Structural studies of the hipersaline adaptation of proteins belonging to halophilic archaea

183 p.

Proteómica dirigida en la cirugía del cáncer gástrico

240 p. + anexos

Influência da ck2 no processo de interação Leishmania donovani-macrófagos

O gênero Leishmania apresenta espécies capazes de desenvolver doenças de grande importância para a saúde pública, as leishmanioses, que apresentam prevalência mundial de 12 milhões de pessoas. Quando os parasitos entram em contato com o hospedeiro humano passam por um processo de metaciclogênese adquirindo capacidade de interagir com os macrófagos. Inúmeras atividades biológicas são desencadeadas pela ativação de sistemas de transdução de sinais, onde as proteínas cinases e fosfatases desempenham papel fundamental. A proteína cinase CK2 parece estar presente em todas as células eucarióticas (núcleo, citoplasma e superfície). É caracterizada como enzima serina/treonina cinase, embora também seja capaz de fosforilar resíduos de tirosina em suas proteínas-alvo. No presente trabalho, demonstramos que o principal inibidor da CK2, TBB, foi capaz de inibir o crescimento de formas promastigotas de L. donovani e mostrou um mecanismo de ação irreversível, entretanto não foi capaz de induzir apoptose nas formas promastigotas de L. donovani. O pré-tratamento dos parasitos e macrófagos, assim como a adição do TBB durante o processo de infecção induziram uma redução significativa no número de amastigotas por macrófagos possivelmente pelo mecanismo de morte celular programada demosntrada pela técnica do TUNEL. O tratamento de macrófagos com TBB não induziram o aumento de óxido nítrico. Ensaios de imunofluorescência demonstraram a presença de CK2α em promastigotas. Macrófagos não infectados demonstraram pouca marcação para CK2α. Após a interação, a enzima mostrou-se distribuída preferencialmente na periferia dos macrófagos. Os dados do trabalho sugerem que a CK2 é uma importante enzima para a atividade biológica da Leishmania donovani, tendo seu estudo importante relevância para a descoberta de novos alvos terapêuticos.

A Tenascina-C e a sinalização celular na diferenciação tubulogênica endotelial

A matriz extracelular (MEC) é capaz de modular a adesão celular, induzindo processos de sinalização celular. No estado de aderência intermediária, induzido por proteínas matricelulares, as células tendem a se diferenciar, migrar e proliferar. A tenascina-C é uma proteína matricelular amplamente secretada em gliomas que está envolvida na proliferação e angiogênese tumoral. A MEC de gliomas, possui elevada incorporação de tenascina-C (TN-C), uma glicoproteína matricelular desadesiva que compete com a glicoproteína adesiva fibronectina (FN), desestabilizando os contatos focais e induzindo proliferação celular em gliomas. Neste trabalho nós nos propusemos a investigar o papel da TN-C tumoral no fenótipo angiogênico de células endoteliais. Recentemente em um trabalho publicado pelo nosso grupo observamos que as células endoteliais semeadas sobre matrizes de glioma (U373 MG) aderem menos e são deficientes na capacidade de formar tubos quando comparadas com àquelas plaqueadas sobre MEC de HUVECs. No entanto, neste trabalho, reproduzimos este fenótipo semeando as células endoteliais em suportes de TN-C /FN miméticos da composição da matriz tumoral nativa. Por western blotting, observamos um aumento na fosforilação em treonina 638 da proteína PKCα, um possível sítio inibitório, e um aumento na ativação de PKCδ. O efeito antagônico na regulação dessas isoformas de PKC foi demonstrado quando usamos inibidores seletivos de PKC α e δ e um ativador de PKCα (PMA). Observamos que quando tratamos as HUVECs plaqueadas sobre MEC de U373 com PMA, resgatamos a capacidade dessas células de formar tubos, o pré-tratamento dessas HUVECs com inibidor de PKC δ (rotlerina) resgatou parcialmente a capacidade tubulogênica dessas células. O pré-tratamento das HUVECs que foram semeadas sobre MEC da HUVEC (que formam tubos normalmente) com um inibidor de PKC α (RO320432) levou a diminuição da capacidade tubulogênica. Além disso, esta matriz também induz ativação de ERK e AKT. Investigamos também se o bloqueio dos diferentes domínios da TN-C na matriz derivada de glioma poderia, de alguma forma, reverter o defeito angiogênico das células, propiciado pela interação com a matriz extracelular de gliomas. O pré-tratamento da matriz extracelular de glioma com anticorpos anti-TN-C (contra os domínios FNIII 1-3, 4-5 FNIII e N-terminal) resgatou parcialmente a capacidade das células endoteliais de formar tubos. Nossos dados sugerem que a indução do fenótipo vascular observado em muitos gliomas, com predomínio de vasos mal formados e sub-funcionais, pode ser parcialmente devido ao comprometido da sinalização mediada por PKCs em células endoteliais, bem como do aumento da ativação das vias de ERK e Akt.

Effect of aureomycin on the behaviour of certain free amino acids in oil sardine (Sardinella longiceps) held in ice storage

The course of development of a few free amino acids under the influence of aureomycin in oil sardine (Sardinella lingiceps) held in ice storage was investigated. The levels of leucines and valine regularly increased in the control and aureomycin treated fush throughout the storage period. Alanines and threonine showed similar trend in both control and fish treated with 20ppm aureomycin. These amino acids however showed a gradual fall in fish treated at 5 ppm level. The changes in tyrosine+tryptophane were found to be irregular. Most of the amino acids studied indicated a remarkable change in trend by about the 16th day of ice storage in the case of fish treated with 50ppm aureimycin.

On the production and nutritive value of fish meal from Silver belly (Leiognathus spp.) landings at Rameswaram

Silver belly (Leiognathus Spp.) forms a major fishery in recent years in the Rameswaram island but fetches for the fishermen very low prices ranging from Rs. 0.03 to 0.12/Kg only, there being practically no demand for the fish. The possibilities of utilizing this cheap fish are discussed and the processing method described. During the glut season the cost of production of Silver belly fish meal works out to competitive prices of Rs. 500 to 700/ton. The silver belly fish meal is of high quality with good protein content averaging 57.71% in commercial samples and 61.90% in laboratory samples and with a high pepsin digestibility of 90.0% to 92.5%. The essential amino acid composition of the Silver belly fish meal compares very favorably with other round fish meals, with high contents of lysine, leucine, arginine, isoleucine, methionine, phenyl alanine, threonine and valine. Since there is good demand for fish meal as poultry and cattle food both in the internal and external markets, there is good scope for large scale production and sale of fish meal.

Squilla protein: chemical composition and nutritive value

Protein extract prepared from squilla (Grato squilla nepa), a commercially unexploited crustacean, was analysed for crude protein and essential amino acids. All the essential amino acids except tryptophan and threonine were present in nutritionally adequate amounts. The protein was evaluated for its nutritional quality in respect of growth rate, protein efficiency ratio (PER) and liver nitrogen content by feeding on rats. Growth rates and protein efficiency ratios were similar in rats fed on casein, squilla protein and a combination of squilla protein and casein (1:1) diet. The weight of liver and kidneys were normal.

Phylogeographic analysis of mitochondrial DNA haplogroup F2 in China reveals T12338C in the initiation codon of the NDS gene not to be pathogenic

In this report, we studied on a homoplasmic T12338C change in mitochondrial DNA (mtDNA), which substituted methionine in the translational initiation codon of the NADH dehydrogenase subunit 5 gene (ND5) with threonine. This nucleotide change was originall

Two cathelicidin genes are present in both rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

Further to the previous finding of the rainbow trout rtCATH_1 gene, this paper describes three more cathelicidin genes found in salmonids: two in Atlantic salmon, named asCATH_1 and asCATH_2, and one in rainbow trout, named rtCATH_2. All the three new salmonid cathelicidin genes share the common characteristics of mammalian cathelicidin genes, such as consisting of four exons and possessing a highly conserved preproregion and four invariant cysteines clustered in the C-terminal region of the cathelin-like domain. The asCATH_1 gene is homologous to the rainbow trout rtCATH_1 gene, in that it possesses three repeat motifs of TGGGGGTGGC in exon IV and two cysteine residues in the predicted mature peptide, while the asCATH_2 gene and rtCATH_2 gene are homologues of each other, with 96% nucleotide identity. Salmonid cathelicidins possess the same elastase-sensitive residue, threonine, as hagfish cathelicidins and the rabbit CAP18 molecule. The cleavage site of the four salmonid cathelicidins is within a conserved amino acid motif of QKIRTRR, which is at the beginning of the sequence encoded by exon W. Two 36-residue peptides corresponding to the core part of rtCATH_1 and rtCATH_2 were chemically synthesized and shown to exhibit potent antimicrobial activity. rtCATH_2 was expressed constitutively in gill, head kidney, intestine, skin and spleen, while the expression of rtCATH_1 was inducible in gill, head kidney, and spleen after bacterial challenge. Four cathelicidin genes have now been characterized in salmonids and two were identified in hagfish, confirming that cathelicidin genes evolved early and are likely present in all vertebrates.

A C-type lectin associated and translocated with cortical granules during oocyte maturation and egg fertilization in fish

Oocyte maturation and egg fertilization in both vertebrates and invertebrates are marked by orchestrated cytoplasmic translocation of secretory vesicles known as cortical granules. It is thought that such redistribution of cellular content is critical for asymmetrical cell division during early development, but the mechanism and regulation of the process is poorly understood. Here we report the identification, purification and cDNA cloning of a C-type lectin from oocytes of a freshwater fish species gibel carp (Carassius auratus gibelio). The purified protein has been demonstrated to have lectin activity and to be a Ca2+-dependent C-type lectin by hemagglutination activity assay. Immunocytochemistry revealed that the lectin is associated with cortical granules, gradually translocated to the cell surface during oocyte maturation, and discharged to the egg envelope upon fertilization. Interestingly, the lectin becomes phosphorylated on threonine residues upon induction of exocytosis by fertilization and returns to its original state after morula stage of embryonic development, suggesting that this posttranslational modification may represent a critical molecular switch for early embryonic development. (C) 2003 Elsevier Inc. All rights reserved.

Differential expression and characterization analysis of a new gene with WD domains in fish oogenesis

A new gene with WD domains is cloned and characterized according to its differential transcription and expression between previtellogenic oocytes (phase I oocytes) and fully-grown oocytes (phase V oocytes) from natural gynogenetic silver crucian carp (Carassius auratus gibelio) by using the combinative methods of suppressive subtraction hybridization, SMART cDNA synthesis and RACE-PCR. The full-length cDNA is 1870 bp. Its 5 ' untranslated region is 210 bp, followed by an open reading frame of 990 bp, which has the typical vertebrate initiator codon of ANNATG. The open reading frame encodes a protein with 329 amino acids. It has 670 bp of 3 ' untranslated region and an AATAAA polyadenylation signal. Because it has 92% homology to STRAP (serine-threonine kinase receptor-associated protein), a recently reported gene, we named it FSTRAP (fish STRAP). Virtual Northern blotting indicated that the FSTRAP was transcribed in fully-grown oocytes (phase V oocytes), but not in previtellogenic oocytes (phase I oocytes). RT-PCR analysis showed that FSTRAP was transcribed in brain, heart, kidney, muscle, ovary, spleen and testis, but not in liver. And its mRNA could be detected in the oocytes from phase II to phase V. Western blotting also showed that FSTRAP protein could be detected in brain, heart, kidney, muscle, ovary, spleen and testis except liver. Results of Western blotting on various oocytes were also similar to the RT-PCR data. FSTRAP protein was not expressed in the previtellogenic oocytes. Its expression initiated from phase II oocytes after vitellogenesis, and was consistent with the mRNA transcription.

Involvement of p90 ribosomal S6 kinase in termination of cell cycle arrest during development of Artemia-encysted embryos

Artemia has evolved a unique developmental pattern of encysted embryos to cope with various environmental threats. Cell divisions totally cease during the preemergence developmental stage from gastrula to prenauplius. The molecular mechanism of this, however, remains unknown. Our study focuses on the involvement of p90 ribosomal S6 kinase (RSK), a family of serine/threonine kinase-mediating signal transduction downstream of mitogen-activated protein kinase cascades, in the termination of cell cycle arrest during the post-embryonic development of Artemia-encysted gastrula. With immunochemistry, morphology, and cell cycle analysis, the identified Artemia RSK was established to be specifically activated during the post-embryonic and early larval developmental stages when arrested cells of encysted embryos resumed mitoses. In vivo knockdown of RSK activity by RNA interference, kinase inhibition, and antibody neutralization consistently induced defective larvae with distinct gaps between the exoskeleton and internal tissues. In these abnormal individuals, mitoses were detected to be largely inhibited in the affected regions. These results display the requirement of RSK activity during Artemia development and suggest its role in termination of cell cycle (G(2)/M phase) arrest and promotion of mitogenesis. Our findings may, thus, provide insights into the regulation of cell division during Artemia post-embryonic development and reveal further aspects of RSK functions.

螺旋藻丝氨酸/苏氨酸激酶基因家族的克隆与表达分析

螺旋藻（Spirulina)，是丝状不形成异型胞的蓝藻，由于其所蕴涵的高品质营养成分而成为一属具有重要经济价值的微藻。丝氨酸/苏氨酸激酶（serine/threonine kinases，STK）系统在螺旋藻的信号转导中发挥了重要作用。螺旋藻生活环境复杂，其中温度是中国北方温带地区螺旋藻大规模养殖的主要限制性因素。本文克隆了一条螺旋藻STK基因，并初步从STK激酶基因家族的角度探索了螺旋藻对高温环境的适应。主要结果如下： 1. 构建了插入片段为1-2kb的螺旋藻基因组文库。基于该文库用简并引物PCR的方法得到了一条螺旋藻STK基因5'端长度为504bp的基因组序列。 2．以本实验室构建的螺旋藻基因组草图为基础，在基因组水平上研究了螺旋藻STK基因家族的特点。发现螺旋藻草图中STK基因家族共包含33个基因，这些基因基于蛋白结构域分析可以分为三大类群。序列保守性分析发现，螺旋藻STK激酶具有与真核STK激酶相同的保守域，此外螺旋藻STK激酶还具有特有的保守氨基酸。 3．在中国北方温带地区，螺旋藻大规模养殖的主要限制性因素是温度。由于北方地区温度较低，螺旋藻规模养殖普遍存在年养殖期较短的问题，造成设备浪费，若采用加温措施又增加养殖成本；而在夏季晴天中午，又存在高温胁迫的问题。跨膜蛋白是信号转导的第一阶段，因此我们设置了不同温度来诱导螺旋藻，对其中7个跨膜STK基因进行半定量RT-PCR分析，结果显示STK2103在不同温度下表达量不同，推测该蛋白参与了螺旋藻温度相关的信号转导过程。 本文将基础研究与实验验证相结合，为螺旋藻的信号转导研究提供了线索。

两种蓝藻真核型Ser/Thr激酶基因功能的初步验证

Ser/Thr蛋白激酶（serine/threonine kinases，STK）在真核生物的信号转导通路中具有重要作用，而且已经成为对抗肿瘤、结核等多种人类疾病的药物作用靶点。上世纪九十年代，有研究发现STK在原核生物的信号转导中也发挥重要作用。本论文以聚球藻PCC7942（Synechococcus sp. PCC7942）和钝顶螺旋藻（Spirulina platensis）为材料，对几个真核型的Ser/Thr蛋白激酶基因的功能进行了初步验证。 蓝藻兼具细菌和植物的特点，具有成熟的转化体系，为真核生物基因功能的研究提供了新的模式宿主。聚球藻PCC7942是一种单细胞的淡水蓝藻，具有天然的外源DNA转化系统，是蓝藻分子遗传学研究的模式生物。通过基因敲除及表达差异分析发现聚球藻PCC7942中的Ser/Thr蛋白激酶stk196参与高温胁迫的信号传递。钝顶螺旋藻是原核丝状蓝藻，由于其蕴涵高品质营养成分而成为一类具有重要经济价值的微藻，该研究利用半定量RT-PCR方法，分析四个具有跨膜结构域的Ser/Thr蛋白激酶在正常生长温度下和经低温、高温诱导后表达量的变化情况，发现stk2103在低温诱导后的表达量降低，高温诱导后的表达量升高，提示该基因的表达可能参与了钝顶螺旋藻对温度的适应。 蓝藻中真核型Ser/Thr蛋白激酶功能的研究为我们进一步研究真核生物的Ser/Thr蛋白激酶功能提供了借鉴，并对植物抗逆胁迫的研究提供重要的理论依据。