Study of Melipona quadrifasciata brain under operant learning using proteomic and phosphoproteomic analysis.

Learning to anticipate events based on the predictive relationship between an action and an outcome (operant conditioning) is a form of associative learning shared by humans and most of other living beings, including invertebrates. Several behavioral studies on the mechanisms of operant conditioning have included Melipona quadrifasciata, a honey bee that is easily manipulated due to lack of sting. In this work, brain proteomes of Melipona bees trained using operant conditioning and untrained (control) bees were compared by two-dimensional gel electrophoresis analysis within pI range of 3-10 and 4-7; in order to find proteins specifically related to this type of associative learning.One protein was detected with differential protein abundance in the brains of trained bees, when compared to not trained ones, through computational gel imaging and statistical analysis. This protein was identified by peptide mass fingerprinting and MS/MS peptide fragmentation using a MALDI-TOF/TOF mass spectrometer as one isoform of arginine kinase monomer, apparently dephosphorylated. Brain protein maps were obtained by 2-DE (Two-dimensional gel electrophoresis) from a total proteins and phosphoproteins extract of the bee Melipona quadrifasciata. One isoform of arginine kinase, probably a dephosphorylated isoform, was significantly more abundant in the brain of trained bees using operant conditioning. Arginine kinase has been reported as an important enzyme of the energy releasing process in the visual system of the bee, but it may carry out additional and unexpected functions in the bee brain for learning process.

Neuroprotective effects of Pouteria ramiflora (Mart.) Radlk (Sapotaceae) extract on the brains of rats with streptozotocin-induced diabetes.

Diabetes mellitus is a chronic disease involving persistent hyperglycemia, which causes an imbalance between reactive oxygen species and antioxidant enzymes and results in damage to various tissues, including the brain. Many societies have traditionally employed medicinal plants to control the hyperglycemia. Pouteria ramiflora, a species occurring in the savanna biome of the Cerrado (Brazil) has been studied because of its possible ability to inhibit carbohydrate digestion. Rats with streptozotocin-induced diabetes treated with an alcoholic extract of Pouteria ramiflora show an improved glycemic level, increased glutathione peroxidase activity, decreased superoxide dismutase activity, and reduced lipid peroxidation and antioxidant status. The extract also restored myosin-Va expression and the nuclear diameters of pyramidal neurons of the CA3 subregion and that of the polymorphic cells of the hilus. We conclude that Pouteria ramiflora extract exerts a neuroprotective effect against oxidative damage and myosin-Va expression and is able to prevent hippocampal neuronal loss in the CA3 and hilus subfields of diabetic rats. However, future studies are needed to understand the mechanism of action of Pouteria ramiflora extract in acute and chronic diabetes.

The streptozotocin-induced rat model of diabetes mellitus evidences significant reduction of myosin-Va expression in the brain.

Diabetes mellitus is a disease characterized by increased glucose levels in the blood. Hyperglycemia causes damage to the brain tissue, and induces significant changes in synaptic transmission. In this investigation, we have found a significant alteration in the expression of the molecular motor involved in the synaptic vesicles transport, myosin-Va, and its distribution in rat brains of streptozotocin-induced diabetes model. Brains were removed after 20 days, homogenized and analysed by Western blotting, qRT-PCR and immunohistochemistry. Myosin-Va presented significantly lower levels of both mRNA and protein in diabetic than those observed in non-diabetic animals. Moreover, neuronal and glial cells of the occipital and frontal cortex exhibited decreased myosin-Va immunostaining in diabetic rat brains. In conclusion, diabetic rat brains displayed altered expression and distribution of myosin-Va, and these finding may contribute to the basic understanding about this myosin role in brain function related to diabetes.

Characterization and localization of dynein and myosins V and VI in the ovaries of queen bees.

The presence of myosin and dynein in the ovaries of both Apis mellifera and Scaptotrigona postica was investigated in extracts and in histological sections. In the ovary extracts, motor proteins, myosins V, VI and dynein were detected by Western blot. In histological sections, they were detected by immunocytochemistry, using a mouse monoclonal antibody against the intermediary chain of dynein and a rabbit polyclonal antibody against the myosin V head domain. The myosin VI tail domain was recognized by a pig polyclonal antibody. The results show that these molecular motors are expressed in the ovaries of both bee species with few differences in location and intensity, in regions where movement of substances is expected during oogenesis. The fact that antibodies against vertebrate proteins recognize proteins of bee species indicates that the specific epitopes are evolutionarily well preserved.

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: myosin-V assay.

The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 ºC, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.

Identification of calmodulin-binding proteins in brain of worker honeybees.

Calmodulin is a Ca(+2)-binding protein important in a variety of cell functions. The Ca(+2)/calmodulin complex interacts with and regulates various enzymes and target proteins, known as calmodulin-binding proteins (CaMBPs). In this study, we revealed a comparative identification of the CaMBPs composition in the worker honeybee (Apis mellifera) brain, considering two different honeybee behaviors in the colony. To this end, the CaMBPs of forager and nurse workers were purified by affinity chromatography, separated in 1D gel, digested and submitted to peptide mass fingerprinting (PMF). In the PMF analysis, 15 different proteins, considered behavior-specific proteins, were identified, one of them exclusively in forager workers and 10 in nurses. All the proteins were classified in terms of their function and cell localization, revealing a greater expression of metabolism-related CaMBPs in both worker subcastes. Protein sequences were then analyzed for the presence of the calmodulin-binding sites. Therefore, the honeybee brain CaMBPs profiles presented differences between worker subcastes. This is the first identification of calmodulin-binding proteins in the brain of A. mellifera upon nursing and foraging behaviors in the colony and this diversity of target proteins for Ca(+2)/CaM may be involved in terms of the function of these proteins in the nervous system.

Saúde Mental, Uso de Álcool e Qualidade do Sono em Estudantes de uma Universidade Pública / Mental Health, Alcohol Use and Sleep Quality by Students in a Public University / Salud Mental, Consumo de Alcohol y Calidad del Sueño en Estudiantes de una Universidad Pública

Este estudo analisou os aspectos cognitivos e comportamentais dos estudantes de uma universidade pública durante a graduação, considerando autopercepção de depressão, ansiedade e estresse, bem como uso de substâncias psicoativas e qualidade do sono. Foi realizado um delineamento longitudinal com 34 estudantes ingressantes em 2015/1 e formandos em 2018/2, aplicando-se questionário online. Do total de universitários, 22 eram mulheres e 12 eram homens, sendo que o consumo de drogas aumentou para ambos os sexos. A frequência da percepção de processos depressivos aumentou no fim do curso, enquanto a de ansiedade diminuiu, para ambos os sexos. Os dados de percepção do estresse mostraram aumento na frequência total. Houve correlação positiva fraca entre depressão e ansiedade ou estresse, mas correlação positiva moderada entre estresse e ansiedade no final do curso. Ainda, obteve-se um aumento expressivo de distúrbio do sono em homens e mulheres, porém de forma significativa apenas para os primeiros. Foi notada correlação positiva fraca entre a percepção de depressão, ansiedade ou estresse no final do curso. Faz-se necessário o acompanhamento multiprofissional do universitário, auxiliando na maneira de lidar com as dificuldades, bem como medidas de melhoria da qualidade de vida e sono.

This study analyzed cognitive and behavioral aspects of students at a public university during undergraduate study, considering self-perception of anxiety, depression and stress, as well as psychoactive substance use and sleep quality. A longitudinal project was conducted with 34 students entering in 2015/1 and graduating in 2018/2, applying an online questionnaire. Of the total of students, 22 were women and 12 were men, and drug use increased for both sexes. The frequency of perception of depressive processes increased at the end of the course, while anxiety decreased, for both sexes. Stress perception data showed an increase in total frequency. There was a weak positive correlation between depression and anxiety or stress, but a moderate positive correlation between stress and anxiety at the end of the course. It is still possible to notice that there was a significant increase in sleep disorders in men and women, but significantly only for the first. A weak positive correlation was noticed between the perception of depression, anxiety or stress at the end of the course. The multidisciplinary follow-up of the university student is necessary in order to assist in dealing with difficulties, as well as measures to improve quality of life and sleep.

Este estudio analizó los aspectos cognitivos y conductuales de los estudiantes en una universidad pública durante la graduación, considerando la autopercepción de depresión, ansiedad y estrés, así como el uso de sustancias psicoactivas y la calidad del sueño. Se realizó un diseño longitudinal con 34 estudiantes que ingresaron en 2015/1 y graduaron en 2018/2 aplicando un cuestionario en línea. El total de estudiantes universitarios, 22 eran mujeres y 12 hombres, con un mayor consumo de drogas para ambos los sexos. La frecuencia de percepción de los procesos depresivos aumentó al final del curso, mientras que la ansiedad disminuyó para ambos os sexos. Los datos para el estrés aumentaron en la frecuencia total. Hubo una correlación positiva débil entre la depresión y la ansiedad o el estrés, pero una correlación positiva moderada entre el estrés y la ansiedad al final del curso. Aun así, el aumento significativo en los trastornos del sueño en hombres y mujeres, sin embargo significado solamente para él primer. Una correlación positiva débil con depresión, ansiedad o estrés fue percibida al final del curso. Se hace necesario el acompañamiento multiprofesional de estudiantes universitarios, a fin de ayudar en la forma de tratar las dificultades, así como medidas para mejorar la calidad de vida y el sueño.

Tracking of risk factors for non-communicable diseases in young shooters, Ituiutaba, MG

The aim of this study was to detect early hypertension, diabetes and obesity, as well as their risk factors, in 105 young shooters from Tiro de Guerra 11-002 in the city of Ituiutaba-MG. This is a cross-sectional mixed study. A semi-structured questionnaire that contained questions related to the sociodemographic context, health status, lifestyle, eating habits, and psychosocial variables was applied. Blood pressure, anthropometric indexes, and lipid and glycemic profiles were measured. Descriptive and inferential statistics, including Pearson's correlation test and Spearman's coefficient, considering significative value (p < 0.05). The shooters had an average of 18.9 years old, most educated and brown. Anthropometric indices indicated a mainly eutrophic population, and normotensive and desirable lipid and glycemic profiles. However, overweight, high blood pressure, and self-declaration of family history for chronic diseases were identified. In addition, most shooters affirmed to ingest alcoholics beverages, to have insufficient time of physical activity, and to be aware of their levels of stress, pressure and anxiety and a few hours of sleep. Overall, the shooters showed good anthropometric and biochemical indicatorsof blood. Risk behavior, such as drinking alcoholic beverages and smoking, low level of physical activity and self-perception of stress, anxiety and pressure were also noted.

Polyploidy Analysis and Attenuation of Oxidative Stress in Hepatic Tissue of STZ-Induced Diabetic Rats Treated with an Aqueous Extract of Vochysia rufa.

Diabetes mellitus (DM) is characterized by hyperglycemia and alterations in the metabolism of lipids, carbohydrates, and proteins. Due to its hypoglycemic effect Vochysia rufa is frequently used in Uberlandia, Brazil, to treat DM. Despite its popularity, there is little information about its effect on hepatic tissue. Therefore, we evaluated the histoarchitecture, oxidative stress parameters, and polyploidy of liver tissue from streptozotocin- (STZ-) induced diabetic rats treated with aqueous extract of Vochysia rufa (AEV). Histology was determined by fixing the livers, processing, and staining with HE. Oxidative stress was determined by evaluating CAT, GPx, and SOD activity in liver homogenates and hepatic mitochondria fraction and by measuring GST, GSH levels and lipid peroxidation (MDA). Polyploidy was determined by subjecting isolated hepatocyte nuclei to flow cytometry. In the diabetic group, GST activity and GSH rates decreased whereas liver homogenate analysis showed that GPx, SOD activity and MDA increased. AEV treatment restored all parameters to normal levels. The oxidative stress analysis of hepatic mitochondria fraction showed similar results. Lower polyploid cell populations were found in the diabetic rat livers, even after glibenclamide treatment. Thus, AEV treatment efficiently reduced hepatic oxidative stress caused by STZ-induced diabetes and produced no morphological changes in the histological analysis.

Aspectos socioeconômicos e eventos de queda entre idosos atendidos no sistema público de saúde / Socioeconomic aspects and falls in older adults served by the public health system / Aspectos socioeconómicos y eventos de caídas en mayores asistidos por el sistema público de salud

Objetivo: Apresentar os aspectos socioeconômicos e a ocorrência de eventos de queda entre idosos atendidos no sistema municipal de saúde. Métodos: Estudo descritivo e transversal realizado com 154 idosos, de ambos os sexos (feminino, n=88; masculino, n=66), atendidos nas unidades de saúde do município de Ituiutaba, Minas Gerais, em 2018. Realizou-se a coleta dos dados a partir da aplicação de questionário semiestruturado contendo questões socioeconômicas (escolaridade, ocupação, origem, renda e situação conjugal) e de condição clínica, incluindo eventos de queda. Analisaram-se as variáveis por estatística descritiva. Resultados: Dos idosos, 80,5% (n=124) era de origem rural, com maior prevalência de mulheres (57,1%; n=68). Sobre as quedas e seus aspectos relacionados, 37,7% (n=58) dos idosos afirmaram ter sofrido algum evento nos últimos 12 meses, sendo mais prevalente entre aqueles com idade entre 70 e 79 anos (55,2%; n=32), do sexo feminino (67,2%; n=39). Em decorrência da queda, 32,7% (n=19) apresentaram fratura e, entre aqueles que caíram, 41,4% (n=24) relataram que a residência apresentava riscos para novos eventos de queda. Conclusão: Os eventos de queda autorreferidos pelos idosos investigados associam-se aos fatores inerentes do envelhecimento biológico, com acentuada feminização do envelhecimento, mas também aos fatores extrínsecos associados ao ambiente onde vivem.

Objective: To present the socioeconomic aspects and the occurrence of falls in older patients served by the municipal health system. Methods: This descriptive cross-sectional study was conducted with 154 older adults of both sexes (women, n=88; men, n=66) served at health care centers in the municipality of Ituiutaba, Minas Gerais, in 2018. Data were collected using a semi-structured questionnaire that addressed socioeconomic issues (education, occupation, origin, income and marital status) and clinical conditions, including occurrence of falls. The variables were analyzed using descriptive statistics. Results: In all, 80.5% (n=124) of the older adults were from rural areas and there was a higher prevalence of women (57.1%; n=68). Regarding falls and related aspects, 37.7% (n=58) of the older adults reported at least one fall in the past 12 months, with a higher prevalence among those aged 70-79 years (55.2%; n=32) and women (67.2%; n=39). Fracture resulting from fall occurred in 32.7% (n=19) of the older adults and 41.4% (n=24) of those who fell said the house presented risks for new falls. Conclusion: Falls self-reported by the older adults analyzed were associated with factors inherent to biological aging and there was a marked feminization of aging. However, they were also associated with extrinsic factors related to the environment where they live.

Objetivo: Apresentar os aspectos socioeconômicos e a ocorrência de eventos de queda entre idosos atendidos no sistema municipal de saúde. Métodos: Estudo descritivo e transversal realizado com 154 idosos, de ambos os sexos (feminino, n=88; masculino, n=66), atendidos nas unidades de saúde do município de Ituiutaba, Minas Gerais, em 2018. Realizou-se a coleta dos dados a partir da aplicação de questionário semiestruturado contendo questões socioeconômicas (escolaridade, ocupação, origem, renda e situação conjugal) e de condição clínica, incluindo eventos de queda. Analisaram-se as variáveis por estatística descritiva. Resultados: Dos idosos, 80,5% (n=124) era de origem rural, com maior prevalência de mulheres (57,1%; n=68). Sobre as quedas e seus aspectos relacionados, 37,7% (n=58) dos idosos afirmaram ter sofrido algum evento nos últimos 12 meses, sendo mais prevalente entre aqueles com idade entre 70 e 79 anos (55,2%; n=32), do sexo feminino (67,2%; n=39). Em decorrência da queda, 32,7% (n=19) apresentaram fratura e, entre aqueles que caíram, 41,4% (n=24) relataram que a residência apresentava riscos para novos eventos de queda. Conclusión: Os eventos de queda autorreferidos pelos idosos investigados associam-se aos fatores inerentes do envelhecimento biológico, com acentuada feminização do envelhecimento, mas também aos fatores extrínsecos associados ao ambiente onde vivem.

Glibenclamide treatment modulates the expression and localization of myosin-IIB in diabetic rat brain.

BACKGROUND: Myosin-IIB is a non-muscle isoform in the brain with increased expression in the brains of diabetic rats. Chronic hyperglycemia caused by diabetes can impair learning and memory. Oral hypoglycemic agents such as glibenclamide have been used to control hyperglycemia. We report changes in the expression and distribution of myosin-IIB in the frontal cortex and hippocampus of diabetic rats treated with glibenclamide. METHODS: The brains were removed after 43 days of treatment with glibenclamide (6 mg/kg bw orally), homogenized and analyzed by Western blotting, qRT-PCR and immunohistochemistry. RESULTS: Myosin-IIB expression increased in the brains of diabetic rats. However, protein expression returned to control levels when treated with glibenclamide. In addition, the expression of MYH10 gene encoding non-muscle myosin heavy chain-B decreased in diabetic rats treated with glibenclamide. Moreover, we found weak myosin-IIB labeling in the hippocampus and frontal cortex of rats treated with glibenclamide. Therefore, the expression of myosin-IIB is affected by diabetes mellitus and may be modulated by glibenclamide treatment in rats. Structural changes in the hippocampus and prefrontal cortex are reversible, and glibenclamide treatment may reduce the patho-physiological changes in the brain. CONCLUSIONS: Our findings can contribute to the understanding of the regulation of myosins in the brains of diabetic rats.

Myosins Are Differentially Expressed under Oxidative Stress in Chronic Streptozotocin-Induced Diabetic Rat Brains.

Diabetes mellitus is a disease characterized by persistent hyperglycemia, which may lead to brain tissue damage due to oxidative stress and also contributes to neuronal death and changes in synaptic transmission. This study evaluated the effect of oxidative stress and the use of antioxidants supplementation on myosins expression levels in the brains of chronic diabetic rats induced by streptozotocin. Lipid peroxidation, antioxidant enzymes activities, and myosins-IIB and -Va expressions at transcriptional and translational levels were examined after 90 days induction. The chronic effect of the diabetes led to the upregulation of superoxide dismutase (SOD) and catalase (CAT) activities, and the downregulation of glutathione peroxidase (GPx), but there was no statistically significant increase in the malondialdehyde (MDA) levels. These alterations were accompanied by high myosin-IIB and low myosin-Va expressions. Although the antioxidant supplementation did not interfere on MDA levels, the oxidative stress caused by chronic hyperglycemia was reduced by increasing SOD and restoring CAT and GPx activities. Interestingly, after supplementation, diabetic rats recovered only myosin-Va protein levels, without interfering on myosins mRNA levels expressed in diabetic rat brains. Our results suggest that antioxidant supplementation reduces oxidative stress and also regulates the myosins protein expression, which should be beneficial to individuals with diabetes/chronic hyperglycemia.

Alterations of antioxidant biomarkers and type I collagen deposition in the parotid gland of streptozotocin-induced diabetic rats.

BACKGROUND AND OBJECTIVE: Acarbose is a competitive inhibitor of intestinal alpha-glycosidases that slows the breakdown of sucrose and starch, thereby reducing glucose and fructose absorption. The aim of this study was to evaluate the effect of acarbose treatment on antioxidant parameters and deposition of type I collagen in the parotid glands of diabetic rats. METHODS: Diabetes mellitus was induced by intravenous injection of streptozotocin, and rats were divided into four groups: non-diabetic (NDM), diabetic (DM), diabetic treated with 25mg/kg acarbose (DMA) and non-diabetic treated with acarbose (NDMA). Changes in enzymatic antioxidant systems, such as the activity of SOD and GPx enzymes, were evaluated, and the specific staining pattern of the type I collagen fibres was investigated in the rat parotid glands. RESULTS: The DM group presented high levels of SOD and GPx enzymes, which were reduced by acarbose treatment. Tissue damage, which was indicated by an increased MDA concentration in the parotid glands of rats in the DM group, was also reversed in the DMA group. Moreover, type I collagen fibres from DM rats were more intensely stained than those of NDM rats. Acarbose treatment was effective in decreasing collagen deposition, which was shown by a decrease in staining intensity of approximately 25%. CONCLUSIONS: These results suggest that the diabetic state influences the type I collagen concentration in the parotid glands of rats. In addition, acarbose treatment was helpful in preventing the deposition of such fibres, as well the increase in oxidative stress induced by hyperglycemia.

Atenção preventiva e educativa em saúde do idoso: uma proposta de integração de saberes e práticas / Preventive care and education in elderly health: a proposal for the integration of knowledges and practices

O envelhecimento da população brasileira trouxe mudanças em seu perfil epidemiológico, como o aumento da ocorrência de doenças crônicas e incapacidades funcionais, além de maior demanda por serviços de saúde, representando um desafio para a Saúde Coletiva no conhecimento das suas especificidades. Nesse sentido, idosos do Programa de Assistência ao Idoso do SESC Minas, na cidade de Governador Valadares (MG), responderam a um questionário semi estruturado para variáveis socioeconômicas, condições de saúde e hábitos de vida a fim de se traçar um diagnóstico desta população e direcionar ações interdisciplinares educativas e preventivas como incentivo ao envelhecimento ativo. Os dados revelaram as principais doenças: hipertensão arterial (63,8%), osteoartrite (25,5%), cardiopatias (17%) e diabetes (10,6%). A poli farmácia foi identificada em 29,8% dos idosos, incluindo o uso de medicamentos alopáticos, fitoterápicos e plantas medicinais. Doze idosos (25,5%) relataram possuir lesões de pele, 72,3% informaram uso de próteses dentárias e 2,1% próteses ortopédicas. A maioria dos idosos classificou sua alimentação como saudável (85,1%), mas todos relataram algum tipo de perda funcional. Os resultados demonstram que o Programa Assistencial conta com idosos com o perfil de saúde semelhante ao da população idosa brasileira, com ampla ocorrência de doenças crônicas, edentulismo, incapacidades funcionais e elevado consumo de medicamentos. O presente estudo reforça a importância do trabalho interdisciplinar como subsídio para o desenvolvimento de intervenções mais sensíveis e eficazes, considerando a perspectiva do próprio indivíduo e suas necessidades.

The aging of the Brazilian population brought changes in their epidemiological profile: increased occurrence of chronic illnesses and functional disabilities; increased demand for health services. The knowledge of the characteristics and elements of such changes have represented a challenge to public health researchers. A preliminary attempt to meet such challenge comes from a program of assistance to the elderly in the city of Governador Valadares, Minas Gerais, Brazil, with the support of an independent agency backed by the commercial sector (SESC-Minas). A sample of individuals aged 60 and above responded to a semi-structured questionnaire to socioeconomic variables, health conditions and life styles. The survey was a research instrument for the proposal of preventive action and active ageing. The data revealed the main diseases: arterial hypertension (63.8 percent); osteoarthritis (25.5 percent); heart diseases (17%) and diabetes (10.6 percent). “`Polipharmacy” was identified in 29.8% of seniors, including the use of allopathic drugs, phytotherapy and medicinal plants. 25.5 percent reported skin lesions, 72.3% wore dentures and 2.1% orthopedic prostheses. Most elderly people claimed to follow a healthy diet (85.1%), but all reported some form of functional loss. The results suggest that the Program relies on the participation of seniors with health profile similar to the general population of Brazilian elderly, with wide occurrence of chronic diseases, edentulism and high consumption of medicines. This study reinforces the importance of interdisciplinary work as subsidy for the development of more effective interventions, taking into account the perspectives of the elderly and their needs.

Overexpression of myosin-IIB in the brain of a rat model of streptozotocin-induced diabetes.

The Ca(2+)/calmodulin complex interacts with and regulates various enzymes and target proteins known as calmodulin-binding proteins (CaMBPs). This group of proteins includes molecular motors such as myosins. In this study, we show that non-muscle myosin-IIB is overexpressed in the brains of diabetic rats. We isolated CaMBPs from the brains of non-diabetic rats and rats with streptozotocin-induced diabetes and purified them by immobilized-calmodulin affinity chromatography. The proteins were eluted with EGTA and urea, separated by SDS-PAGE, digested and submitted to peptide mass fingerprinting analysis. Thirteen intense bands were found in both types of brains, two were found exclusively in non-diabetic brains and four were found exclusively in diabetic brains. A large fraction of the eluted proteins contained putative IQ motifs or calmodulin-binding sites. The results of the myosin-IIB affinity chromatography elution, western blot and RT-PCR analyses suggest that myosin-IIB protein and mRNA are expressed at high levels in diabetic brains. This is the first study that has demonstrated differential expression of CaMBPs in diabetic and non-diabetic brain tissue through a comparative proteomic analysis, and it opens up a new approach to studying the relationship between the expression of myosins in the brain, hyperglycemia and intracellular calcium regulation.

Myosins and DYNLL1/LC8 in the honey bee (Apis mellifera L.) brain.

Honey bees have brain structures with specialized and developed systems of communication that account for memory, learning capacity and behavioral organization with a set of genes homologous to vertebrate genes. Many microtubule- and actin-based molecular motors are involved in axonal/dendritic transport. Myosin-Va is present in the honey bee Apis mellifera nervous system of the larvae and adult castes and subcastes. DYNLL1/LC8 and myosin-IIb, -VI and -IXb have also been detected in the adult brain. SNARE proteins, such as CaMKII, clathrin, syntaxin, SNAP25, munc18, synaptophysin and synaptotagmin, are also expressed in the honey bee brain. Honey bee myosin-Va displayed ATP-dependent solubility and was associated with DYNLL1/LC8 and SNARE proteins in the membrane vesicle-enriched fraction. Myosin-Va expression was also decreased after the intracerebral injection of melittin and NMDA. The immunolocalization of myosin-Va and -IV, DYNLL1/LC8, and synaptophysin in mushroom bodies, and optical and antennal lobes was compared with the brain morphology based on Neo-Timm histochemistry and revealed a distinct and punctate distribution. This result suggested that the pattern of localization is associated with neuron function. Therefore, our data indicated that the roles of myosins, DYNLL1/LC8, and SNARE proteins in the nervous and visual systems of honey bees should be further studied under different developmental, caste and behavioral conditions.

Identification of major royal jelly proteins in the brain of the honeybee Apis mellifera.

The consumption of royal jelly (RJ) determines the differences between castes and behavioral development in the honeybee Apis mellifera. However, it is not known whether the proteins of RJ are related to these differences, or which proteins are responsible for the changes. To understand the functions of RJ proteins that are present in other tissues of the bee, in addition to hypopharyngeal gland, we used a polyclonal antibody anti-MRJP1 to investigate the presence of this protein in nervous system of honeybee. This study showed the presence of three polypeptides (p57, p70 and p128) in specific tissues of bee brain. Mushroom body, optic lobe and antennal lobe neuropils all contained proteins recognized by anti-MRJP1. Proteomic analysis showed that the three polypeptides are correlated with proteins of the MRJP family. p57 is correlated with MRJP1, p70 with MRJP3, while p128 may be an oligomeric form or a new polypeptide. Immunostaining of the brain and hypopharyngeal gland revealed differential expression of MRJPs in various brain regions and in different honeybee castes and subcastes. The identification and localization of these MRJPs contribute to the elucidation of the biological roles of this protein family.

Proteomic analysis of honey bee brain upon ontogenetic and behavioral development.

The honey bee (Apis mellifera) is a social insect that shows complex and integrated behaviors. Its ability to read and respond to several sets of extrinsic and intrinsic signals is fundamental for the modulation of individual activities and social systems. For instance, A. mellifera behavior changes upon the ontogenetic differentiation from nurse to forager worker subcastes. In this work, brain proteomes of nurses and foragers were compared by two-dimensional gel electrophoresis within pH range of 4-7 in order to find proteins related to such an ontogenetic and behavioral development. Twenty differentially expressed proteins were detected by gel image computational analysis, and identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Nurse brain showed increased expression of major royal jelly proteins (MRJP1, MRJP2 and MRJP7), which are related to determination of castes during the honey bee larvae differentiation. Immunocytochemistry and electron microscopy showed that MRJP1 was localized in the cytoplasm of brain cells, seemingly along filaments of the cytoskeleton, in the antennal lobe, optical lobe and mushroom body. Also, MRJP1 was deposited on the rhabdom, a structure of the retinular cells, composed of numerous tubules. Such evidence suggests that MRJP1 could be associated to proteins of filamentous structures. MRJP1 was also found in intercellular spaces between cells in mushrooms bodies, indicating that it is a secreted protein. Other proteins implicated in protein synthesis and putative functions in the olfactory system were also up-regulated in the nurse brain. Experienced foragers overexpressed proteins possibly involved in energy production, iron binding, metabolic signaling and neurotransmitter metabolism. Such differential expression of proteins may be related to ontogenetic and behavior changes in A. mellifera.

Glicogenoses: uma revisão geral / Glycogenoses: an overeview

Compreender o metabolismo dos diferentes tipos de glicogênio no organismo humano torna-se de suma importância, pois além da sua relevância no fornecimento energético e no controle da glicemia, o glicogênio pode estar relacionado com diversos tipos de doenças que comprometem a saúde do ser humano, especialmente pela deficiência em enzimas de vias anabólicas e catabólicas. Em um contexto de avanços no desenvolvimento de pesquisas relacionadas às áreas da saúde, uma série de estudos busca entender fisiologicamente os caminhos do glicogênio em situações de exercício, repouso, jejum, dentre outras, além de analisar os mais variados transtornos decorrentes da deficiência no metabolismo desse polissacarídeo. Um exemplo são as glicogenoses, doenças hereditárias, em sua maioria de caráter recessivo, relacionadas com o armazenamento de glicogênio. Dentre alguns dos treze tipos de glicogenoses podemos citar a glicogenose tipo 0, uma doença rara que se desenvolve na infância e implica na produção defeituosa da enzima glicogênio sintase; e a glicogenose tipo I, também conhecida como Doença de Von Gierke, que se caracteriza pela deficiência no complexo enzimático glicose-6-fosfatase, responsável pela catalisação da hidrólise de glicose-6-fosfato na metabolização do glicogênio. Apesar de todas essas doenças serem caracterizadas por glicogenoses, elas possuem diferenças quanto ao órgão afetado, à gravidade de suas manifestações, o perfil etário que cada uma atinge e no efeito enzimático. Por isso, a necessidade de estudos que correlacionam as principais causas e sintomas, e visam proporcionar uma visão global dessas desordens de hereditariedade.

The comprehension of the metabolism of different types of glycogen in the human organism becomes extremely important since, other than its relevance in providing energy and controlling glycemia, glycogen can be related to many types of diseases that compromise the human health, especially when it comes to the deficiency in enzyme anabolic and catabolic pathways. In the context of advances in the development of researches related of health area, many studies inquire a physiological understanding of the glycogen pathways exercising, resting, fasting and other conditions, as well as analyzing the most varied disorders arising from hereditary deficiencies in the carbohydrate metabolism, in polysaccharide specially. The glycogenoses are hereditary disorders, which present mainly recessive feature, related with the glycogen storage. Among the thirteen types of glycogenoses, type 0 is a rare disease that develops in early stages of life and implies in the production of defective glycogen synthase enzyme; and type I is characterized by the deficiency of the glucose-6-phosphatase enzyme complex, responsible for catalyzing the hydrolysis of glucose-6- phosphate in glycogen metabolism. Although all these diseases are characterized as being glycogenoses, they possess differences as to the organ affected, the gravity of their manifestations, the age it begins to manifest, and in which way it affects enzymatic properties. Therefore, there is a necessity of studies that correlates the main causes and symptoms, and aim to provide a global vision of these hereditary disorders.

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: Myosin-v assay

The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 ºC, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.