IMMUNOHISTOCHEMICAL DETECTION OF L CELLS IN GASTROINTESTINAL TRACT MUCOSA OF PATIENTS AFTER SURGICAL TREATMENT FOR CONTROL OF TYPE 2 DIABETES MELLITUS.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) is a disease of global impact that has led to an increase in comorbidities and mortality in several countries. Immunoexpression of the incretin hormones such as glucagon-like peptide-1 (GLP-1) and peptide YY (3-36) (PYY3-36) can be used as a scorer in the gastrointestinal tract to analyze L-cell activity in response to T2DM treatment. This study aimed to investigate the presence, location, and secretion of L cells in the small intestine of patients undergoing the form of bariatric surgery denominated adaptive gastroenteromentectomy with partial bipartition. METHODS: Immunohistochemical assays, quantitative real-time polymerase chain reaction (qPCR), and Western blot analysis were performed on samples of intestinal mucosa from patients with T2DM in both the preoperative and postoperative periods. RESULTS: All results were consistent and indicated basal expression and secretion of GLP-1 and PYY3-36 incretins by L cells. A greater density of cells was demonstrated in the most distal portions of the small intestine. No significant difference was found between GLP-1 and PYY3-36 expression levels in the preoperative and postoperative periods because of prolonged fasting during which the samples were collected. CONCLUSION: The greater number of L cells in activity implies better peptide signaling, response, and functioning of the neuroendocrine system.

Molecular data reveal a complex population genetic structure for Psalidodon scabripinnis (Teleostei: Characidae) in the Atlantic Rainforest, Brazil.

Recently renamed, Psalidodon scabripinnis populations of Serra da Mantiqueira, previously known as Astyanax scabripinnis have been deeply studied in the last years. These populations are small and isolated and occur very close to the watershed between Paraíba do Sul River basin and Upper Paraná River basin, in Serra da Mantiqueira region in the Atlantic Rainforest. These conditions arouse the interest in knowing theor genetic conservation status and how they responded to the separation between the two rivers basins. Therefore, we accessed the genetic diversity of five P. scabripinnis populations of this region with microsatellites and mitochondrial data. The results showed a complex structure pattern that doesn't match the simple basin separation and a reasonably conservation status when compared with other populations of the same family or with similar natural history.

Immunohistochemical detection of l cells in gastrointestinal tract mucosa of patients after surgical treatment for control of type 2 diabetes mellitus / Detecção imunohistoquímica de células l na mucosa do trato gastrointestinal de pacientes após tratamento cirúrgico para controle de diabetes mellitus tipo 2

ABSTRACT - BACKGROUND: Type 2 diabetes mellitus (T2DM) is a disease of global impact that has led to an increase in comorbidities and mortality in several countries. Immunoexpression of the incretin hormones such as glucagon-like peptide-1 (GLP-1) and peptide YY (3-36) (PYY3-36) can be used as a scorer in the gastrointestinal tract to analyze L-cell activity in response to T2DM treatment. OBJECTIVE: This study aimed to investigate the presence, location, and secretion of L cells in the small intestine of patients undergoing the form of bariatric surgery denominated adaptive gastroenteromentectomy with partial bipartition. METHODS: Immunohistochemical assays, quantitative real-time polymerase chain reaction (qPCR), and Western blot analysis were performed on samples of intestinal mucosa from patients with T2DM in both the preoperative and postoperative periods. RESULTS: All results were consistent and indicated basal expression and secretion of GLP-1 and PYY3-36 incretins by L cells. A greater density of cells was demonstrated in the most distal portions of the small intestine. No significant difference was found between GLP-1 and PYY3-36 expression levels in the preoperative and postoperative periods because of prolonged fasting during which the samples were collected. CONCLUSION: The greater number of L cells in activity implies better peptide signaling, response, and functioning of the neuroendocrine system.

RESUMO - RACIONAL: O diabetes tipo 2 (DM2) é uma doença de impacto mundial que tem levado ao aumento de comorbidades e mortalidade em vários países. A imunoexpressão dos hormônios incretínicos glp-1 e pyy3-36, pode ser usada como marcador no trato gastrointestinal para analisar a atividade da célula L em resposta ao tratamento do DM2. OBJETIVO: O presente estudo teve como objetivo investigar a presença, localização e secreção de células L no intestino delgado de pacientes submetidos à forma de cirurgia bariátrica denominada gastroenteromentectomia adaptativa com bipartição parcial. MÉTODOS: Ensaios imunohistoquímicos, reação quantitativa em cadeia de polimerase em tempo real (qPCR) e análise de manchas ocidentais foram realizados em amostras de mucosa intestinal de pacientes com diabetes tipo 2 nos períodos pré- e pós-operatório. RESULTADOS: Todos os resultados foram consistentes e indicaram expressão basal e secreção de peptídeos glucagon-1 (GLP-1) e peptídeos YY (PYY3-36) incretinas por células L. Uma maior densidade de células foi demonstrada nas porções mais distais do intestino delgado. Não foi encontrada diferença significativa entre os níveis de expressão GLP-1 e PYY3-36 nos períodos pré-operatório e pós-operatório, provavelmente devido ao estado de jejum prolongado durante o qual as amostras foram coletadas CONCLUSÃO: O maior número de células L em atividade implica melhor sinalização de peptídeo, resposta e funcionamento do sistema neuroendócrino.

Dynamics of Replication and Nuclear Localization of the B Chromosome in Kidney Tissue Cells in Astyanax scabripinnis (Teleostei: Characidae).

B chromosomes are extra genomic compounds found in different taxonomic groups, including plants and animals. Obtaining patterns of resolutive chromosomal bands is necessary to understand the nuclear organization, variability and nature of B chromosome chromatin and possible transcriptional regions. In this study, we analyzed 35 Astyanax scabripinnis specimens sampled from Fazenda Lavrinha, a stream in the Paraíba do Sul river basin, Brazil. Through the incorporation of the thymidine analog 5'-bromo-2'-deoxyuridine (5-BrdU) in vivo, it was possible to recognize the replicating regions of the B chromosome at the beginning of the S phase, differentially characterized in relationship to the regions of late replication. In this perspective, it is possible to suggest that the B chromosome of this species possesses a territory and the chromatin accessible for transcription, especially in the light (i.e., early replicating) bands (p1.1; p1.3; and p2.1 and q1.1, q1.3, q2.1, and q2.2). The late-replicating regions are corresponding to the blocks of constitutive heterochromatin. They show a preferential accumulation of satellite DNA As51. By the use of the fluorochrome chromomycin A3 (CMA3), it was possible to identify GC-rich chromosomal regions, corresponding to late-replicating parts of genome, confirming the revealed data by the replication banding and C-banding. In addition, the analysis by confocal microscopy in kidney cells indicates the location of a peripheral anchorage of this chromosome in the nuclear lamina, reinforcing the idea of downregulation of the associated regions.

Differential Expression of Genes Related to Sexual Determination Can Modify the Reproductive Cycle of Astyanax scabripinnis (Characiformes: Characidae) in B Chromosome Carrier Individuals.

The species complex Astyanax scabripinnis is one of the most studied with respect to origin, distribution, and frequency of B chromosomes, and is considered a model organism for evolutionary studies. Research using population inferences about the occurrence and frequency of the B chromosome shows seasonal variation between sexes, which is associated with the presence of this supernumerary element. We hypothesized that the B chromosome could influence the sex ratio of these animals. Based on this assumption, the present work aimed to investigate if differences exist among levels of gene expression with qRT-PCR of the amh (associated with testicular differentiation) and foxl2a (associated with ovarian differentiation) genes between B-carrier and non-B-carrier individuals. The results showed that for the amh gene, the difference in expression between animals with B chromosomes was not accentuated compared to that in animals without this chromosome. Expression of foxl2a in B-carrier females, however, was reduced by 73.56% compared to females that lacked the B chromosome. Males had no difference in expression of the amh and foxl2a genes between carriers and non-carriers of the B chromosome. Results indicate that the presence of B chromosomes is correlated with the differential expression of sex-associated genes. An analysis of these results integrated with data from other studies on the reproductive cycle in the same species reveals that this difference in expression may be expanding the reproductive cycle of the species.

Genetic variability in a population of Astyanax scabripinnis: recent bottleneck and the possible influence of individuals with B chromosomes

Access the genetic variability of endangered and isolated populations has become an important conservation tool. Astyanax scabripinnis is a well-known fish model for genetic studies, forming very isolated populations in headwaters. Besides that, this species frequently presents supernumerary chromosomes, which elevates the interest on genetic studies. Genetic diversity of an Astyanax scabripinnispopulation from the Atlantic Forest (Serra da Mantiqueira region, Brazil) was assessed with microsatellite markers for the first time. Since microsatellite markers are not described for this species, we tested markers described for a related species for transferability to A. scabripinnis. Six polymorphic loci were sufficiently reliable for population genetic analysis. We found that this population passed through a recent bottleneck because of the presence of an excess of heterozygotes, low allelic diversity, heterozygosity excess, and small effective population size. Individuals with and without B chromosomes were previously identified in this population and our study found private alleles in the individuals without B chromosomes. Furthermore, when individuals without B chromosomes were removed from the analysis, the population did not present heterozygosity excess, suggesting that the bottleneck event was driven by individuals with B chromosomes. Our results provide an insight into the value of microsatellite markers as molecular tools and is the first genetic study using molecular data of A. scabripinnis from this area.

Swim training and the genetic expression of adipokines in monosodium glutamate-treated obese rats.

OBJECTIVE: The aim of this study was to evaluate the genetic expression of adipokines in the adipocytes of monosodium glutamate (MSG)-treated obese rats submitted to physical activity. MATERIALS AND METHODS: Obesity was induced by neonatal MSG administration. Exercised rats (MSG and control) were subjected to swim training for 30 min for 10 weeks, whereas their respective controls remained sedentary. Total RNA was obtained from sections of the mesenteric adipose tissue of the rats. mRNA levels of adiponectin (Adipoq), tumor necrosis factor alpha (Tnf), peroxisome proliferator-activated receptor alpha (Ppara), and peroxisome proliferator-activated receptor gamma (Pparg) adipokines were quantified by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). RESULTS: In the exercise-trained control group, the expression of Adipoq increased compared to the sedentary control, which was not observed in the MSG-obese rats. Increased levels of Tnf in MSG-obese rats were not reversed by the swim training. The expression of Ppara was higher in sedentary MSG-obese rats compared to the sedentary control. Swimming increased this adipokine expression in the exercise-trained control rats compared to the sedentary ones. mRNA levels of Pparg were higher in the sedentary MSG-rats compared to the sedentary control; however, the exercise did not influenced its expression in the groups analyzed. CONCLUSIONS: In conclusion, regular physical activity was not capable to correct the expression of proinflammatory adipokines in MSG-obese rat adipocytes.

Swim training and the genetic expression of adipokines in monosodium glutamate-treated obese rats

Objective The aim of this study was to evaluate the genetic expression of adipokines in the adipocytes of monosodium glutamate (MSG)-treated obese rats submitted to physical activity.Materials and methods Obesity was induced by neonatal MSG administration. Exercised rats (MSG and control) were subjected to swim training for 30 min for 10 weeks, whereas their respective controls remained sedentary. Total RNA was obtained from sections of the mesenteric adipose tissue of the rats. mRNA levels of adiponectin (Adipoq), tumor necrosis factor alpha (Tnf), peroxisome proliferator-activated receptor alpha (Ppara), and peroxisome proliferator-activated receptor gamma (Pparg) adipokines were quantified by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).Results In the exercise-trained control group, the expression of Adipoq increased compared to the sedentary control, which was not observed in the MSG-obese rats. Increased levels of Tnf in MSG-obese rats were not reversed by the swim training. The expression of Ppara was higher in sedentary MSG-obese rats compared to the sedentary control. Swimming increased this adipokine expression in the exercise-trained control rats compared to the sedentary ones. mRNA levels of Pparg were higher in the sedentary MSG-rats compared to the sedentary control; however, the exercise did not influenced its expression in the groups analyzed.Conclusions In conclusion, regular physical activity was not capable to correct the expression of proinflammatory adipokines in MSG-obese rat adipocytes.

Relação do polimorfismo C825T do gene GNB3 e hipertensão arterial sistêmica de difícil controle / Relationship between C825T polymorphism of the GNB3 gene and dificult-to-treat hypertension

Fundamentos: O polimorfismo C825T do gene GNB3 está associado à hipertensão arterial sistêmica (HAS) em algumas populações já analisadas, porém alguns estudos se mostram controversos no que se refere a esta relação. Objetivo: Avaliar a relação do polimorfismo C825T do gene GNB3 com a HAS de difícil controle medicamentoso em hipertensos de Campos Gerais, PR - Brasil. Métodos: Em relação ao polimorfismo C825T de GNB3, foram determinados os genótipos de 60 hipertensos, os quais foram estratificados em dois grupos (fácil e difícil controle medicamentoso), por meio da técnica de PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Lenght Polymorphism). Foram avaliadas as frequências alélicas e genotípicas, utilizando-se o teste do qui-quadrado de Pearson, com correção de Yates e odds ratio (OR). Resultados: Não houve diferenças entre os grupos, quando comparadas as frequências alélicas e genotípicas, indicando que a população está em equilíbrio. A probabilidade de o paciente possuir o polimorfismo e a HAS de difícil controle foi 53,5 % (OR=1,15; IC95 % = 0,41-3,26), analisando-se os genótipos. Já a análise dos alelos, separadamente, mostrou uma associação de 55,4 % (OR=1,24; IC95 % = 0,59-2,57). Conclusão: Nesta população não foi encontrada relação entre o polimorfismo C825T do gene GNB3 e a HAS de difícil controle, indicando que outros fatores estão influenciando a manifestação dessa doença nestes pacientes.

Background: C825T polymorphism of the GNB3 gene is associated with systemic arterial hypertension (SAH) in some studied populations, although certain studies are controversial in terms of this relationship. Objective: To evaluate the relationship between C825T polymorphism of the GNB3 gene and difficult-to-treat SAH among hypertensive patients in Campos Gerais, Paraná State, Brazil. Methods: With regard to C825T polymorphism of the GNB3 gene, the genotypes were defined for sixty hypertensive patients divided in 2 groups (easy and difficult-to-treat with drugs), using the Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) technique. The allele and genotype frequencies were assessed through the Pearson chi-square test, with Yates correction and odds ratio (OR). Results: There were no differences between the groups when comparing the allele and genotype frequencies, indicating that the population is in equilibrium. The probability that a patient has polymorphism with difficult-to-treat SAH reached 53.5% (OR=1.15, 95%CI = 0.41-3.26), analyzing the genotypes. A separate allele analysis showed an association of 55.4% (OR=1.24, 95%CI = 0.59-2.57). Conclusion: No relationship was found in this population between C825T polymorphism of the GNB3 gene and difficult-to-treat SAH, indicating that other factors are influencing the appearance of this disease among these patients.

Karyotype variability in neotropical catfishes of the family pimelodidae (Teleostei: Siluriformes)

Dados cariotípicos são apresentados para quatro espécies da família Pimelodidae. Todas apresentaram o mesmo número diploide, 2n = 56 cromossomos, com diferenças nas fórmulas cariotípicas. As espécies aqui analisadas mostraram pouca quantidade de heterocromatina localizada preferencialmente na região centromérica e telomérica de alguns cromossosmos do complemento cariotípico. As regiões organizadoras de nucléolo (Ag-RONs) e a localização dos genes ribossomais pela hibridização in situ fluorescente (FISH), com sondas 18S e 5S, evidenciaram somente um par cromossômico marcado portador de genes ribossomais, à exceção de Pimelodus britskii que apresentou NORs múltiplas e localização sintênica das sondas 18S e 5S. Eventos não-Robertsonianos, como inversão pericêntrica e duplicação das NORs são requeridos para explicar a diversificação cariotípica em Pseudoplatystoma do rio Paraguai (MS), Pimelodus do rio Iguaçu (PR), Sorubim do rio Paraguai (MS) e Steindachneridion do rio Paraíba do Sul (SP). Os dados obtidos para a macroestrutura cariotípica destas espécies corrobora um padrão conservado observado na família Pimelodidae. Por outro lado, evidências de variações interespecíficas pelos marcadores de citogenética molecular empregados possibilitam inferências citotaxonômicas e diferenciação das espécies aqui analisadas.

Karyotypic data are presented for four species of fish belonging to the Pimelodidae family. These species show a conserved diploid number, 2n = 56 chromosomes, with different karyotypic formulae. The analyzed species showed little amount of heterochromatin located preferentially in the centromeric and telomeric regions of some chromosomes. The nucleolus organizer regions activity (Ag-NORs) and the chromosomal location of ribosomal genes by fluorescent in situ hybridization (FISH), with 18S and 5S probes, showing only one chromosome pair marked bearer of ribosomal genes, the only exception was Pimelodus britskii that presented multiple NORs and syntenic location of the 18S and 5S probes. Non-Robertsonian events, as pericentric inversion and NORs duplication are requested to explain the karyotype diversification in Pseudoplatystoma from the rio Paraguay (MS), Pimelodus from the rio Iguaçu (PR), Sorubim from the rio Paraguay (MS) and Steindachneridion from the rio Paraíba do Sul (SP). The obtained data for the karyotype macrostructure of these species corroborates a conserved pattern observed in Pimelodidae. On the other hand, interspecific variations detected by molecular cytogenetics markers made possible cytotaxonomic inferences and differentiation of the species here analyzed.

Karyotype characterization, constitutive heterochromatin and nucleolus organizer regions of Paranaita opima (Coleoptera, Chrysomelidae, Alticinae)

Species of the subtribe Oedionychina not only have a highly uniform diploid number of 2n = 22 (20+X+y) but have the karyotypic peculiarity of possessing extremely large sex chromosomes. We analyzed Paranaita opima embryos and gonadal cells to determine their diploid number, chromosomal morphology, type of sex determination system, constitutive heterochromatin pattern and which chromosomes bear nucleolus organizer regions (NORs). The diploid number of P. opima was 2n = 22 (20+XY/XX) with all chromosomes being metacentric. Chromosome pair 6 showed an interstitial secondary constriction on the short arm. The C-banding technique revealed centromeric constitutive heterochromatin in all chromosomes, which, in pair 6, extended up to the secondary constriction of the short arm, additional C-bands also being present on the Y chromosome. Silver nitrate nucleolar organizer region (Ag-NOR) staining showed NORs on the secondary constriction of pair 6. Fluorochrome analysis with chromomycin A3 (CMA3), 4'-6-diamidino-2-phenylindole (DAPI) and the distamycin A (DA) counterstain showed that the short arm of chromosome pair 6 exhibited a GC-rich block extending from the proximal to the median region, including part of the secondary constriction. The same techniques also showed AT-rich blocks at the centromeric region of all chromosomes and at the terminal region of the short arm of pair 6. The basic karyotype characteristics and C band pattern of P. opima are similar to those described for other species in the subtribe Oedionychina. The pattern of autosomal NORs observed in P. opima corresponds to that registered in the majority of the Chrysomelidae species.

Cytogenetic analysis and chromosomal characteristics of the polymorphic 18S rDNA in the fish Prochilodus lineatus (Characiformes, Prochilodontidae)

We used differential staining techniques (BSG, GTG, AgNO3, DAPI and CMA3 banding) and fluorescent in situ hybridization (FISH) with 5S and 18S probes to investigated the karyotypic and cytogenetic chracteristics of Prochilodus lineatus specimens from a population in Vila Velha state park (Parque Estadual de Vila Velha, Ponta Grossa, Paraná state, southern Brazil). The specimens studied showed the same karyotype as that found in other P. lineatus populations, i.e. 2n = 54 biarmed chromosomes (40m + 14 sm) and c-positive heterochromatin preferentially located pericentromerically in all chromosomes. The presence of partial or totally heterochromatic supernumerary chromosomes with numeric intra-individual variation was confirmed in the analyzed population. The nucleolar organizing regions (NORs) were interstitially situated on the long arm of chromosome pair 4 directly beneath the centromere. The differential banding techniques and FISH revealed NOR size polymorphism due to structural events such as breaks and duplication of the larger rDNA site cluster. We also observed syntenic localization of the 5S ribosomal genes in the distal segment of the 45S cluster.