Gene expression and immunolocalization of chitin deacetylase BmCDA2 in silkworm / 生物工程学报

Deacetylation of chitin is closely related to insect development and metamorphosis. Chitin deacetylase (CDA) is a key enzyme in the process. However, to date, the CDAs of Bombyx mori (BmCDAs), which is a model Lepidopteran insect, were not well studied. In order to better understand the role of BmCDAs in the metamorphosis and development of silkworm, the BmCDA2 which is highly expressed in epidermis was selected to study by bioinformatics methods, protein expression purification and immunofluorescence localization. The results showed that the two mRNA splicing forms of BmCDA2, namely BmCDA2a and BmCDA2b, were highly expressed in the larval and pupal epidermis, respectively. Both genes had chitin deacetylase catalytic domain, chitin binding domain and low density lipoprotein receptor domain. Western blot showed that the BmCDA2 protein was mainly expressed in the epidermis. Moreover, fluorescence immunolocalization showed that BmCDA2 protein gradually increased and accumulated with the formation of larval new epidermis, suggesting that BmCDA2 may be involved in the formation or assembly of larval new epidermis. The results increased our understandings to the biological functions of BmCDAs, and may facilitate the CDA study of other insects.

Evaluation of survival and metamorphosis of larvae of Caligus rogercresseyi (Boxshall and Bravo, 2000) (Crustacea, Copepoda) in Chile, depending on temperature, salinity and oxygen / Avaliação de sobrevivência e metamorfose de larvas de Caligus rogercresseyi (Boxshall and Bravo, 2000) (Crustacea, Copepoda) no Chile, dependendo da temperatura, salinidade e oxigênio

Abstract The present study assessed the percentage of survival and metamorphosis of larval stages of Caligus rogercresseyi (Boxshall and Bravo, 2000) nauplius I, nauplius II to and nauplius II to copepodite, conducting bioassays in triplicate with 50 larvae each, nauplius I or nauplius II, at temperature of 12 °C, 15 °C and 18 °C; salinity 20, 23, 25, 2729, 31, 33 and 35 g/L and oxygen saturation with ranges between 30-60%, 90-100% and 190 - 200%. Bioassays were performed in station Quillaipe of Fundación Chile, Puerto Montt, Chile. It is concluded that the temperature is inversely proportional to the time of metamorphosis and survival of the larvae of Caligus rogercresseyi . In salinity is observed that increased this, greater is the percentage of survival and metamorphosis is faster, while the larvae do not survive less than 20g/L. Oxygen saturation ranges indicate that the larvae do not survive at saturations between 30-60%, and it had no differences between 90-100% saturation and 190-200%.

Resumo O presente estudo avaliou a percentagem de sobrevivência e metamorfose dos estádios larvais de Caligus rogercresseyi (Boxshall and Bravo, 2000) nauplio I, nauplio II ae nauplio II a copepodita, realizando bioensaios em triplicado com 50 larvas cada, nauplio I ou nauplio II, À temperatura de 12 °C, 15 °C e 18 °C; Salinidade 20, 23, 25, 2729, 31, 33 e 35 g / L e saturação de oxigénio com variações entre 30-60%, 90-100% e 190-200%. Os bioensaios foram realizados na estação Quillaipe da Fundação Chile, Puerto Montt, Chile. Conclui - se que a temperatura é inversamente proporcional ao tempo de metamorfose e sobrevivência das larvas de Caligus rogercresseyi. Na salinidade observa-se que aumentou esta, maior é a percentagem de sobrevivência e metamorfose é mais rápida, enquanto as larvas não sobrevivem menos de 20g / L. Os intervalos de saturação de oxigénio indicam que as larvas não sobrevivem a saturações entre 30-60% e não têm diferenças entre 90-100% de saturação e 190-200%.

There is no pulp necrosis or calcific metamorphosis of pulp induced by orthodontic treatment: biological basis

ABSTRACT To biologically explain why the orthodontic treatment does not induce pulp necrosis and calcific metamorphosis of the pulp, this paper presents explanations based on pulp physiology, microscopy and pathology, and especially the cell and tissue phenomena that characterize the induced tooth movement. The final reflections are as follows: 1) the orthodontic movement does not induce pulp necrosis or calcific metamorphosis of the pulp; 2) there is no literature or experimental and clinical models to demonstrate or minimally evidence pulp alterations induced by orthodontic movement; 3) when pulp necrosis or calcific metamorphosis of the pulp is diagnosed during orthodontic treatment or soon after removal of orthodontic appliances, its etiology should be assigned to concussion dental trauma, rather than to orthodontic treatment; 4) the two pulp disorders that cause tooth discoloration in apparently healthy teeth are the aseptic pulp necrosis and calcific metamorphosis of the pulp, both only induced by dental trauma; 5) the concussion dental trauma still requires many clinical and laboratory studies with pertinent experimental models, to increasingly explain its effects on the periodontal and pulp tissues.

RESUMO Para fundamentar biologicamente por que o tratamento ortodôntico não induz necrose pulpar e metamorfose cálcica da polpa, apresentou-se explicações com base na fisiologia, microscopia e patologia pulpar, bem como, e principalmente, nos fenômenos celulares e teciduais que caracterizam a movimentação dentária induzida. As reflexões finais foram: 1) o movimento ortodôntico não induz necrose pulpar ou metamorfose cálcica da polpa; 2) não há literatura e modelos experimentais e clínicos que comprovem ou minimamente evidenciem alterações pulpares induzidas pelo movimento ortodôntico; 3) quando a necrose pulpar ou metamorfose cálcica da polpa for diagnosticada durante o tratamento ortodôntico ou logo após a remoção dos aparelhos ortodônticos, a sua etiologia deve ser atribuída ao traumatismo dentário do tipo concussão, e não ao tratamento ortodôntico; 4) as duas doenças pulpares que levam ao escurecimento coronário em dentes aparentemente hígidos são a necrose pulpar asséptica e a metamorfose cálcica da polpa, ambas induzidas exclusivamente pelo traumatismo dentário; 5) o traumatismo dentário do tipo concussão requer, ainda, muitos estudos clínicos e laboratoriais, com modelos experimentais pertinentes, para fundamentar cada vez mais os seus efeitos sobre os tecidos periodontais e pulpares.

Transitando de embrión a feto: la metamorfosis de los cordados / Transiting from embryo to fetus: the metamorphosis of the chordata

Durante el período del desarrollo conocido como prefetal, el embrión cambia sus características ictiomórficas comunes a todos los vertebrados y adquiere gradualmente las formas propias de la especie que representa. Durante este período se forma la cara, involucionan los arcos faríngeos (branquiales) formándose el cuello, y aparecen los miembros. Se constituye, además, la hernia umbilical fisiológica, que consiste en la presencia de asas intestinales dentro del cordón umbilical. El sistema nervioso origina las vesículas telencefálicas, el diencéfalo, mesencéfalo, metencéfalo, y mielencéfalo. Este periodo corresponde a una etapa de máxima susceptibilidad ante los teratógenos que pueden generar malformaciones en todas las especies animales. El objetivo del presente trabajo es presentar los principales eventos acontecidos durante el periodo prefetal, además de una visión y opinión de los autores, proponiendo una nueva denominación a la etapa: periodo metamórfico.

During the period of development known as prefetal, the embryo changes its ictiomorphic characteristics common to all vertebrates and gradually acquires the proper forms of the species it represents. During this period the face is formed, the pharyngeal arches (branchial) involute forming the neck, and the limbs appear. In addition, the physiological umbilical hernia is constituted, which consists of the presence of intestinal loops inside the umbilical cord. The nervous system originates the telencephalic vesicles, the diencephalon, mesencephalon, metencephalon, and myelencephalon. This period corresponds to a stage of maximum susceptibility to teratogens that can generate malformations in all animal species. The objective of this paper is to present the main events that took place during the preferential period, as well as a vision and opinion of the authors, proposing a new name for the stage: metamorphic period.

Intra-puparial development of Lucilia eximia (Diptera, Calliphoridae) / Desenvolvimento Intra-pupal de Lucilia eximia (Diptera: Calliphoridae)

There are few studies about the intra-puparial development in Diptera, nonetheless its importance has been increased because several dipteran species are of forensic interest. Studies on the life cycles of flies often disregard the changes that occur inside the pupae. The objectives of this research were to analyze the intra-puparial development of Lucilia eximia, and to describe chronological and morphological changes that occur during this stage. Around 1.600 specimens were laboratory-reared. The pre-pupae were identified by the reduction of their size and change in coloration, and 10 individuals were sampled every three hours (n=1.000) until adult emergence. The specimens were fixed in 96% alcohol, subsequently immersed in Canoy solution for 24 hours and in formic acid (5%) for 48 hours, to facilitate dissection and analysis of the morphological changes of the individuals. Four stages of the intra-pupal development of L. eximia were observed: 1) Larva-pupa apolysis, which lasted 23 ± 1.08 h; 2) Cryptocephalic pupa, 5 ± 0.53 h; 3) Phanerocephalic pupa, 92 ± 1.94 h; and 4) Pharate adult: Transparents eyes, 125 ± 2.15 h; Yellow eyes, 23 ± 0.89 h; Pink eyes, 14 ± 0.73 h; and Red eyes, 20 ± 0.60 h. The pharate adult is completely formed after 296 hours and the emergence of the adult occurred after 302 ± 3.81 h. In addition, there were included the stage of pre-pupa, pupariation and the beginning of the adult stage, Imago and adult emergence. Each stage is described and compared with those described for Chrysomya albiceps (Calliphoridae).

No mundo existem poucos estudos sobre o desenvolvimento intrapupal de Diptera. Comumente, os estudos dos ciclos de vida das espécies de moscas são feitos eliminando o desenvolvimento embrionário que é muito importante, principalmente nas espécies de interesse forense. O principal objetivo foi o de analisar o desenvolvimento intrapupal de Lucilia eximia, descrevendo as mudanças morfológicas que ocorrem durante o desenvolvimento intrapupal. Foram analisados cerca de 1.600 espécimes cultivados em laboratório. Foram analisadas as fases de pré-pupa, pupa, o início da fase adulta, a imago e a emergência. Pré-pupas foram identificadas de acordo com a redução no tamanho e na alteração da coloração. Dez espécimes foram coletados a cada três horas (n=1.000), sem interrupção, até a emergência dos adultos. As pupas foram fixadas em etanol a 96%. Em seguida foram imersas em solução Carnoy por 24 horas e ácido fórmico a 5% por 48 horas para facilitar a dissecação e análises das alterações morfológicas. Foram determinados 4 estágios de desenvolvimento intrapupal. 1) Apólise larva-pupa com duração de 23 ± 1,08 h; 2) Pupa criptocefálica: 5 ± 0,53 h; 3) Pupa fanerocefálica: 92 ± 1,94 h e; 4) Adulto farado: olhos transparentes: 125 ± 2,15 h; olhos amarelos: 23 ± 0,89 h; olhos róseos: 14 ± 0,73 h e olhos vermelhos 20 ± 0,60 h. O adulto farado estava completo após 296 horas e a emergência dos adultos ocorreu com 302 ± 3,81 h. Cada fase foi descrita e comparada com o descrito para Chrysomya albiceps (Calliphoridae).

Cloning and expression profile of Bmlin-41 and its regulation by the silkworm microRNA let-7 / 生物工程学报

The heterochronic genes regulate cell proliferation and switch development stage transitions. Heterochronic genes might also play important roles in regulating the development of silkworm, but very few of their expression profiles, functions and their relationship with microRNAs are available so far. Firstly, in this work, the primers for cloning Bmlin-41 were designed based on the homologous sequence of known Drosophila melanogaster lin-41, which was used as the query to blast against SilkDB. The obtained full CDS (2 166 bp) of Bmlin-41 encodes 721 amino acids and contains B-box and NHL domains. Then, the spatiotemporal expression patterns of Bmlin-41 were characterized by RT-PCR, quantitative real time PCR as well as our lab's previous silkworm genome microarray data. Bmlin-41 was increasingly expressed from embryonic to adult stage. In diverse tissues of day-3 fifth instar, Bmlin-41 showed the highest accumulation in ovary, secondly in testis and midgut, but very low expression was observed in other tissues. Finally, 3'UTR of Bmlin-41 1 434 bp was cloned by rapid-amplification of cDNA ends (3'RACE) and was predicted to bare two binding sites of bmo-let-7 by using online RNAhybrid. To verify the binding effect, 3'UTR was cloned into psi-CHECK-2 vector and submitted to dual luciferase assay in the S2 cells in vitro. The dual luciferase assay demonstrated that Bmlin-41 was down-regulated by bmo-let-7 mimics and upregulated by bmo-let-7 antagomir, thus confirming the Bmlin-41 is negatively regulated by bmo-let-7. Our work might help further study on the roles of Bmlin-41 and bmo-let-7 and their regulation relationship involved in controlling metamorphosis of silkworm.

Cloning and expression profile of Bmyan in the silkworm (Bombyx mori) and experimental validation as one target of microRNA 7 / 生物工程学报

microRNAs (miRNAs) are an extensive class of -22-nucleotide (nt) endogenous noncoding RNAs regulating life activities ofmetazoans through binding to 3'-untranslated regions (3'-UTRs) of their target genes. This work aimed to identify yan gene in the silkworm, reveal its expression profile and confirm if it is one target of bmo-miR-7 and, as such, have potential for contributing to better understanding of the molecular mechanisms involved in the metamorphosis of silkworm. Based on homolog searching and PCR amplification, we cloned the coding sequence (CDS) of Bmyan, which encodes 476 amino acid residues and contains SAM-PNT and ETs domains. Quantitative PCR (q-PCR), RT-PCR and microarray data revealed high expression of Bmyan in the head, body wall and ovary of day-3 fifth instar larval silkworm, low or no expression in other tissues. It was lowly expressed in the early larval stages, but highly expressed from late spinning to day 4 pupa. The 3'-UTR of Bmyan was obtained by rapid-amplification of cDNA ends (3'RACE) and predicted to contain two potential recognition sites of bmo-miR-7. The luciferase reporter vector containing the 3'-UTR of Bmyan was constructed and co-transfected into BmE cell line with the mimic of bmo-miR-7 and the decreased relative activity of luciferase showed that Bmyan is one target of bmo-miR-7. This work helps further functional analysis of bmo-miR-7 and Bmyan in the silkworm.

Morphometry of the midgut of Melipona quadrifasciata anthidioides (Lepeletier) (Hymenoptera: Apidae) during metamorphosis

In Hymenoptera, midgut changes begin in the last instar. At this stage, the larval epithelial digestive cells degenerate, leaving only the basal membrane and the regenerative cells which will develop into a new epithelium during the pupal stage and in the adult. Epithelium renewal is followed by changes in volume and shape of the midgut. Morphometric analysis of digestive cells and total midgut volume of Melipona quadrifasciata anthidioides (Lepeletier) were conducted to verify whether cell volume increase are sufficient to account for the total midgut volume increase that occurs during metamorphosis. An increase in midgut volume was verified in spite of the scarcity of cell proliferation found during metamorphosis. At the end of metamorphosis, the increase in cell volume was not sufficient to explain the increase in volume of the midgut, indicating that an increase in the number of digestive cells is apparently necessary. Nevertheless, the mechanism by which regenerative cells reconstitute the epithelium during metamorphosis remains unknown.

Larval settlement and spat recovery rates of the oyster Crassostrea brasiliana (Lamarck, 1819) using different systems to induce metamorphosis / Taxas de assentamento larval e recuperação de sementes da ostra Crassostrea brasiliana (Lamarck, 1819) com uso de diferentes sistemas de indução à metamorfose

This study aimed at the assessment, in the laboratory, of the larval settlement and spat recovery rates of oysters of the species Crassostrea brasiliana using plastic collectors, epinephrine (C9H13NO3 C4H6O6) and shell powder in settlement tanks. Polypropylene was used attached to bamboo frames. The material was chosen due to its pliability - that favours the spat detachment. Two experiments were carried out; the first between February and April 2008, and the second between November and December 2008 at the Marine Mussel Laboratory of Santa Catarina Federal University (Laboratório de Moluscos Marinhos da Universidade Federal de Santa Catarina). In the first experiment, the scratched plastic collectors were tested consorting them with shell powder; on the second, the plastic collectors were tested consorted with shell powder, only shell powder and epinephrine as the metamorphosis stimulator. The quantification was carried out of the larvae settled in the plastic collectors, and of the recovery and integrity of the spats after their detachment. The first experiment has shown a recovery rate of 48.83 percent of the spats in comparison with the D larvae used. From this percentage, 4.9 percent settled in the plastic collectors and 43.93 percent in shell powder. The second experiment revealed 55.78 percent regarding the settled spats in comparison with the total of larvae used (using epinephrine), 78.62 percent in the treatment with the collector plus shell powder and 58.33 percent in the treatment only with shell powder. Thus, the use of the collector plus shell powder resulted in a greater spat recovery when compared to the other treatments.

O presente estudo teve como objetivo avaliar a taxa de assentamento larval e recuperação de sementes de ostras da espécie Crassostrea brasiliana, em laboratório, através do uso de coletores plásticos, epinefrina (C9H13NO3 C4H6O6) e pó de concha em tanques de assentamento. Foram utilizados coletores plásticos de polipropileno, presos a armações de bambu. O material foi escolhido devido à boa maleabilidade, o que facilita o destacamento das sementes. Foram realizados dois experimentos, o primeiro entre fevereiro e abril de 2008, e o segundo entre novembro e dezembro de 2008 no Laboratório de Moluscos Marinhos da Universidade Federal de Santa Catarina. No primeiro experimento, testaram-se coletores de plástico arranhado consorciado com pó de concha em um tanque de assentamento, enquanto que no segundo foram utilizados dois tanques de assentamento, um contendo os coletores de plástico consorciado com pó de concha e apenas pó de concha, e, no outro tanque, utilizou-se epinefrina como estimulador da metamorfose. Foi realizada a quantificação das larvas assentadas nos coletores plásticos e a taxa de recuperação e integridade das sementes após o destacamento. No primeiro experimento, recuperaram-se 48,83 por cento de sementes em relação às larvas D utilizadas. Deste percentual, 4,9 por cento assentaram em coletores plásticos e 43,93 por cento em pó de concha. No segundo experimento, a porcentagem de sementes assentadas em relação ao total de larvas utilizadas foi de 55,78 por cento com o uso de epinefrina, 78,62 por cento no tratamento com coletor mais pó de concha e de 58,33 por cento no tratamento só com pó de concha. Assim, verifica-se que o uso de coletor mais pó de concha resulta em maior recuperação de sementes se comparado com os demais tratamentos.

Influence of natural settlement cues on the metamorphosis of fiddler crab megalopae, Uca vocator (Decapoda: Ocypodidae)

Megalopae of many decapod crab species accelerate their development time to metamorphosis (TTM) when exposed to natural physical and/or chemical cues characteristic of the parental habitat. In the present study, the influence of natural settlement cues on the moulting rates and development TTM in megalopae of the fiddler crab Uca vocator was investigated. The effects of mud from different habitats (including well-preserved and degraded-polluted mangrove habitats) and conspecific adult 'odours' (seawater conditioned with crabs) on the induction of metamorphosis were compared with filtered pure seawater (control). 95 to 100 percent of the megalopae successfully metamorphosed to first juvenile crab stage in all treatments, including the control. However, the development TTM differed significantly among treatments. Settlement cues significantly shortened development, while moulting was delayed in their absence. The fact that megalopae responded to metamorphosis-stimulating cues originating from both adult and non-adult benthic habitats demonstrates that settlement in this species may occur in a wider range of habitats within the mangrove ecosystem, including impacted areas.

Megalopas de muitas espécies de caranguejos decápodes aceleram seu período de desenvolvimento até a metamorfose (TOM) quando são expostas a estímulos naturais físicos e/ou químicos característicos do habitat parental. No presente estudo, a influência de estímulos naturais sobre as taxas de muda e sobre o PDM foi investigada nas megalopas do caranguejo violinista Uca vocator. Os efeitos da (i) lama de diferentes habitats (incluindo habitats de um manguezal bem preservado e de um degradado e poluído) e (ii) 'odores' dos adultos conspecíficos (água do mar acondicionada com caranguejos) sobre a indução da metamorfose foram comparados com (iii) água do mar pura e filtrada (controle). 95 a 100 por cento das megalopas realizaram a metamorfose com sucesso para o primeiro estágio de caranguejo juvenil em todos os tratamentos, incluindo o controle. No entanto, o PDM diferiu significativamente entre os tratamentos. Os estímulos encurtaram significativamente o desenvolvimento, enquanto que a muda foi retardada na ausência deles. O fato de que as megalopas responderam aos estímulos indutores da metamorfose oriundos de ambos os habitats bentônicos dos adultos e de habitats onde eles são ausentes, demonstra que o assentamento nesta espécie pode ocorrer em uma grande variedade de habitats dentro do ecossistema de manguezal, incluindo áreas impactadas.

Perissophlebiodes flinti Savage (Ephemeroptera; Leptophlebiidae: Atalophlebiinae): novo registro, distribuição e comentários sobre sua identificação / Perissophlebiodes flinti Savage (Ephemeroptera; Leptophlebiidae: Atalophlebiinae): new occurrence, distribution and comments about its identification

Perissophlebiodes flinti Savage 1982, described from the State of Rio de Janeiro, and without new records since 1977, is now recorded for the State of São Paulo. Comments on distribution and suggestions for identification of two-winged neotropical leptophlebiids are made.

Perissophlebiodes flinti Savage 1982, descrita para o Estado do Rio de Janeiro, e sem registros desde 1977, é agora registrada para o Estado de São Paulo. São feitos comentários sobre a distribuição e sugestões para identificação das ninfas de Leptophlebiidae neotropicais com duas asas.

Ultradiluição homeopática de triiodotironina altera a apoptose celular da cauda de girinos de Rana catesbeiana: in vitro / Ultra high dilution of triiodothyronine modifies the cellular apoptosis of Rana catesbeiana tadpole tail: in vitro

Ultradiluição (UHD) é o efeito de uma solução, diluída acima do número de Avogrado, que na dependência da sua dinamização (diluição com sucussão) induz um efeito celular supressivo ou estimulante, com conseqüente obtenção de uma curva dose-efeito oscilatória. Por outro lado, a 3,3,5 Triiodo-L-Tironina (T3) é o hormônio mais importante na indução e manutenção das mudanças metamórficas dos girinos, nelas incluídas a absorção da cauda. O presente estudo, cego e randomizado, tem como objetivo comprovar que o T3 5.10-24M (10ª cH) altera a apoptose induzida pelo T3 100 nM na cauda de girinos de Rana catesbeiana, in vitro. Foram distribuídos 60 explantes em três grupos: Grupo A: sem o estímulo do T3 em dose farmacológica e em UHD; Grupo B (teste): sob a ação de T3 100 nM e T3 10ª cH (5.10-24 M); Grupo C (controle): sob a ação do T3 100 nM e etanol 70% sem sucussão. A análise estatística da área dos explantes, no primeiro e ultimo dia do experimento, e do índice apoptótico foi realizado através do teste t Student e foi considerado estatisticamente significante quando p<0,05. Embora sem diferenças significativas na área dos explantes do grupo teste e no grupo controle, um maior e significante índice apoptótico foi identificado nos explantes do grupo teste. Este resultado confirma que o T3 na 10ª cH altera a ação do T3 em dose farmacológica. Futuros experimentos serão realizados, com diferentes dinamizações, com o objetivo da parametrização da curva dose-efeito.

Ultra High Dilution (UHD) is the effect of a solution, beyond the Avogadro limits, that in the dependence of the applied dinamization (dilution with succussion) elicits a suppressive or a stimulant effect on a living cell, with a consequent generation of an oscillatory dose-effect curve. The entire process of anuran amphibian metamorphosis is under thyroid hormones control, included the complete resorption of the tadpole tail. A random and blind study was performed, with the intent to prove that T3 5.10-24 M (10ª cH) modifies the apoptosis induction of T3 100 nM in Rana catesbeiana tadpoles tail tips, in vitro. 60 Explants were distributed in three ways: Group A: without T3 action, at pharmacological and UHD dose; Group B (test): under the action of T3 100 nM and treated with T3 10ª cH (UHD); Group C (control): under the action of T3 100 nM and treated with ethanol 70% unsuccussed. In order to identify significant differences in the area of the remainder explants, at the first and final day of the experiment, and in the apoptotic index we used a student t-test. Although we didnt find statistical difference in macroscopic tadpoles tail tips area from test and control groups, a high and significant (p<0,05) index of apoptosis in histology was found in explants of test group. This data confirms that T3 10 cH modifies the effect of T3 at pharmacological dose. More studies will be necessary, using different dinamizations, to the parameterization of the dose-effect curve proceeding from these experiments.

Plasma matrix metalloproteinases-2 and -9 levels are elevated in patients with acute coronary syndrome and coronary chronic total occlusion / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the changes in plasma matrix metalloproteinases-2 and -9 (MMP2 and MMP9, respectively) levels in patients with different types of coronary heart diseases (CHD), and assess the value of MMP2/MMP9 detection in predicting acute coronary syndrome (ACS).</p><p><b>METHODS</b>According to the findings by coronary angiography and the clinical manifestations, 118 patients were divided in ACS group including 30 patients with unstable angina pectoris (UAP) and 19 with acute myocardial infarction (AMI) and non-ACS group including 23 patients with stable angina pectoris (SAP) and 21 with chronic total occlusion (CTO) of the coronary artery. Twenty-five individuals with normal coronary artery (NCA) served as the control group. Plasma levels of MMP9 and MMP2 were determined in these subjects using enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Both the ACS and non-ACS groups showed significantly higher MMP9 and MMP2 levels than the NCA group (P<0.05), and MMP2 and MMP9 levels were significantly higher in ACS group than in non-ACS group (P<0.05). Compared with the NCA group, the UAP, AMI and CTO subgroups showed obvious increases in plasma MMP2 and MMP9 levels (P<0.01). Significantly increased MMP9, but not MMP2 level was noted in AMI subgroup in comparison with SAP (P<0.01) and UAP subgroups (P<0.05); both MMP2 and MMP9 levels were elevated in CTO subgroup in comparison with those in SAP (P<0.001), UAP (P<0.01), and AMI subgroups (P<0.05).</p><p><b>CONCLUSION</b>Increased MMP2 and MMP9 levels in patients with CHD suggest the instability of the atherosclerotic plaque in correlation to the severity of ACS, and may serve as good indicators for the prediction of ACS and diagnosis of CTO of the coronary artery.</p>

Prokaryotic expression and polyclonal antibody preparation of TRalphaA in Japanese flounder Paralichthys olivaceus / 生物工程学报

To study the role of the thyroid hormone receptor TRalphaA involved in the process of the metamorphic development of Japanese flounder, we firstly cloned the TRalphaA gene, then ligated into the fusion expression vector pET30a and expressed in Escherichia coli DE3 (BL21) host cells. After induced for 4 h with 1 mmol/L Isopropyl beta-D-Thiogalactoside, the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. The recombinant protein was denatured and purified by His-Bind resin, then renatured through gradient washing on His-bind resin column. After that, polyclonal antibody was prepared by immunizing New Zealand rabbits with purified protein. Dot blotting analysis showed the antibody with the titer of 1:200 000 reacted specifically to the expressed recombinant protein. Furthermore, a chromatin immunoprecipitation assay was performed to identify the specific binding between the antibody and TRalphaA in living cells of Japanese flounder. The result showed that thyroid hormone was involved in the alkaline phosphatase (ALP) gene transcriptional regulation through TRalphaA in vivo.

Effects of the coastal sediment elutriates containing persistent organic pollutants (POPs) on early reproductive outputs of the Pacific oyster, Crassostrea gigas.

We previously found that embryonic development of the bivalve species was highly vulnerable to xenobiotic chemicals, damaging the coastal ecosystem integrity To further assess their potential damage to ecosystem, the xenobiotic composition of the sediment elutriates from two representative industrialized Korean coasts, Pohang and Ulsan, were determined with gas chromatography/mass spectrometry (GC/MS). The presumed critical dilution of the elutriate was then exposed to early life stages of the Pacific oyster (Crassostrea gigas), embryonic development and metamorphic stage to first spat, at which they were believably more vulnerable by the chemical exposure. The early life damage by the xenobiotic exposure was apparently significant by the significant degree of pollution. Here, we indicated their potential damages to the Pacific oyster

The tadpole of Hypsiboas leptolineatus (Braun and Braun, 1977), a species in the Hypsiboas polytaenius clade (Anura; Hylidae)

The larval morphology of Hypsiboas leptolineatus was studied. The tadpole has an ovoid body in lateral view, wider than deep; snout rounded with dorsal reniform nostrils; spiracle sinistral with lateral wall attached to body; anal tube dextral; tail fins convex with acuminate tip; oral disc ventral; labial tooth row formula is 2(2)/3(1); moderately developed beaks with serrated jaw sheaths. These external oral features are compared with those of the known tadpoles in the Hypsiboas polytaenius clade. The oral cavity was studied using an electron microscope. Life history aspects are commented.

A morfologia larval de Hypsiboas leptolineatus foi estudada. O girino possui corpo ovóide em vista lateral, mais largo que alto; focinho redondo com narinas dorsais; espiráculo esquerdo, com parede lateral junto ao corpo; tubo anal destro; nadadeiras convexas, com ponta acuminada; disco oral ventral; fórmula dentária 2(2)/3(1); bico córneo moderadamente desenvolvido serrilhado. Estas características externas são comparadas com as dos outros girinos conhecidos do clado Hypsiboas polytaenius. A cavidade oral foi estudada usando microscopia eletrônica. Aspectos de história natural são comentados.

Programmed cell death in the larval salivary glands of Apis mellifera (Hymenoptera, Apidae).

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine,the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy.Tissue sections were subsequently stained with haematoxylin -eosin,bromophenol blue,silver,or a variant of the critical electrolyte concentration (CEC) method.Ultrathin sections were contrasted with uranyl acetate and lead citrate.Glands were processed for the histochemical and cytochemical localization of acid phosphatase,as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed.The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and,additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen.The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed.The nuclei were pyknotic,with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.

[On the] geochemistry and mineral chemistry of the Gabbroic rocks at Wadi Um Khors area, central eastern desert, Egypt

The studied metagabbro-diorite complex comprises hornblende metagabbro, quartz-metagabbro, appinitic-metagabbro, and quartz diorite beside the fresh gabbro relics. They are calc-alkaline and were emplaced at subduction zones related to island arc magma. They were subjected to metamorphism but still carry minor relics of fresh gabbro. Mineralogically, the metagabbroic rocks are composed of plagioclase [An 30-45], Ca-rich clinopyroxenes [diopside and augite] and hornblende of green and olive brown colour]. The obtained crystallization temperature for the coexisting plagioclase- olive brown hornblende mineral pair was 600 - 700 °C and for the coexisting plagioclase- green hornblende mineral pair was 800 - 900 °C. The Ca-rich clinopyroxenes are low in alumina content suggesting crystallization between 500 and 1100 °C at shallow depth. Also, the analyzed clinopyroxenes pertain to those found in sub- alkaline rocks. The studied metagabbroic rocks show relative enrichments in LILE [Sr, Rb, Ba] over the HFSE [Nb, Zr, Y], a feature characteristic of subduction- related magmas