Expression and activities of cyclins and cyclin-dependent kinases in developing rat ventricular myocytes

The molecular mechanisms responsible for the alterations in proliferative capacity of cardiac myocytes during development remain unknown; however, cell cycle dependent molecules may be involved. We have determined the expression of cyclins A, D1–3and E, and cyclin-dependent kinases (CDKs) 2, 4, 5 and 6 and cdc2 in freshly isolated rat cardiac myocytes from fetal (18 days gestation), neonatal (2 days post-natal) and adult animals by immunoblotting. Our results show a dramatic decrease in expression of these proteins during normal cardiac development, such that levels are highest in fetal myocytes but are significantly down-regulated in adult cells (P<0.05, in each case). We also have determined thein vitrokinase activities of cdc2, CDK2, CDK4, CDK5 and CDK6 immunocomplexes in fetal, neonatal and adult myocytes. There was a consistent and significant loss of cdc2, CDK2, CDK4 and CDK6 kinase activities in adult cardiac cell lysates (5.3-, 10.6-, 1.5- and 1.9-fold decreases, respectively) when compared to neonatal samples (P<0.05); CDK5 activity showed a similar trend but failed to reach significance. In conclusion, our results show that the expression and activities of various positive regulators of the cell cycle are down-regulated significantly during development of the cardiac myocyte, concomitant with the loss of proliferative capacity in adult myocytes. Down-regulation of these proteins may be pivotal in the withdrawal of the cardiac myocyte from the cell cycle.

Discovery of a mammalian splice variant of myostatin that stimulates myogenesis

Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P,0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product.

Convergence and accommodation development is pre-programmed in premature infants

Purpose This study investigated whether vergence and accommodation development in pre-term infants is pre-programmed or is driven by experience. Methods 32 healthy infants, born at mean 34 weeks gestation (range 31.2-36 weeks) were compared with 45 healthy full-term infants (mean 40.0 weeks) over a 6 month period, starting at 4-6 weeks post-natally. Simultaneous accommodation and convergence to a detailed target were measured using a Plusoptix PowerRefII infra-red photorefractor as a target moved between 0.33m and 2m. Stimulus/response gains and responses at 0.33m and 2m were compared by both corrected (gestational) age and chronological (post-natal) age. Results When compared by their corrected age, pre-term and full-term infants showed few significant differences in vergence and accommodation responses after 6-7 weeks of age. However, when compared by chronological age, pre-term infants’ responses were more variable, with significantly reduced vergence gains, reduced vergence response at 0.33m, reduced accommodation gain, and increased accommodation at 2m, compared to full-term infants between 8-13 weeks after birth. Conclusions When matched by corrected age, vergence and accommodation in pre-term infants show few differences from full-term infants’ responses. Maturation appears pre-programmed and is not advanced by visual experience. Longer periods of immature visual responses might leave pre-term infants more at risk of development of oculomotor deficits such as strabismus.  

Frequency of infant stroking reported by mothers moderates the effect of prenatal depression on infant behavioural and physiological outcomes

Animal studies find that prenatal stress is associated with increased physiological and emotional reactivity later in life, mediated via fetal programming of the HPA axis through decreased glucocorticoid receptor (GR) gene expression. Post-natal behaviours, notably licking and grooming in rats, cause decreased behavioural indices of fear and reduced HPA axis reactivity mediated via increased GR gene expression. Post-natal maternal behaviours may therefore be expected to modify prenatal effects, but this has not previously been examined in humans. We examined whether, according to self-report, maternal stroking over the first weeks of life modified associations between prenatal depression and physiological and behavioral outcomes in infancy, hence mimicking effects of rodent licking and grooming. From a general population sample of 1233 first time mothers recruited at 20 weeks gestation we drew a stratified random sample of 316 for assessment at 32 weeks based on reported inter-partner psychological abuse, a risk to child development. Of these 271 provided data at 5, 9 and 29 weeks post delivery. Mothers reported how often they stroked their babies at 5 and 9 weeks. At 29 weeks vagal withdrawal to a stressor, a measure of physiological adaptability, and maternal reported negative emotionality were assessed. There was a significant interaction between prenatal depression and maternal stroking in the prediction of vagal reactivity to a stressor (p = .01), and maternal reports of infant anger proneness (p = .007) and fear (p = .043). Increasing maternal depression was associated with decreasing physiological adaptability, and with increasing negative emotionality, only in the presence of low maternal stroking. These initial findings in humans indicate that maternal stroking in infancy, as reported by mothers, has effects strongly resembling the effects of observed maternal behaviours in animals, pointing to future studies of the epigenetic, physiological and behavioral effects of maternal stroking.

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To Study this, pregnant once were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved Lip to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocyres, in addition to a slight delay in endochondral and intramembranous ossification. This Suggests that LIP to E 16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly (similar to 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast. D2 mRNA expression increased significantly by E18.5 and markedly (similar to 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time Suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development. (c) 2008 Elsevier Inc. All rights reserved.

Seguimento clinoceletrográfico de criançcas e adolescentes com crise epiléptica única não-provocada

Os fenômenos convulsivos despertaram o interesse de estudiosos e pensadores já na Antigüidade, quando aspectos mágicos e sobrenaturais eram a eles associados. No século XIX foram lançadas as bases dos conceitos atuais sobre a desestruturação funcional cerebral na epilepsia, e Berger, em 1929, marcou definitivamente a história com a descoberta dos ritmos cerebrais. Crise epiléptica e epilepsia não são sinônimos, já que o último termo refere-se a crises recorrentes espontâneas. Ela costuma iniciar na infância, daí a preocupação com o risco de repetição do primeiro episódio e com a decisão de instituir tratamento medicamentoso. Fatores prognósticos são apontados, mas não há consenso. No Brasil existem poucas pesquisas nesta linha, tanto de prevalência da epilepsia como de fatores envolvidos na recorrência de crises. Este estudo teve como objetivo geral avaliar aspectos clinicoeletrográficos capazes de auxiliar no prognóstico e no manejo da epilepsia da criança e do adolescente. Foram objetivos específicos determinar a incidência de crise epiléptica não provocada recorrente; identificar fatores remotos implicados na ocorrência de crise epiléptica; relacionar tipo de crise com achados eletrencefalográficos; relacionar tipo de crise, duração da crise, estado vigília/sono no momento da crise e achados eletrencefalográficos com possibilidade de recorrência; e identificar os fatores de risco para epilepsia. Foram acompanhados 109 pacientes com idades entre 1 mês e 16 anos, com primeira crise não-provocada, em média por 24 meses, a intervalos trimestrais, no Hospital de Clínicas de Porto Alegre (HCPA). Foram realizados eletrencefalogramas (EEG) após a primeira crise; depois, solicitados anualmente. Não foram incluídos casos com epilepsia ou síndrome epiléptica bem definida, ou que fizeram uso prévio de drogas antiepilépticas. A média de idade foi 6 anos, com predomínio da faixa etária de 6 a 12 anos. Setenta eram meninos e 39, meninas. Os indivíduos brancos eram 92, e os não-brancos, 17. O nível de escolaridade dos casos esteve de acordo com a distribuição da idade e, entre os responsáveis, predominaram 8 anos de escolaridade. Foi possível concluir que as crises únicas não-provocadas mais freqüentes foram generalizadas, e sem predomínio significativo do tipo de EEG. A incidência de crise não-provocada recorrente foi 51,4%. História de intercorrências pré-natais maternas aumentou em 2 vezes o risco de repetição de crises. Via de nascimento, escore de Apgar no 5º minuto, relação peso ao nascer/idade gestacional, intercorrências no período pós-natal imediato e desenvolvimento neuropsicomotor não tiveram influência na recorrência. História familiar de crises mostrou tendência à significância estatística para repetição dos episódios, com risco de 1,7. Não foi encontrada associação entre tipo de crise e achado eletrencefalográfico. A maioria das crises foi de curta duração (até 5 minutos), mas este dado não esteve relacionado com a recorrência. Estado de vigília teve efeito protetor na recorrência. Se a primeira crise foi parcial, o risco de repetição foi 1,62, com tendência à significância. Quando o primeiro EEG foi alterado, houve relação significativa com primeira crise tanto generalizada como parcial. O primeiro EEG com alterações paroxísticas focais apontou risco de repetição de 2,90. Quando as variáveis envolvidas na repetição de crises foram ajustadas pelo modelo de regressão de Cox, EEG alterado mostrou risco de 2,48, com riscos acumulados de 50%, 60%, 62% e 68%; com EEG normal, os riscos foram 26%, 32%, 34% e 36% em 6, 12, 18 e 24 meses respectivamente.

The Developing Left Superior Cervical Ganglion of Pacas (Agouti Paca)

In this study the main question investigated was the number and size of both binucleate and mononucleate superior cervical ganglion (SCG) neurons and, whether post-natal development would affect these parameters. Twenty left SCGs from 20 male pacas were used. Four different ages were investigated, that is newborn (4 days), young (45 days), adult (2 years), and aged animals (7 years). By using design-based stereo-logical methods, that is the Cavalieri principle and a physical disector combined with serial sectioning, the total volume of ganglion and total number of mononucleate and binucleate neurons were estimated. Furthermore, the mean perikaryal (somal) volume of mononucleate and binucleate neurons was estimated using the vertical nucleator. The main findings of this study were a 154% increase in the SCG volume, a 95% increase in the total number of mononucleate SCG neurons and a 50% increase in the total volume of SCG neurons. In conclusion, apart from neuron number, different adaptive mechanisms may coexist in the autonomic nervous system to guarantee a functional homeostasis during ageing, which is not always associated with neuron losses. Anat Rec, 292:966-975, 2009. (C) 2009 Wiley-Liss, Inc.

Caracterização comportamental e distribuição de neurônios inibitórios em um modelo animal de autismo induzido por ácido valpróico

Autism comprises a heterogeneous group of neurodevelopmental disorders that affects the brain maturation and produces sensorial, motor, language and social interaction deficits in early childhood. Several studies have shown a major involvement of genetic factors leading to a predisposition to autism, which are possibly affected by environmental modulators during embryonic and post-natal life. Recent studies in animal models indicate that alterations in epigenetic control during development can generate neuronal maturation disturbances and produce a hyper-excitable circuit, resulting in typical symptoms of autism. In the animal model of autism induced by valproic acid (VPA) during rat pregnancy, behavioral, electrophysiological and cellular alterations have been reported which can also be observed in patients with autism. However, only a few studies have correlated behavioral alterations with the supposed neuronal hyper-excitability in this model. The aim of this project was to generate an animal model of autism by pre-natal exposure to VPA and evaluate the early post-natal development and pre-puberal (PND30) behavior in the offspring. Furthermore, we quantified the parvalbumin-positive neuronal distribution in the medial prefrontal cortex and Purkinje cells in the cerebellum of VPA animals. Our results show that VPA treatment induced developmental alterations, which were observed in behavioral changes as compared to vehicle-treated controls. VPA animals showed clear behavioral abnormalities such as hyperlocomotion, prolonged stereotipies and reduced social interaction with an unfamiliar mate. Cellular quantification revealed a decrease in the number of parvalbumin-positive interneurons in the anterior cingulate cortex and in the prelimbic cortex of the mPFC, suggesting an excitatory/inhibitory unbalance in this animal model of autism. Moreover, we also observed that the neuronal reduction occurred mainly in the cortical layers II/III and V/VI. We did not detect any change in the density of Purkinje neurons in the Crus I region of the cerebellar cortex. Together, our results strengthens the face validity of the VPA model in rats and shed light on specific changes in the inhibitory circuitry of the prefrontal cortex in this autism model. Further studies should address the challenges to clarify particular electrophysiological correlates of the cellular alterations in order to better understand the behavioral dysfunctions

Fatores que influenciam a neofobia alimentar em fêmeas e filhotes de sagüis (Callithrix jacchus)

Considering the constant environmental changes, the ability to introduce new food items in the diet is crucial to omnivore animal survival. For optimal nourishment and lessening of intoxication risks, the animals must detect signs that indicate which items are adequate for their intake. We investigate some factors that interfere in the responses to non familiar food, modulating their neophobic behavior, of marmosets Callithrix jacchus, an omnivore and generalist primate, native to Northeast Brazil, known for being cautious in ingesting not known food. We analyzed the influence of food taste (sweet or salty), pregnancy and sex in feeding behavior and neophobic responses in these animals. 10 captive females were first selected, 5 of them being then pregnant. The females, pregnant or not, ate more when presented to the sweet items than to the salty ones. Pregnant females, however, themselves were less neophobic to both tastes, being also strongly neophilic to the sweets. We verified then the influence of nourishment during pregnancy on young males and females post natal feeding behavior. We observed 10 young divided in two groups, one whose mother ate that food item during pregnancy and one whose mother had no contact to it. In the first group that food was more easily accepted by the young, suggesting that neofobia and feeding behavior had a pre natal influence. Female young also ingested more food and were less neophobic than males, a difference already observed in behavior of adults of these specie. These results suggest that the low neophobic behavior to sweet food showed by females can be adaptive, and might have bestowed more fitness to those who presented it

Mortalidade infantil em município do interior do estado de São Paulo

Considerando que a mortalidade infantil é indicador dos níveis de saúde da população, realizamos este trabalho, cujo objetivo foi identificar as causas de mortalidade infantil no ano de 1998 em Botucatu. O coeficiente de mortalidade infantil obtido foi 12/1000 NV, com maior participação dos óbitos neonatais - 8,3/1000 NV A maior parte dos óbitos foi classificada como reduzível ou parcialmente reduzível, mas a atenção necessária para viabilizar tal redução foi variada. Dos óbitos ocorridos, 21,7% eram inevitáveis, evidenciando que para redução dos índices de mortalidade infantil deveremos continuar investindo na qualidade da assistência à saúde e melhoria das condições de vida da população.

Is urine interleukin-8 level a reliable laboratory test for diagnosing late onset sepsis in premature infants?

The present study is aimed to determine serum and urine interleukin-8 (IL-8) levels in premature infants with late onset sepsis (LOS) and to evaluate if urine IL-8 is a useful test for LOS diagnosis. Fifty-six premature infants admitted to the NICU over 1 year had serum and urine IL-8 determined by ELISA. They were divided into three groups: I definite sepsis, II probable sepsis and III non-infected. Results were expressed as mean or median. Differences between groups were assessed by ANOVA, Kruskal-Wallis ANOVA and Dunns Method. Sensitivity, specificity and positive and negative predictive values were calculated and a receiver operator characteristic curve was constructed to determine serum and urine IL-8 accuracy. There were no differences between groups for birth weight, and gestational and post-natal age. Median serum and urine IL-8 levels were significantly higher in GI and GII: 929 x 906 x 625pg/ml; P=0.024, and 249 x 189 x 42pg/mgCr; P< 0.001. Optimal cut-off point was 625pg/ml for serum IL-8 with 69 sensitivity and 75pg/mgCr for urine IL-8 with 92 sensitivity. IL-8 can be determined in urine from premature infants with LOS and is an accurate and feasible diagnosis method.

Crescimento de prematuros de extremo baixo peso nos primeiros dois anos de vida

OBJETIVO: Analisar o padrão de crescimento de prematuros de extremo baixo peso (EBP) até 24 meses de idade corrigida, a influência da displasia broncopulmonar (DBP) e os fatores de risco para falha de crescimento. MÉTODOS: Coorte de prematuros <1.000g de gestação única, nascidos e acompanhados em um centro terciário. O crescimento foi avaliado por meio de escores-z para peso, comprimento e perímetro cefálico ao nascimento, com 40 semanas, aos 3, 6, 12, 18 e 24 meses de idade corrigida. Dentre 81 sobreviventes, 70 foram estudados e estratificados em dois grupos: DBP (n=41) e sem DBP (n=29). Foi realizada análise bivariada com teste t ou Mann-Whitney, qui-quadrado ou Exato de Fisher, e análise multivariada com regressão logística. RESULTADOS: em ambos os grupos, o escore-z de peso diminuiu significantemente entre o nascimento e 40 semanas. Houve um pico de incremento nos escores-z de peso, comprimento e perímetro cefálico entre 40 semanas e três meses. No grupo sem DBP, os escores-z atingiram a faixa normal a partir dos seis meses e assim permaneceram até 24 meses de idade corrigida. Crianças com DBP tiveram menores escores-z de peso e perímetro cefálico no primeiro ano, mas equipararam-se às sem DBP no segundo ano de vida. A regressão logística mostrou que catch-down no escore-z de peso com 40 semanas foi fator de risco para falha de crescimento. CONCLUSÕES: Prematuros EBP apresentam catch-up precoce do crescimento nos primeiros dois anos. Crianças com DBP têm pior crescimento ponderal. A restrição do crescimento pós-natal prediz a falha de crescimento nos primeiros anos.

Paternal treatment with cisplatin impairs reproduction of adult male offspring in rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise quantitativa e molecular de hemoglobina fetal em indivíduos da população brasileira

A hemoglobina fetal - Hb F, formada por duas cadeias gama e duas cadeias alfa, é característica do período fetal do desenvolvimento, tendo sua síntese diminuída no período pós-natal. em algumas alterações hereditárias, a Hb F permanece aumentada, como nas delta-beta talassemia, beta talassemia e persistência hereditária de Hb F (PHHF). A síntese da globina gama também pode ser estimulada por fatores externos como leucemias, transplantes de medula óssea, induções químicas, dentre outros. Através da observação de Hb F aumentada em doadores de sangue por procedimentos eletroforéticos objetivou-se avaliar a quantidade de Hb F em amostras de sangue de candidatos à doação, visando estabelecer seus limites de normalidade na população de São José do Rio Preto e região, por meio de desnaturação alcalina e cromatografia líquida de alta pressão (HPLC), comparar as metodologias aplicadas e, nos indivíduos com Hb F aumentada, realizar estudos moleculares para identificar as mutações que alteram a expressão dos genes gama. Foram analisadas 208 amostras de sangue, sendo 119 de candidatos à doação e 89 de indivíduos sem sintomas de anemia ou achados hematológicos e com Hb F aumentada como grupo comparativo. Das 119 amostras de candidatos à doação, 110 foram utilizadas para traçar o perfil de normalidade de Hb F, comparando-se as metodologias de desnaturação alcalina e HPLC, onde se obteve a média de 1,48% e de 0,6%, respectivamente. A análise estatística por regressão linear mostrou diferença significativa na comparação entre as duas metodologias aplicadas, sendo a HPLC mais precisa para a quantificação de Hb F. Foram observados nos testes de rastreamento de hemoglobinas anormais nestas 110 amostras de sangue: 16,4% de alfa talassemia, 0,9% com Hb F aumentada, 0,9% com beta talassemia e 0,9% com hemoglobina variante de cadeia delta. Os outros nove doadores de sangue apresentaram Hb F acima de 10% em eletroforese e observou-se média de 32,28% para desnaturação alcalina e de 26,4% para HPLC. As análises moleculares por PCR-ASO foram realizadas na tentativa de se identificar um defeito genético que pudesse explicar o aumento de Hb F, pelo rastreamento de 16 mutações que originam talassemias do tipo beta. Encontraram-se 5,3% de heterozigotos para mutação CD6-A e 1,75% para as mutações CD 39, IVS1:6, -87 e IVS2:654, todas em heterozigose. Os resultados encontrados neste estudo evidenciam a necessidade de melhor caracterização dos perfis de hemoglobina obtidos pelos métodos clássicos e a importância de sua caracterização molecular.

Effect of thermal conditioning during embryonic development on aspects of physiological responses of broilers to heat stress

The purpose of the present study was to investigate the effect of thermal conditioning, (through exposure to heat stress), during pre-hatch development on some physiological responses of post-hatch broilers to a post-natal heat stress challenge. Exposure to heat stress at this stage, we hope, may possibly induce epigenetic heat adaptation. Incubating eggs were exposed to temperature of 39.0degreesC for 2 h from Day 13 to 17 of incubation. At 33, 35, 37, 39, 41 and 43 d of age, the broilers hatched from these eggs were housed individually in open-circuit respiration cells. The climatic chambers were set to 22degreesC and increased to 30degreesC for 4 h. O-2 consumption and CO2 production of each chicken was monitored continuously in order to calculate the heat production. Blood samples were obtained before and during the 4 h heat stress. Thermal conditioning during incubation did not affect the plasma T-4, corticosterone, glucose, uric acid and CK concentrations. Temperature challenge, decreased plasma T-3 of broilers of both groups but the decrease was greater in pre-conditioned broilers compared with controls. A similar trend was observed for triglycerides. These changes did not affect total heat production. Since decreased T3 and triglyceride levels are part of the mechanisms for thermoregulation, these suggest that thermal conditioning during incubation can improve the broiler chicken capability for thermotolerance at later post-hatch age. (C) 2002 Elsevier B.V. Ltd. All rights reserved.