Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A

Chk1 kinase coordinates cell cycle progression and preserves genome integrity. Here, we show that chemical or genetic ablation of human Chk1 triggered supraphysiological accumulation of the S phase-promoting Cdc25A phosphatase, prevented ionizing radiation (IR)-induced degradation of Cdc25A, and caused radioresistant DNA synthesis (RDS). The basal turnover of Cdc25A operating in unperturbed S phase required Chk1-dependent phosphorylation of serines 123, 178, 278, and 292. IR-induced acceleration of Cdc25A proteolysis correlated with increased phosphate incorporation into these residues generated by a combined action of Chk1 and Chk2 kinases. Finally, phosphorylation of Chk1 by ATM was required to fully accelerate the IR-induced degradation of Cdc25A. Our results provide evidence that the mammalian S phase checkpoint functions via amplification of physiologically operating, Chk1-dependent mechanisms.

Growth hormone receptor and insulin-like growth factor-I immunoreactivity in osteoclast-like cells during tooth eruption in the toothless (Osteopetrotic) rat following treatment with colony-stimulating factor-1

In the toothless (tl/tl) osteopetrotic rat, teeth form but fail to erupt. Treatment of tl/tl rats with colony-stimulating factor-1 (CSF-1) activates bone resorption by osteoclasts, permits tooth eruption, and up-regulates the immunoreactivity of bone marrow mononuclear cells to growth hormone receptor (GHr) and insulin-like growth factor (IGF)-I. This study examined the distribution of tartrate-resistant acid phosphatase (TRAP) and immunoreactivity for GHr and IGF-I in osteoclast-like cells located on the alveolar bone margin, adjacent to the lower first molar crown, in 14-day-old normal and tl/tl rats, following treatment with CSF-1. Osteoclast-like cells demonstrated a positive reaction for TRAP, GHr, and IGF-I in all groups. However, in tl/tl tissue, osteoclast-like cells were generally negative for GHr. There was no significant difference in the total number of TRAP, GHr, and IGF-I-positive osteoclast-like cells on the adjacent bone margin in normal, normal treated with CSF-1, and tl/tl rats. CSF-1 treatment of the tl/tl rat significantly increased the total number of osteoclast-like cells, which were positive for TRAP (p < 0.001), GHr (p < 0.05) and IGF-I (P < 0.01).

14-3-3 acts as an intramolecular bridge to regulate cdc25B localization and activity

One of the major regulators of mitosis in somatic cells is cdc25B. cdc25B is tightly regulated at multiple levels. The final activation step involves the regulated binding of 14-3-3 proteins. Previous studies have demonstrated that Ser-323 is a primary 14-3-3 binding site in cdc25B, which influences its activity and cellular localization. 14-3-3 binding to this site appeared to interact with the N-terminal domain of cdc25B to regulate its activity. The presence of consensus 14-3-3 binding sites in the N-terminal domain suggested that the interaction is through direct binding of the 14-3-3 dimer to sites in the N-terminal domain. We have identified Ser-151 and Ser-230 in the N-terminal domain as functional 14-3-3 binding sites utilized by cdc25B in vivo. These low affinity sites cooperate to bind the 14-3-3 dimer bound to the high affinity Ser-323 site, thus forming an intramolecular bridge that constrains cdc25B structure to prevent access of the catalytic site. Loss of 14-3-3 binding to either N-terminal site relaxes cdc25B structure sufficiently to permit access to the catalytic site, and the nuclear export sequence located in the N-terminal domain. Mutation of the Ser-323 site was functionally equivalent to the mutation of all three sites, resulting in the complete loss of 14-3-3 binding, increased access of the catalytic site, and access to nuclear localization sequence.

Site-directed mutagenesis of the ATM promoter: Consequences for response to proliferation and ionizing radiation

Although ATM, the protein defective in ataxia-telangiectasia (A-T), is activated primarily by radiation, there is also evidence that expression of the protein can be regulated by both radiation and growth factors. Computer analysis of the ATM promoter proximal 700-bp sequence reveals a number of potentially important cis-regulatory sequences. Using nucleotide substitutions to delete putative functional elements in the promoter of ATM, we examined the importance of some of these sites for both the basal and the radiation-induced activity of the promoter. In lymphoblastoid cells, most of the mutations in transcription factor consensus sequences [Sp1(1), Sp1(2), Cre, Ets, Xre, gammaIre(2), a modified AP1 site (Fse), and GCF] reduced basal activity to various extents, whereas others [gammaIre(1), NF1, Myb] left basal activity unaffected. In human skin fibroblasts, results were generally the same, but the basal activity varied up to 8-fold in these and other cell lines. Radiation activated the promoter approximately 2.5-fold in serum-starved lymphoblastoid cells, reaching a maximum by 3 hr, and all mutated elements equally blocked this activation. Reduction in Sp1 and AP1 DNA binding activity by serum starvation was rapidly reversed by exposure of cells to radiation. This reduction was not evident in A-T cells, and the response to radiation was less marked. Data provided for interaction between ATM and Sp1 by protein binding and co-immunoprecipitation could explain the altered regulation of Sp1 in A-T cells. The data described here provide additional evidence that basal and radiation-induced regulation of the ATM promoter is under multifactorial control. (C) 2003 Wiley-Liss, Inc.

ATP activates ataxia-telangiectasia mutated (ATM) in vitro - Importance of autophosphorylation

Ataxia-telangiectasia Mutated (ATM), mutated in the human disorder ataxia-telangiectasia, is rapidly activated by DNA double strand breaks. The mechanism of activation remains unresolved, and it is uncertain whether autophosphorylation contributes to activation. We describe an in vitro immunoprecipitation system demonstrating activation of ATM kinase from unirradiated extracts by preincubation with ATP. Activation is both time- and ATP concentration-dependent, other nucleotides fail to activate ATM, and DNA is not required. ATP activation is specific for ATM since it is not observed with kinase-dead ATM, it requires Mn2+, and it is inhibited by wortmannin. Exposure of activated ATM to phosphatase abrogates activity, and repeat cycles of ATP and phosphatase treatment reveal a requirement for autophosphorylation in the activation process. Phosphopeptide mapping revealed similarities between the patterns of autophosphorylation for irradiated and ATP-treated ATM. Caffeine inhibited ATM kinase activity for substrates but did not interfere with ATM autophosphorylation. ATP failed to activate either A-T and rad3-related protein (ATR) or DNA-dependent protein kinase under these conditions, supporting the specificity for ATM. These data demonstrate that ATP can specifically induce activation of ATM by a mechanism involving autophosphorylation. The relationship of this activation to DNA damage activation remains unclear but represents a useful model for understanding in vivo activation.

Expression of ephs and ephrins in developing mouse inner ear

Levels of expression of mRNAs encoding the different Ephs and ephrins were measured by semi-quantitative reverse-transcription polymerase chain reaction in developing mouse whole inner ears, and in dissected fractions of the neonatal mouse inner ear. Nineteen of the 24 known Ephs and ephrins were surveyed. The results showed that between embryonic age (E) 11.5 days and E12.5, levels increased 10-300 times per unit of tissue. In neonatal mice, the fraction containing combined organ of Corti and spiral ganglion showed relatively strong expression of EphA4, EphB3, ephrin-A3, ephrin-B2 and ephrin-B3. In the lateral wall, EphA4, ephrin-A3 and ephrin-B2 were strongly expressed, while ephrin-A3 was particularly strongly expressed in utricular and saccular sensory epithelia. The results suggest that the Ephs and ephrins are likely to play a part in the differentiation of the structures of the inner ear, and show which Ephs and ephrins are most likely to play important roles in the different structures. (C) 2003 Elsevier Science B.V. All rights reserved.

Abnormalities of the PTH-vitamin D axis and bone turnover markers in children, adolescents and adults with cystic fibrosis: comparison with healthy controls

Abnormalities of calcium and vitamin D metabolism in cystic fibrosis (CF) are well documented. We tested the hypothesis that alterations in calcium metabolism are related to vitamin D deficiency, and that bone resorption is increased relative to accretion in patients with CF. Calcitropic hormones, electrolytes, osteocalcin (OC) and bone alkaline phosphatase (BAP), (markers of bone mineralisation), urinary deoxypyridinoline [total (t) Dpd, a marker of bone resorption] and lumbar spine bone mineral density (LS BMD), expressed as a z-score, were measured in 149 (81 M) CF and 141 (61 M) control children aged 5.3-10.99 years, adolescents aged 11-17.99 years and adults aged 18-55.9 years. Data were analysed by multiple regression to adjust for age. In patients, FEV1% predicted and CRP (as disease severity markers), genotype and pancreatic status (PS) were recorded. The distribution of PTH differed between groups (P

Avaliação da qualidade do leite e caracterização de laticínios do estado do Espírito Santo

O leite é um alimento de grande importância na alimentação humana e amplamente consumido. Desta forma, justifica-se o estudo de suas características e a avaliação de procedimentos higiênicos durante toda a sua cadeia produtiva, desde a ordenha até o seu processamento. O objetivo do trabalho foi caracterizar laticínios localizados no estado do Espírito Santo, bem como avaliar as características de qualidade do leite cru e do leite pasteurizado de quatro estabelecimentos. O estudo foi dividido em quatro etapas: 1) seleção de dois laticínios com Selo de Inspeção Federal (SIF) e dois laticínios com Selo de Inspeção Estadual (SIE); 2) Elaboração de questionário para coleta de dados; 3) coleta de amostras de leite cru refrigerado e leite pasteurizado nos laticínios selecionados e avaliação da qualidade da matéria-prima e; 4) caracterização dos quatro laticínios e avaliação das condições higiênico-sanitárias dos estabelecimentos (aplicação questionário elaborado e da Lista de Verificação de Boas Práticas de Fabricação - check-list – presente na RDC nº 275 / 2002 da Anvisa).Os resultados obtidos com as análises de composição centesimal, acidez titulável, pH e Contagem de Células Somáticas (CCS) das amostras dos laticínios SIF 1, SIF 2, SIE 1 e SIE 2 indicaram conformidade com o padrão exigido pela Instrução Normativa nº 62/2011 do MAPA. Com relação ao teste do alizarol, todas as amostras analisadas apresentaram coloração parda avermelhada sem coagulação, indicando conformidade com a exigência da legislação. Para o teste de detecção de antibiótico da classe β-lactâmicos, todas as amostras de leite dos quatro laticínios analisadas nas três coletas tiveram ausência pelo método utilizado. Em uma das amostras coletadas da indústria SIF 1 foi verificada a presença da enzima fosfatase alcalina em leite pasteurizado, indicando que o tratamento térmico não foi adequado e que, portanto, poderia haver presença de microrganismos patogênicos na amostra, ou que a enzima se renaturou, apresentando um resultado falso positivo para o teste. Além disso, foi verificado que duas amostras de leite coletadas do laticínio SIE 1 apresentaram ausência da enzima lactoperoxidase,O leite é um alimento de grande importância na alimentação humana e amplamente consumido. Desta forma, justifica-se o estudo de suas características e a avaliação de procedimentos higiênicos durante toda a sua cadeia produtiva, desde a ordenha até o seu processamento. O objetivo do trabalho foi caracterizar laticínios localizados no estado do Espírito Santo, bem como avaliar as características de qualidade do leite cru e do leite pasteurizado de quatro estabelecimentos. O estudo foi dividido em quatro etapas: 1) seleção de dois laticínios com Selo de Inspeção Federal (SIF) e dois laticínios com Selo de Inspeção Estadual (SIE); 2) Elaboração de questionário para coleta de dados; 3) coleta de amostras de leite cru refrigerado e leite pasteurizado nos laticínios selecionados e avaliação da qualidade da matéria-prima e; 4) caracterização dos quatro laticínios e avaliação das condições higiênico-sanitárias dos estabelecimentos (aplicação questionário elaborado e da Lista de Verificação de Boas Práticas de Fabricação - check-list – presente na RDC nº 275 / 2002 da Anvisa).Os resultados obtidos com as análises de composição centesimal, acidez titulável, pH e Contagem de Células Somáticas (CCS) das amostras dos laticínios SIF 1, SIF 2, SIE 1 e SIE 2 indicaram conformidade com o padrão exigido pela Instrução Normativa nº 62/2011 do MAPA. Com relação ao teste do alizarol, todas as amostras analisadas apresentaram coloração parda avermelhada sem coagulação, indicando conformidade com a exigência da legislação. Para o teste de detecção de antibiótico da classe β-lactâmicos, todas as amostras de leite dos quatro laticínios analisadas nas três coletas tiveram ausência pelo método utilizado. Em uma das amostras coletadas da indústria SIF 1 foi verificada a presença da enzima fosfatase alcalina em leite pasteurizado, indicando que o tratamento térmico não foi adequado e que, portanto, poderia haver presença de microrganismos patogênicos na amostra, ou que a enzima se renaturou, apresentando um resultado falso positivo para o teste. Além disso, foi verificado que duas amostras de leite coletadas do laticínio SIE 1 apresentaram ausência da enzima lactoperoxidase,O leite é um alimento de grande importância na alimentação humana e amplamente consumido. Desta forma, justifica-se o estudo de suas características e a avaliação de procedimentos higiênicos durante toda a sua cadeia produtiva, desde a ordenha até o seu processamento. O objetivo do trabalho foi caracterizar laticínios localizados no estado do Espírito Santo, bem como avaliar as características de qualidade do leite cru e do leite pasteurizado de quatro estabelecimentos. O estudo foi dividido em quatro etapas: 1) seleção de dois laticínios com Selo de Inspeção Federal (SIF) e dois laticínios com Selo de Inspeção Estadual (SIE); 2) Elaboração de questionário para coleta de dados; 3) coleta de amostras de leite cru refrigerado e leite pasteurizado nos laticínios selecionados e avaliação da qualidade da matéria-prima e; 4) caracterização dos quatro laticínios e avaliação das condições higiênico-sanitárias dos estabelecimentos (aplicação questionário elaborado e da Lista de Verificação de Boas Práticas de Fabricação - check-list – presente na RDC nº 275 / 2002 da Anvisa).Os resultados obtidos com as análises de composição centesimal, acidez titulável, pH e Contagem de Células Somáticas (CCS) das amostras dos laticínios SIF 1, SIF 2, SIE 1 e SIE 2 indicaram conformidade com o padrão exigido pela Instrução Normativa nº 62/2011 do MAPA. Com relação ao teste do alizarol, todas as amostras analisadas apresentaram coloração parda avermelhada sem coagulação, indicando conformidade com a exigência da legislação. Para o teste de detecção de antibiótico da classe β-lactâmicos, todas as amostras de leite dos quatro laticínios analisadas nas três coletas tiveram ausência pelo método utilizado. Em uma das amostras coletadas da indústria SIF 1 foi verificada a presença da enzima fosfatase alcalina em leite pasteurizado, indicando que o tratamento térmico não foi adequado e que, portanto, poderia haver presença de microrganismos patogênicos na amostra, ou que a enzima se renaturou, apresentando um resultado falso positivo para o teste. Além disso, foi verificado que duas amostras de leite coletadas do laticínio SIE 1 apresentaram ausência da enzima lactoperoxidase,indicando a sua desnaturação devido à superpasteurização do leite. Para as análises microbiológicas de contagem bacteriana total e bactérias psicrotróficas, foi verificado uma contagem acima do estabelecido pela legislação. Além disso, os maiores valores médios de Contagem Bacteriana Total (CBT), contagem de microrganismos psicrotróficos e coliformes totais nas amostras de leite cru refrigerado foram verificados entre os laticínios com SIF, podendo ter como causa o uso de tanques comunitários pelos produtores, tempo de transporte para coleta de leite maior do que a média dos laticínios com SIE, a falta de adoção das Boas Práticas de Ordenha e o maior volume de leite coletado de diferentes produtores. Com relação à CBT e à contagem de coliformes totais em leite pasteurizado, os maiores valores médios foram verificados também nos laticínios SIF 1 e SIF 2. Os laticínios que apresentaram maior porcentagem de adequação aos requisitos das BPF foram os laticínios SIF 1 (87,82 %) e SIF 2 (80,66 %), os quais já possuíam os POP’s (Procedimento Operacional Padronizado), CIP (Controle Integrado de Pragas) e BPF (Boas Práticas de Fabricação) implantados ou em fase final de implantação. A análise dos resultados das análises microbiológicas, da aplicação do check-list e da aplicação do questionário permitiu a conclusão de que as empresas que possuíam SIF, apesar de apresentarem uma maior porcentagem de adequação aos requisitos de boas práticas de fabricação, possuíam uma qualidade da matéria-prima menor do que as indústrias com SIE. A partir dos resultados obtidos, pode-se concluir que o estudo de laticínios no estado do Espírito Santo possibilitou o conhecimento do setor e de seus problemas, contribuindo para o emprego de ações de melhoria e prevenção de futuros problemas.

Two-way MANCOVA: an application to public health

The aim of this work is to use the MANCOVA model to study the influence of the phenotype of an enzyme - Acid phosphatase - and a genetic factor - Haptoglobin genotype - on two dependent variables - Activity of Acid Phosphatase (ACP1) and the Body Mass Index (BMI). Therefore it's used a general linear model, namely a multivariate analysis of covariance (Two-way MANCOVA). The covariate is the age of the subject. This covariate works as control variable for the independent factors, serving to reduce the error term in the model. The main results showed that only the ACP1 phenotype has a significant effect on the activity of ACP1 and the covariate has a significant effect in both dependent variables. The univariate analysis showed that ACP1 phenotype accounts for about 12.5% of the variability in the activity of ACP1. In respect to this covariate it can be seen that accounts for about 4.6% of the variability in the activity of ACP1 and 37.3% in the BMI.

Modulation of osteoclastogenesis by antihypertensive drugs

Despite its rigid structure, bone is a dynamic tissue that is in constant remodeling. This process requires the action of the bone-resorbing osteoclasts and the bone-synthesing osteoblasts. One of the adverse effects attributed to some antihypertensive agents is the ability to alter normal bone metabolism. However, their effective actions on human bone cells remain to be clarified. In this work, the effects of five calcium channel blockers, a class of antihypertensive drugs (AHDs), were investigated on osteoclastic differentiation. Osteoclastic cell cultures were established from precursor cells isolated from human peripheral blood, and were maintained in the absence (control) or in the presence of 10-8-10-4 M of different AHDs (amlodipine, felodipine, diltiazem, lercanidipine and nifedipine). Cell cultures were characterized throughout a 21 day period for tartrate-resistant acid phosphatase (TRAP) activity, number of TRAP+ multinucleated cells, presence of cells with actin rings and expressing vitronectin and calcitonin receptors, and apoptosis rate. Also, the involvement of several signaling pathways on the cellular response was addressed. It was observed that the tested AHDs had the ability to differentially affect osteoclastogenesis. At low doses, amlodipine and felodipine caused an increase on osteoclastic differentiation, while the other drugs inhibited it. At higher doses, all the molecules caused a decrease on the process. The tested AHDs also showed different effects on the analysed signaling pathways. In conclusion, AHDs appeared to have a direct effect on human osteoclast precursor cells, affecting their differentiation. Interestingly, some of them increased while others inhibited the process. Unraveling the mechanisms beneath these observations might help to explain the adverse effects on bone tissue described for this drug class.

Negative modulation of human osteoclastogenesis by antiepileptic drugs

Bone is constantly being molded and shaped by the action of osteoclasts and osteoblasts. A proper equilibrium between both cell types metabolic activities is required to ensure an adequate skeletal tissue structure, and it involves resorption of old bone and formation of new bone tissue. It is reported that treatment with antiepileptic drugs (AEDs) can elicit alterations in skeletal structure, in particular in bone mineral density. Nevertheless, the knowledge regarding the effects of AEDs on bone cells are still scarce, particularly on osteoclastic behaviour. In this context, the aim of this study was to investigate the effects of five different AEDs on human osteoclastic cells. Osteoclastic cell cultures were established from precursor cells isolated from human peripheral blood, and were maintained in the absence (control) or in the presence of 10-8-10-4 M of different AEDs (valproate, carbamazepine, gabapentin, lamotrigine and topiramate). Cell cultures were characterized throughout a 21-day period for tartrate-resistant acid phosphatase (TRAP) activity, number of TRAP+ multinucleated cells, presence of cells with actin rings and expressing vitronectin and calcitonin receptors, and apoptosis rate. Also, the involvement of several signaling pathways on the cellular response was addressed. All the tested drugs were able to affect osteoclastic cell development, although with different profiles on their osteoclastogenic modulation properties. Globally, the tendency was to inhibit the process. Furthermore, the signaling pathways involved in the process also seemed to be differentially affected by the AEDs, suggesting that the different drugs may affect osteoclastogenesis through different mechanisms. In conclusion, the present study showed that the different AEDs had the ability to negatively modulate the osteoclastogenesis process, shedding new light towards a better understanding of how these drugs can affect bone tissue.

Protein-ligand docking study: diterpenes from Juniperus brevifolia as anticancer and antimicrobial agentes

REDCAT: Natural Products and related Redox Catalysts: Basic Research and Applications in Medicine and Agriculture, Aveiro, 25-27 Novembro de 2012.

Efeito da combinação de antibióticos e sinvastatina sobre microrganismos de interesse endodôntico e na expressão de marcadores odontoblásticos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Celiac disease detection using a transglutaminase electrochemical immunosensor fabricated on nanohybrid screen-printed carbon electrodes

Celiac disease is a gluten-induced autoimmune enteropathy characterized by the presence of tissue tranglutaminase (tTG) autoantibodies. A disposable electrochemical immunosensor (EI) for the detection of IgA and IgG type anti-tTG autoantibodies in real patient’s samples is presented. Screen-printed carbon electrodes (SPCE) nanostructurized with carbon nanotubes and gold nanoparticles were used as the transducer surface. This transducer exhibits the excellent characteristics of carbon–metal nanoparticle hybrid conjugation and led to the amplification of the immunological interaction. The immunosensing strategy consisted of the immobilization of tTG on the nanostructured electrode surface followed by the electrochemical detection of the autoantibodies present in the samples using an alkaline phosphatase (AP) labelled anti-human IgA or IgG antibody. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with a commercial ELISA kit indicating that the electrochemical immunosensor is a trustful analytical screening tool.

Voltammetric immunosensor for the diagnosis of celiac disease based on the quantification of anti-gliadin antibodies

Antibodies against gliadin are used to detect celiac disease (CD) in patients. An electrochemical immunosensor for the voltammetric detection of human anti-gliadin antibodies (AGA) IgA and AGA IgG in real serum samples is proposed. The transducer surface consists of screen-printed carbon electrodes modified with a carbon nanotube/gold nanoparticle hybrid system, which provides a very useful surface for the amplification of the immunological interactions. The immunosensing strategy is based on the immobilization of gliadin, the antigen for the autoantibodies of interest, onto the nanostructured surface. The antigen–antibody interaction is recorded using alkaline phosphatase labeled anti-human antibodies and a mixture of 3-indoxyl phosphate with silver ions (3-IP/Ag+) was used as the substrate. The analytical signal is based on the anodic redissolution of the enzymatically generated silver by cyclic voltammetry. The electrochemical behavior of this immunosensor was carefully evaluated assessing aspects as sensitivity, non-specific binding and matrix effects, and repeatability and reproducibility. The results were supported with a commercial ELISA test.