l é suficiente para estabelecer o diagnóstico?

Abstract Fabry disease is an X-linked lysosomal storage disease due to alpha-galactosidase A (α-Gal A) deficient activity which leads to the accumulation of glucoesphingolipids, such as globotriaosilceramide. There are over 700 known mutations of the enzyme gene, and most of them cause Fabry Disease. This case report describes a hemodialysis patient with a rare and controversial GLA gene mutation, the D313Y. The medecial investigation confirmed that D313Y is an alpha-galactosidase A sequence variant that causes pseudo deficient enzyme activity in plasma but not Fabry disease. Thus, clinical symptoms that prompted Fabry disease investigation could not be attributable to Fabry disease and therefore enzyme replacement therapy was not indicated.

Resumo Doença de Fabry (DF) é uma doença de depósito lisossômico ligada ao cromossomo X, causada pela deficiência da enzima alfa-galactosidase A (α-Gal A) que leva ao acúmulo de glicoesfingolipídeos, principalmente globotriaosilceramide. Existem mais de 700 mutações conhecidas do gene da enzima, a maioria delas são causadoras de DF. Este relato de caso descreve sobre um paciente em hemodiálise com uma mutação do gene GLA rara e controversa, a D313Y. A investigação médica confirmou que D313Y é uma variante que leva à pseudodeficiência plasmática da enzima, mas não ocasiona DF. Assim, os sintomas clínicos que induziram a investigação da doença não devem ser atribuídos à DF e, portanto, não foi indicada a terapia de reposição enzimática.

Prevalencia de intento de suicidio adolescente y factores de riesgo asociados en una comuna rural de la provincia de Concepción / Suicide attempts among Chilean adolescents

Background: Suicide mortality rates are increasing among teenagers. Aim: To study the prevalence and predictive factors of suicide attempts among Chilean adolescents. Material and Methods: A random sample of 195 teenagers aged 16 ± 1 years (53% males) answered an anonymous survey about their demographic features, substance abuse, the Osaka suicidal ideation questionnaire, Smilksten familial Apgar. Beck hopelessness scale, Beck depression scale and Coppersmith self-esteem inventory. Results: Twenty five percent of respondents had attempted suicide at least in one occasion during their lives. These attempts were significantly associated with female gender, absent parents, family dysfunction, drug abuse, smoking, low self-esteem, hopelessness, depression and recent suicidal ideation. A logistic regression analysis accepted female gender, smoking and recent suicidal ideation as significant independent predictors of suicide attempt. Conclusions: Suicide attempted is common among teenagers and its predictors are female sex, smoking and previous suicidal ideation.

Estratégias para o enfrentamento do absenteísmo em consultas odontológicas nas Unidades de Saúde da Família de um município de grande porte: uma pesquisa-ação / Strategies for tackling absenteeism in dental appointments in the Family Health Units of a large municipality: action research

O objetivo deste trabalho foi analisar os motivos das faltas às consultas odontológicas em Unidades de Saúde da Família (USF) e implementar estratégias para sua redução por meio da pesquisa-ação. O estudo foi realizado em 12 USF de Piracicaba/SP, de 01 de janeiro a 31 de dezembro de 2010. A amostra se consistiu de 385 usuários, entrevistados por telefone, sobre os motivos das faltas, além de 12 cirurgiões-dentistas e 12 enfermeiras. Realizaram-se duas oficinas com os profissionais: uma para problematização dos dados coletados nas entrevistas e elaboração de estratégias; e outra após 4 meses, para avaliação. O maior motivo de faltas foi a coincidência do horário de funcionamento das unidades com o de trabalho dos usuários. Dentre as estratégias ressaltou-se a realização de palestras sobre saúde bucal, educação permanente nas reuniões de equipe, capacitação dos Agentes Comunitários de Saúde, participação em grupos terapêuticos e parcerias entre Equipe de Saúde Bucal e equipamentos sociais da comunidade. A adoção de prontuário único foi a estratégia desafiadora encontrada pelos profissionais. Concluiu-se que as estratégias implementadas levaram à diminuição das faltas em 66,6% e o caráter motivador das oficinas possibilitou a reflexão crítica para o redirecionamento da prática em saúde.

The aim of this study was to analyze the reasons for missed appointments in dental Family Health Units (FHU) and implement strategies to reduce same through action research. This is a study conducted in 12 FHUs in Piracicaba in the State of São Paulo from January, 1 to December, 31 2010. The sample was composed of 385 users of these health units who were interviewed over the phone and asked about the reasons for missing dental appointments, as well as 12 dentists and 12 nurses. Two workshops were staged with professionals: the first to assess the data collected in interviews and develop strategy, and the second for evaluation after 4 months. The primary cause for missed appointments was the opening hours of the units coinciding with the work schedule of the users. Among the strategies suggested were lectures on oral health, ongoing education in team meetings, training of Community Health Agents, participation in therapeutic groups and partnerships between Oral Health Teams and the social infrastructure of the community. The adoption of the single medical record was the strategy proposed by professionals. The strategies implemented led to a 66.6% reduction in missed appointments by the units and the motivating nature of the workshops elicited critical reflection to redirect health practices.

Human glutathione S-transferase polymorphisms associated with prostate cancer in the Brazilian population

Objective To evaluate the influence of polymorphisms in GSTA1, GSTM1, GSTT1, and GSTP1 in the risk of developing Prostate Cancer (PCa) in a population of Rio de Janeiro and compare the distribution of allele and genotype frequencies of the polymorphisms analyzed in the present study population with other regions in the country and different ethnic groups. Materials and Methods We analyzed a sample of the Brazilian population, comprising 196 patients with PCa treated by the urology services of the Brazilian National Cancer Institute (INCA) and Mario Kroeff Hospital (HMK), and 208 male blood donors from the Clementino Fraga Filho Hospital, Federal University of Rio de Janeiro (UFRJ). The polymorphisms were determined in DNA, extracted from peripheral blood leucocytes using the Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP). Results Our results showed that the distribution of polymorphisms can vary significantly according to the Brazilian region and ethnic groups. The distribution of allele and genotype frequencies of the polymorphism GSTA1 was statistically different between cases and controls. Genotypes (A / B + B / B) were associated with protection (OR = 0.61, 95 % CI = 0.40-0.92) for PCa in comparison to genotype A / A. Conclusion The distribution of genotype frequencies of the polymorphism GSTA1 was statistically different between the case and control groups (p = 0.023), and the presence of genotypes A / B and B / B suggests a protective role against the risk of PCa compared to genotype A / A. This is the first study that reports the genotypic frequency of this polymorphism and its association with PCa in a Brazilian population sample. .

Inhibitory effects of osteoprotegerin on osteoclast formation and function under serum-free conditions

The purpose of this study was to determine whether osteoprotegerin (OPG) could affect osteoclat differentiation and activation under serum-free conditions. Both duck embryo bone marrow cells and RAW264.7 cells were incubated with macrophage colony stimulatory factor (M-CSF) and receptor activator for nuclear factor kappaB ligand (RANKL) in serum-free medium to promote osteoclastogenesis. During cultivation, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells. Osteoclast differentiation and activation were monitored via tartrate-resistant acid phosphatase (TRAP) staining, filamentous-actin rings analysis, and a bone resorption assay. Furthermore, the expression osteoclast-related genes, such as TRAP and receptor activator for nuclear factor kappaB (RANK), that was influenced by OPG in RAW264.7 cells was examined using real-time polymerase chain reaction. In summary, findings from the present study suggested that M-CSF with RANKL can promote osteoclast differentiation and activation, and enhance the expression of TRAP and RANK mRNA in osteoclasts. In contrast, OPG inhibited these activities under serum-free conditions.

Judicialização da saúde na garantia do acesso ao medicamento / The judicialisation of health as a means ensuring access to medicines / La Judicialización de la salud como garantía de acceso a medicamentos

Objetivo Visando conhecer o impacto das demandas judiciais sobre a organização dos serviços públicos de saúde, realizou-se uma revisáo sistemática com enfoque na "judicialização da saúde" para fornecimento de medicamentos. Métodos Foram analisados artigos originais publicados no período de 2007 a 2011, na literatura nacional e internacional, resultando no total de 49239 artigos disponíveis nas bases de dados Science Direct e BIREME. Resultados A pesquisa indicou predominância da bibliografia proveniente do Brasil, principalmente do sudeste, bem como de estudo realizado na Colômbia. Discursáo Dentre os pleitos, configuraram-se como principais agravos relatados as doenças crônicas, podendo-se citar: diabetes, hipertensáo, cânceres e artrite reumatóide. Por serem afecções parte de programas específicos do Sistema Único de Saúde, a dificuldade de acesso a esses fármacos e consequente judicialização da saúde demonstrou a fragilidade das políticas públicas existentes. Conclusão Por fim, conclui-se que a via judicial, apesar de ser uma estratégia para garantir o acesso ao medicamento, apresenta inabilidade para lidar com o julgamento das ações e gera, dessa forma, distorções no fluxo dos sistemas públicos.

Objective A systematic review, focusing on the judicialisation of health regarding gaining access to medicines, was aimed at understanding the impact of lawsuits on the organisation of public health services. Method Original articles published between 2007 and 2011 in the pertinent national and international literature were analysed, resulting in 49,239 articles being found in Science Direct and BIREME databases. Results The survey indicated a predominance of literature from Brazil, mainly the southeast, as well as a study from Colombia. Discussion The aforementioned chronic disease-related claims involved diabetes, high blood pressure, cancer and rheumatoid arthritis. Forming part of specific Unified Healthcare System programmes highlighted the difficulty in gaining access to the appropriate medicine and consequent health judicialisation demonstrated the fragility of existing public policy. Conclusion It was concluded that the courts (despite being a strategy for ensuring access to medicine) were unable to deal with the current spate of lawsuits, thereby leading to disruption regarding the flow of public systems.

Objetivo El estudio tiene como objetivo evaluar el impacto de las demandas judiciales sobre la organización de los servicios públicos de salud, mediante la realización de una revisión sistemática centrada en el uso de los tribunales para el suministro de medicamentos. Método Fueron identificados 49239 artículos en las bases de datos Science Direct e BIREME. Resultado El estudio indicó que la mayor parte de la bibliografía es de Brasil, con uno estudio en Colombia. Discusión Aparecen como los principales trastornos de salud relatados a las enfermedades crónicas, se pueden citar: la diabetes, la hipertensión, el cáncer y la artritis reumatoide. Debido a que son parte de los programas específicos de lo sistema de salud, la dificultad de acceso a estos fármacos y la consiguiente judicialización de la salud de manifiesto la fragilidad de las políticas públicas existentes. Conclusiones Por último, está la conclusión de que los tribunales, a pesar de ser una estrategia para garantizar el acceso a la medicina, presenta incapacidad para hacer frente al juicio de las acciones y por lo tanto genera distorsiones en el flujo de los sistemas públicos.

Simvastatin inhibits osteoclast differentiation by scavenging reactive oxygen species

Osteoclasts, together with osteoblasts, control the amount of bone tissue and regulate bone remodeling. Osteoclast differentiation is an important factor related to the pathogenesis of bone-loss related diseases. Reactive oxygen species (ROS) acts as a signal mediator in osteoclast differentiation. Simvastatin, which inhibits 3-hydroxy-3-methylglutaryl coenzyme A, is a hypolipidemic drug which is known to affect bone metabolism and suppresses osteoclastogenesis induced by receptor activator of nuclear factor-kappaB ligand (RANKL). In this study, we analyzed whether simvastatin can inhibit RANKL-induced osteoclastogenesis through suppression of the subsequently formed ROS and investigated whether simvastatin can inhibit H2O2-induced signaling pathways in osteoclast differentiation. We found that simvastatin decreased expression of tartrate-resistant acid phosphatase (TRAP), a genetic marker of osteoclast differentiation, and inhibited intracellular ROS generation in RAW 264.7 cell lines. ROS generation activated NF-kappaB, protein kinases B (AKT), mitogen-activated protein kinases signaling pathways such as c-JUN N-terminal kinases, p38 MAP kinases as well as extracellular signal-regulated kinase. Simvastatin was found to suppress these H2O2-induced signaling pathways in osteoclastogenesis. Together, these results indicate that simvastatin acts as an osteoclastogenesis inhibitor through suppression of ROS-mediated signaling pathways. This indicates that simvastatin has potential usefulness for osteoporosis and pathological bone resorption.

GSTM1 Tissue Genotype as a Recurrence Predictor in Non-muscle Invasive Bladder Cancer

Tissue genotyping is more useful approach than using blood genomic DNA, which can reflect the effects of the somatic mutations in cancer. Although polymorphisms in glutathione S-transferase (GST) have been associated with the risk of bladder cancer (BC) development, few reports provide information about the prognosis of BC. We investigated glutathione S-transferase mu (GSTM1) and glutathione S-transferase theta (GSTT1) genotypes using genomic DNA from primary 165 BC tissue samples to assess the association with disease prognosis. DNA samples from tumor were analyzed by multiplex polymerase chain reaction (PCR). The results were compared with clinicopathological parameters. The prognostic significance of the GSTs was evaluated by Kaplan-Meier and multivariate Cox regression model. Kaplan-Meier estimates revealed significant differences in time to tumor recurrence according to the GSTM1 tissue genotype (P = 0.038) in non-muscle invasive bladder cancer (NMIBC). Multivariate Cox regression analysis also revealed that the tissue GSTM1 genotype (hazards ratio [HR]: 0.377, P = 0.031) was an independent predictor of bladder tumor recurrence in NMIBC. This identification of GSTM1 tissue genotype as a prognosticator for determining recurrence in NMIBC should prove highly useful in a clinical setting.

Genetic variability among Anopheles species belonging to the Nyssorhynchus and Anopheles subgenera in the Amazon region / Variabilidade genética entre espécies de Anopheles dos subgêneros Nyssorhynchus e Anopheles da região Amazônica

INTRODUCTION: Isoenzymatic analyses were performed involving species of the Nyssorhynchus and Anopheles subgenera in order to estimate the intra and interspecies genetic variability. METHODS: Mosquitoes were caught at different localities in the Amazon region. The collection and rearing of mosquitoes in the laboratory followed specific protocols. For the genetic variability analyses, the technique of horizontal electrophoresis on starch and starch-agarose gel with appropriate buffer systems was used. The alloenzyme variation was estimated using the Biosys-1 software. RESULTS: Out of the 13 loci, eight were polymorphic. Anopheles nuneztovari presented the largest number of alleles per locus, while the smallest number was detected in Anopheles marajoara from Macapá. The largest number of polymorphic loci was found for Anopheles marajoara from Maruanum and the smallest for Anopheles benarrochi (Guayará Mirim). Anopheles darlingi (Macapá) presented the greatest heterozygosity (Ho = 0.167 ± 0.071), while the lowest heterozygosity (Ho = 0.045 ± 0.019) was observed in Anopheles intermedius (Pacoval) of the subgenus Anopheles. Wright's F coefficient revealed considerable genetic structuring between the populations of Anopheles darlingi (Fst = 0.110) and between the populations of Anopheles marajoara (Fst = 0.082). CONCLUSIONS: Considering all the species studied, the genetic distance ranged from 0.008 to 1.114. The greatest distance was between Anopheles mattogrossensis and Anopheles oswaldoi, while the smallest was between the Anopheles benarrochi populations.

INTRODUÇÃO: Análises isoenzimáticas foram realizadas envolvendo espécies dos subgêneros Nyssorhynchus e Anopheles para estimar a variabilidade genética intra e interespecífica. MÉTODOS: Os mosquitos foram capturados em diferentes localidades da região Amazônica. A coleta e a criação dos mosquitos em laboratório foram conforme protocolos específicos. Na análise da variabilidade genética empregou-se a técnica de eletroforese horizontal em géis de amido e amido-agarose com sistemas tampões apropriados. A variação alozímica foi estimada pelo Programa Biosys-1. RESULTADOS: Dos 13 loci, oito foram polimórficos. Anopheles nuneztovari apresentou o maior número de alelos por loco, sendo o menor detectado para Anopheles albitarsis (Macapá). O maior número de loci polimórficos foi detectado em Anopheles marajoara (Maruanum) e o menor, em Anopheles benarrochi (Guayará Mirim). Anopheles darlingi (Macapá) apresentou a maior heterozigosidade (Ho = 0,167 ± 0.071) e a menor (Ho = 0,045 ± 0.019) foi observada em Anopheles intermedius (Pacoval) do subgênero Anopheles. O coeficiente F de Wright evidenciou considerável estruturação genética entre populações de Anopheles darlingi (Fst= 0,110) e entre as populações de Anopheles marajoara (Fst= 0,082). CONCLUSÕES: Considerando todas as espécies estudadas, a distância genética variou de 0,008 a 1,144, onde a maior distância foi entre Anopheles mattogrossensis e Anopheles oswaldoi e a menor, entre as populações de Anopheles benarrochi.

Isoenzymatic polymorphism in the leaf-cutting ant Atta capiguara Gonçalves (hymenoptera: formicidae)

This study was carried out to analyze the genetic population structure of Atta capiguara from 12 nests collected in Tapejara in the state of Paraná, Brazil, using isoenzyme polymorphisms. The analyzed isoenzymes were esterases (EST - EC 3.1.1.1), acid phosphatase (ACP - EC 3.1.3.2) and carbonic anhydrase (CA - EC 4.2.1.1). Ten loci were found in A.capiguara and four polymorphic loci were detected. The observed heterozigosity (0.0296) was low when compared to the expected heterozigosity (0.1461). The high value of F IS (0.7954) shows an excess of homozygous genotypes probably caused by inbreeding.

Rebamipide-induced downregulation of phospholipase D inhibits inflammation and proliferation in gastric cancer cells

Rebamipide a gastroprotective drug, is clinically used for the treatment of gastric ulcers and gastritis, but its actions on gastric cancer are not clearly understood. Phospholipase D (PLD) is overexpressed in various types of cancer tissues and has been implicated as a critical factor in inflammation and carcinogenesis. However, whether rebamipide is involved in the regulation of PLD in gastric cancer cells is not known. In this study, we showed that rebamipide significantly suppressed the expression of both PLD1 and PLD2 at a transcriptional level in AGS and MKN-1 gastric cancer cells. Downregulation of PLD expression by rebamipide inhibited its enzymatic activity. In addition, rebamipide inhibited the transactivation of nuclear factor kappa B (NFkappaB), which increased PLD1 expression. Rebamipide or PLD knockdown significantly suppressed the expression of genes involved in inflammation and proliferation and inhibited the proliferation of gastric cancer cells. In conclusion, rebamipide-induced downregulation of PLD may contribute to the inhibition of inflammation and proliferation in gastric cancer.

Paraoxonases : Structure, gene polymorphism & role in coronary artery disease.

Paraoxonases (PONs) i.e. PON1, PON2, PON3 are basically lactonases. Of these, PON1 in addition has an efficient esterase activity and can hydrolyze organophosphates. The PONs prevent low density lipoprotein cholesterol (LDL-C) from peroxidation, thereby preventing atherosclerosis. The PON1 is exclusively associated with high density lipoprotein cholesterol (HDL-C) and its antioxidant activity is largely attributed to PON1 located on it. At present, PON1 status i.e. its activity and concentration, is considered to be more important than polymorphism alone, in prevention of coronary artery disease (CAD). Its activity has been found to be affected by a number of pharmacological agents, diet and other factors, thereby becoming a promising target for pharmacological intervention. The PON2 prevents cell mediated lipid peroxidation. However, little is known about the role of PON3. This review describes the structure, gene polymorphism, and factors affecting the activity of PONs, and their role in prevention of CAD.

Mating system and seed variation of Acacia hybrid (A. mangium x A. auriculiformis).

The mating system and seed variation of Acacia hybrid (A. mangium x A. auriculiformis) were studied using allozymes and random amplified polymorphic DNA (RAPD) markers, respectively. Multi-locus outcrossing rate estimations indicated that the hybrid was predominantly outcrossed (mean+/- s.e. tm = 0.86+/-0.01). Seed variation was investigated using 35 polymorphic RAPD fragments. An analysis of molecular variance (AMOVA) revealed the highest genetic variation among seeds within a pod (66%-70%), followed by among pods within inflorescence (29%-37%), and the least variation among inflorescences within tree (1%). In addition, two to four RAPD profiles could be detected among seeds within pod. Therefore, the results suggest that a maximum of four seeds per pod could be sampled for the establishment of a mapping population for further studies.

Postconditioning ameliorates lipid peroxidation in liver ischemia-reperfusion injury in rats / Pós-condicionamento melhora a peroxidação lipídica na lesão de isquemia-reperfusão hepática em ratos

PURPOSE: Liver ischemia-reperfusion injury is a phenomenon presents in events like liver resections and transplantation. The restoration of blood flow may leads to local and systemic injury. Several techniques have been developed in order to avoid or ameliorate ischemia-reperfusion injury in clinical situations. The application of a sttuter reperfusion after the ischemic event (postconditioning) could alters the hydrodynamics and stimulates endogenous mechanisms that attenuate the reperfusion injury. The present study was designed to evaluate the potential protective effect of postconditioning in a model of ischemia-reperfusion in rats. METHODS: Hepatic anterior pedicle of median and left anterolateral segments were exposed and clamped for 1 hour. Two hours later, clamp was released in two different ways: Control Group (n=7): clamp was release straightforward; Postconditioning Group (n=7): clamp was released intermittently. Lipid peroxidation (malondialdehyde) and expression of the glutathione-s-transferase-α-3 gene were studied. RESULTS: Lipid peroxidation was significantly decreased in ischemic and non-ischemic liver by postconditioning. GST- α3 gene was overexpressed in postconditioned group, but not significantly. CONCLUSION: Postconditioning induced hepatoprotection by reducing lipid peroxidation in the ischemic and non-ischemic liver.

OBJETIVO: A lesão de isquemia-reperfusão hepática é um fenômeno presente em eventos tais como ressecções hepáticas e transplante de fígado. A restauração do fluxo sangüíneo após a isquemia gera lesões locais e sistêmicas. Várias técnicas foram desenvolvidas com o objetivo de evitar ou diminuir a lesão de isquemia-reperfusão hepática em situações clínicas. A utilização da reperfusão intermitente após o evento isquêmico (pós-condicionamento) pode alterar a hidrodinâmica e estimular mecanismos endógenos que atenuam o dano da reperfusão. O presente estudo foi realizado para avaliar o potencial efeito protetor do pós-condicionamento em um modelo de isquemia-reperfusão em ratos. MÉTODOS: O pedículo dos lobos mediano e ântero-lateral foi isolado e clampeado por 1 hora. Após 2 horas, o pedículo foi liberado de duas maneiras diferentes: Grupo Controle (n=7): clampe liberado de uma só vez; Grupo Pós-condicionamento (n=7): clampe liberado de maneira intermitente. Malondialdeído (MDA) e expressão do gene GST- α3 foram estudadas nos grupos. RESULTADOS: A peroxidação lipídica foi significativamente diminuída no fígado isquêmico e no fígado não isquêmico pelo pós-condicionamento. A expressão do gene GST- α3 aumentou, porém não significativamente, no grupo pós-condicionamento. CONCLUSÃO: O pós-condicionamento induziu hepatoproteção pela redução da peroxidação lipídica nos fígados isquêmico e não isquêmico.

Biochemical comparison of two Hypostomus populations (Siluriformes, Loricariidae) from the Atlântico Stream of the upper Paraná River basin, Brazil

Two syntopic morphotypes of the genus Hypostomus - H. nigromaculatus and H. cf. nigromaculatus (Atlântico Stream, Paraná State) - were compared through the allozyme electrophoresis technique. Twelve enzymatic systems (AAT, ADH, EST, GCDH, G3PDH, GPI, IDH, LDH, MDH, ME, PGM and SOD) were analyzed, attributing the score of 20 loci, with a total of 30 alleles. Six loci were diagnostic (Aat-2, Gcdh-1, Gpi-A, Idh-1, Ldh-A and Mdh-A), indicating the presence of interjacent reproductive isolation. The occurrence of few polymorphic loci acknowledge two morphotypes, with heterozygosity values He = 0.0291 for H. nigromaculatus and He = 0.0346 for H. cf. nigromaculatus. F IS statistics demonstrated fixation of the alleles in the two morphotypes. Genetic identity (I) and distance (D) of Nei (1978) values were I = 0.6515 and D = 0.4285. The data indicate that these two morphotypes from the Atlântico Stream belong to different species.

Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli

The present work aims to study a new NADH-cytochrome b5 reductase (cb5r) from Mucor racemosus PTCC 5305. A cDNA coding for cb s r was isolated from a Mucor racemosus PTCC 5305 cDNA library. The nucleotide sequence of the cDNA including coding and sequences flanking regions was determined. The open reading frame starting from ATG and ending with TAG stop codon encoded 228 amino acids and displayed the closest similarity (73 percent) with Mortierella alpina cb s r. Lack of hydrophobic residues in the N-terminal sequence was apparent, suggesting that the enzyme is a soluble isoform. The coding sequence was then cloned in the pET16b transcription vector carrying an N-terminal-linked His-Tag® sequence and expressed in Escherichia coli BL21 (DE3). The enzyme was then homogeneously purified by a metal affinity column. The recombinant Mucor enzyme was shown to have its optimal activity at pH and temperature of about 7.5 and 40 °C, respectively. The apparent Km value was calculated to be 13 μM for ferricyanide. To our knowledge, this is the first report on cloning and expression of a native fungal soluble isoform of NADH-cytochrome b5 reductase in E. coli.

Estructura genética en cinco especies de flebótomos (Lutzomyia spp.) de la serie townsendi, grupo verrucarum, en Colombia (Diptera: Prychodidae)

Genetic structure in five Phlebotominae (Lutzomyia spp.), townsendi series, verrucarum group, in Colombia (Diptera: Prychodidae). Sixteen isoenzyme patterns were analyzed for five Colombian Lutzomyia species. The average unbiased expected heterozygosity levels ranged from 0.098 (Lu. youngi) to 0.215 (Lu. torvida). The five species samples, taken all the isoenzymes employed, were significantly deviated from the Hardy-Weinberg equilibrium by homozygous excess with classical as well as Markov chain exact tests. Possible causes: (1) Wahlund effect within populations due to subdivision and/or sampling. Endogamy could be discarded because these loci were affected by highly different levels of homozygous excess. (2) Null alleles could be not discarded, at least for some isoenzymes. The hierarchical Wright´s F analysis showed high and significant values for each parameter. The average F IT value was 0.655 with a conspicous homozygous excess at a global level (all species taken together); the average F IS value was significantly positive (0.515) as well, with homozygous excess within each species. The genetic heterogeneity between the fives species was noteworthy (F ST = 0.288), indicating clear genetic differentiation. The more related species pairs were Lu. longiflocosa-Lu. torvida (0.959) and Lu torvida-Lu. spinicrassa (0.960); while Lu. torvida-Lu. youngi (0.805) and Lu. quasitownsendi-Lu. youngi (0.796) were the most divergent (Nei´s genetic identity matrix). UPGMA and Wagner algorithms showed that the most divergent species was Lu. youngi, whereas the most related were Lu. longiflocosa-Lu. torvida and Lu torvida-Lu. spinicrassa. A spatial autocorrelation analysis (Moran´s I index) revealed a very weak, or inexistent spatial structure, which means that the speciation events between these species were independent from the geographic distances from where they currently live. Rev. Biol. Trop. 56 (4): 1717-1739. Epub 2008 December 12.

Se analizaron 16 sistemas isoenzimáticos para cinco especies colombianas del género Lutzomyia. Los niveles de heterocigosis media esperada insesgada oscilaron entre 0.098 (Lu. youngi) y 0.215 (Lu. torvida). Las cinco muestras estudiadas de forma global, para todos los marcadores analizados, presentaron desviación respecto al equilibrio Hardy-Weinberg por un exceso de homocigotos, tanto al utilizar algunas pruebas clásicas como tests exactos con cadenas de Markov. Este hecho puede estar favorecido por diversas causas: (1) la más probable es la existencia de efecto Wahlund en el seno de cada población debido a subdivisión y/o a la técnica de muestreo empleada. La endogamia puede descartarse ya que no todos los loci están afectados por el mismo tipo de exceso de homocigotos. (2) Sin embargo, no se puede descartar la existencia de alelos nulos, al menos, para algunos de los marcadores isoenzimáticos utilizados. El análisis jerarquizado con las F de Wright mostró valores elevados y significativos para cada uno de los estadísticos. El estadístico promedio F IT mostró un valor de 0.655 existiendo un conspicuo exceso de homocigotos a nivel total de todas las especies, el estadístico promedio F IS fue altamente positivo (0.515) mostrando exceso de homocigotos a nivel individual en cada una de las especies estudiadas. La heterogeneidad genética entre las cinco especies fue notable (F ST = 0.288). Esto muestra que esas especies están bien diferenciadas a nivel isoenzimático y que en el interior de cada especie también hay una subdivisión genética. La matriz de identidades genéticas de Nei muestra que las especies más relacionadas fueron Lu. longiflocosa-Lu. torvida (0.959) y Lu torvida-Lu. spinicrassa (0.960) mientras que las genéticamente más distantes fueron Lu. torvida-Lu. youngi (0.805) y Lu. quasitownsendi-Lu. youngi (0.796). Con los algoritmos UPGMA y Wagner, se observó que la especie más divergente fue Lu. youngi, mientras que las relaciones más conspicuas se observaron entre Lu. longiflocosa-Lu. torvida y Lu torvida-Lu. spinicrassa. Adicionalmente, con un análisis de autocorrelación espacial (índice de Moran) la mayoría de los alelos utilizados presentaron una estructura espacial muy débil o inexistente, lo que significa que los eventos de especiación entre las especies estudiadas se dieron en forma independiente de las distancias geográficas existentes actualmente entre ellas.

Genetic diversity in some perennial plant species with-in short distances.

Distinct morphophysiological variations observed for over 2 years with-in short distances among four perennial plants indicated genetic diversity among the lines growing at three places. The isozyme and SDS polyacrylamide gel banding patterns as genetic markers were used to investigate four perennial species, namely Dalbergia sissoo Roxb., Delonix regia (Boj.) Refin., Cassia fistula L. and Calotropis procera R. Br. Plant materials collected from three locations (Agra, Gwalior and Lucknow) differing in climo-edaphic variables were examined for 4 enzyme systems, viz., esterase, polyphenol oxidase, peroxidase and superoxide dismutase (EST, PPO, PRX and SOD). Among the four isozymes SOD and PRX revealed best discriminating power. Protein banding patterns as well as zymogram revealed that Dalbergia sissoo growing at Gwalior was closer to that of Agra; Delonix regia depicted highest similarity between Lucknow and Agra and Calotropis procera of Lucknow location was more closer to Gwalior than Agra. The results confirm genetic diversity in the species as a means of adaptation to differing climo-edaphic variables.