Assembly of the protoplasm of Codium fragile (Bryopsidales, Chlorophyta) into new protoplasts

The cell organelles of the coenocytic alga Codium fragile (Sur.) Hariot aggregated rapidly and protoplasts were formed when its protoplasm was extruded out in seawater. Continuous observation showed that there were long and gelatinous threads connecting the cell organelles. The threads contracted, and thus the cell organelles aggregated into protoplasmic masses. The enzyme digestion experiments and Coomassie Brilliant Blue and Anthrone stainings showed that the long and gelatinous threads involved in the formation of the protoplasts might include protein and saccharides as structure components. Nile Red staining indicated that the protoplast primary envelope was non-lipid at first, and then lipid materials integrated into its surface gradually. The fluorescent brightener staining indicated that the cell wall did not regenerate in the newly formed protoplasts and they all disintegrated within 72 h after formation. Transmission electron microscopy of the cell wall of wild C. fragile showed electron-dense material embedded in the whole cell wall at regular intervals. The experiments indicated that C. fragile would be a suitable model alga for studying the formation of protoplasts.

鮸（Miichthys miiuy）早期生长存活过程和消化生理机制的研究

鮸Miichthys miiuy是中国南方重要的经济养殖种类，早期发育阶段的高死亡率和高畸形率严重制约着鮸养殖的工业化和商品化进程。为了更好的了解鮸早期发育规律，提高其人工养殖技术和管理策略，本论文在实验条件下对鮸早期生长和存活过程、消化生理发生机制及其在胁迫条件下的生态对策进行了研究。 鮸早期发育阶段分为仔鱼期和稚鱼期。个体发育的形态学变化和组织器官分化主要集中在仔鱼期，胚胎期所具有的特征在仔鱼期被具体的功能系统所代替，如呼吸、摄食、运动和消化系统。鮸个体的头、尾部快于躯干部的生长，表明其早期发育过程中摄食和运动器官发育的优先性。鱼类早期是优先发育对其生存起首要作用的器官，然后是对其生存作用次之的器官。 在24℃，鮸仔鱼在3日龄开口摄食，4日龄卵黄囊吸收完毕，6日龄仔鱼如不能建立外源性营养即进入饥饿死亡不可逆点(PNR)，而延迟投饵3天（6日龄）以上的仔鱼在8日龄全部死亡。在7日龄时，正常投喂仔鱼的SGR明显高于延迟投饵仔鱼。延迟3天和饥饿的仔鱼在6日龄后出现明显的负增长。在36日龄时，正常投喂仔鱼与延迟投饵1天仔鱼之间的生长差异消失，与延迟投饵2天的仔鱼之间的生长差异显著。饥饿显著影响开口仔鱼的生长存活，但对后期存活仔鱼的生长存活及体成分的影响不显著。仔鱼在12L条件下存活率（9-16%）要小于其他的各组（12-39%），仔鱼的存活率随着密度的增加逐渐减小。SGR与存活率具有相同的变化趋势，在18L条件下仔鱼的生长（3-10%）要大于12L（2-7%）和24L（2.8-8.7%）条件下仔鱼的生长。 鮸消化系统发育分为三个阶段：从孵化到初次摄食之前，消化道为一细长管道；从外源营养开始到胃腺出现，在此阶段，液泡、杯状细胞开始出现在消化道中，在6日龄胃出现，消化道明显分为口咽腔、食管、胃、前肠和后肠；从20日龄胃腺出现开始，胃在25日龄分为贲门胃、胃体和幽门胃三个部分，幽门盲囊出现，消化系统趋于完善。饥饿严重影响鮸消化器官及消化腺的正常发育过程。饥饿使其组织学的结构和功能明显衰退，肝组织变得疏松，细胞缩小，细胞核解体，稚鱼肝组织内没有脂质积累，细胞质中液泡减少；胰脏组织变得致密，腺泡萎缩，分泌物减少；胃腺细胞收缩，结构不完整，肠微绒毛退化，肠粘膜褶减少，肠上皮细胞遭到破坏。 鮸主要消化酶分为三个发育时期：从初孵到外源营养开始的迅速增长期；继而是从3日龄到25日龄波动期；25日龄后处于相对稳定期。各种消化酶总活性随着个体的发育逐渐增加，30日龄后总活性显著增加。延迟1天投饵的仔鱼和正常摄食的仔鱼其消化酶的活性没有显著差异（P>0.05），但是延迟2天投饵的仔鱼和正常摄食及延迟1天投饵的仔鱼消化酶的活性存在显著差异(P<0.05)，无论是延迟1天还是延迟2天仔鱼消化酶与正常摄食的仔鱼具有相同的发育方式。延迟3天投饵的仔鱼和饥饿仔鱼发育方式类似，从3日龄开始，消化酶活性显著下降。 饥饿对鮸仔稚鱼胰蛋白酶、淀粉酶和脂肪酶的活性有显著的影响。在初次摄食期，摄食仔鱼和饥饿2天的仔鱼各种消化酶活性均存在显著差异（P<0.05）；但是仔鱼后期，经过3天饥饿的仔鱼其消化酶的活性和摄食仔鱼之间存在显著差异（P<0.05），饥饿4天的仔鱼恢复摄食5天，即可恢复到和摄食仔鱼相同水平。饥饿6天的鮸稚鱼其消化酶与正常摄食的稚鱼之间存在显著差异（P<0.05），在恢复摄食5天后其消化酶活性可以恢复到和正常摄食仔鱼相同水平。 光照和饲养密度显著影响了鮸消化酶的活性。胰蛋白酶、淀粉酶和脂肪酶在18L低密度饲养条件下其活性明显高于（P<0.05）其他各组；12L高密度饲养条件下各种消化酶活性明显低于（P<0.05）其他各组；在0L条件下，各种消化酶活性在开口摄食之前明显低于其他各组，开口摄食之后，各种消化酶活性迅速下降。延迟投饵、光照周期和饲养密度虽然对鮸消化酶的活性产生了重要影响，但是对消化酶的发育方式没有显著影响，因此，鮸消化酶的具体发育方式是由内部遗传机制来控制，但受外界环境因素（食物组成、光照和饲养密度）的调节。

Determination of Total Arsenic and Arsenic Metabolites in Human Liver Hepatocytes

The effects of direct sampling and three digestion methods were investigated on the determination of arsenic in Chang liver hepatocytes after ultrasonic disintegration were investigated. The results showed that the efficiency of microwave digestion and obturator digestion was better than cold digestion and direct sampling. The day precision (present as RSD) of microwave digestion and obturator digestion were 2.1% and 1.2% the inter-day precision were 1.2% and 2.0%, respectively. The spike recovery for the total As in the sample is 95.7% - 108.1%. The As detection limits with these four sample treatment methods (including direct sampling) were 0.74 - 0.93 mu g/L. In addition, arsenic speciation in Chang liver hepatocytes was also analyzed using the hyphenated technique of high performance liquid chromatography coupled with inductively coupled plasma-mass spectrometry. The experimental results indicated that dimethylarsinic acid (DMA) and an intermediate metabolite of DMA were found lit Chang liver hepatocytes besides inorganic arsenic (As(III) and As(V)).

Effect of water temperature on digestive enzyme activity and gut mass in sea cucumber Apostichopus japonicus (Selenka), with special reference to aestivation

The effect of water temperature on gut mass and digestive enzyme activity in sea cucumber Apostichopus japonicus, including relative gut mass (RGM), amylase, lipase, pepsin and trypsin activities were studied at temperatures of 7, 14, 21, and 28A degrees C over a period of 40 days. Results show that RGM significantly decreased after 40 days at 21A degrees C and markedly decreased over the whole experiment period at 28A degrees C; however, no significant effect of duration was observed at 7 or 14A degrees C. At 14A degrees C, trypsin activity significantly decreased over 10 and 20 days, then increased; amylase and trypsin activity significantly decreased after 40 days at 28A degrees C. However, no significant effect of duration was found on amylase, pepsin or trypsin activities in the other temperature treatment groups. At 28A degrees C, lipase activity peaked in 20 days and then markedly decreased to a minimum at the end of the experiment. On the other hand, pepsin activity at 28A degrees C continuously increased over the whole experimental period. Principle component analysis showed that sea cucumbers on day 40 in the 21A degrees C group and in the previous 20 days in the 28A degrees C group were in the prophase of aestivation. At 28A degrees C, sea cucumbers aestivated at 30-40 days after the start of the experiment. It is concluded that the effect of temperature on the digestion of A. japonicus is comparatively weak within a specific range of water temperatures and aestivation behavior is accompanied by significant changes in RGM and digestive enzyme activities.

Identification of Crassostrea ariakensis and related oysters by multiplex species-specific PCR

Genetic markers are needed for rapid and reliable identification of oysters. In this study, we developed multiplex genus- and species-specific PCR markers for the identification of oysters from China. We used the mitochondrial cytochrome oxidase I (COI) and nuclear 28S ribosomal RNA genes for marker development. DNA sequences from different species were obtained from GenBank or by direct sequencing. Sequences were aligned, and genus- and species-specific nucleotides were identified. Primers were designed for genus/species-specific amplification to generate fragments of different sizes. A multiplex set of genus- and species-specific primers from the 28S gene was able to separate C. ariakensis and C. hongkongensis from other species and assign oysters to four genera. A set of species-specific COI primers provided positive identification of all five Crassostrea species from China, C. ariakensis, C. hongkongensis, C. angulata, C. gigas, and C. sikamea in a single PCR. The multiplex PCR assays do not require fluorescence-labeling or post-PCR enzyme digestion, providing a simple, fast and reliable method for the identification of oysters from China.

Proteomic detection of changes in protein expression induced by cordycepin in human hepatocellular carcinoma BEL-7402 cells

The nucleoside analogue cordycepin (3'-deoxyodenosine, 3'-dA), one of the components of cordyceps militaris, has been shown to inhibit the growth of various tumor cells. However, the probable mechanism is still obscure. In this study, the inhibition of cell growth and changes in protein expression induced by cordycepin were investigated in BEL-7402 cells. Using the MTT assay and flow cytometry, we found that cordycepin inhibits cell viability and induces apoptosis in BEL 7402 cells. Additionally. the proteins were separated using two-dimensional polyacrylamide gel electrophoresis, and eight proteins were found to be significantly, affected by cordycepin compared to untreated control; among them, two were downregulated and six were upregulated. Of the eight proteins, six were identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after in-gel trypsin digestion. These proteins are involved in various aspects of cellular metabolism. It is suggested that the effect of cordycepin on the growth of tumor cells is significantly related to the metabolism-associated protein expression induced by cordycepin. Copyright 2008 Prous Science, S.A.U. or its licensors. All rights reserved.

Effects of azadirachtin on hemolymph protein expression in Ostrinia furnacalis (Lepidoptera : Crambidae)

The botanical insecticide azadirachtin affects a variety of biological processes. Our early work indicated that protein level and type are significantly influenced by azadirachtin in pupae of Osttiniafumacalis (Guenee) (Lepidoptera: Crambidae) because a correlation exists between protein content and azadiraebtin concentration. By use of proteomic techniques, we analyzed changes in hemolymph protein expression of 48-h-old pupae in O. furnacalis induced by azadirachtin treatment. After feeding by third instars on an artificial diet containing 10 ppm azadirachtin until pupation, 48-b-old pupae were collected, and hemolymph protein samples were prepared. They were separated by two-dimensional polyacrylamide gel electrophoresis, and six proteins were significantly affected by azadiracbtin treatment compared with an untreated control. Two of these proteins were identified by database searching with peptide mass fingerprinting by using matrix-assisted laser desorption/ time-of-flight mass spectrometry after in-gel trypsin digestion. They belong to the insect apolipophorin-III and phospboribosyltransferase family, respectively. These two proteins may function on lipid metabolism in insect hemolymph. Furthermore, fat body is the center of synthesis and secretion of hemolymph proteins. We suggest that the azadirachtin exerts its insecticidal effects on the fat body of O. furnacalis by interfering with protein expression related to hemolymph lipid metabolism.

Digestibility, nutrient balance and urinary purine derivative excretion in dry yak cows fed oat hay at different levels of intake

A feeding trial A as conducted at the farm of Qinghai Academy of Animal and Veterinary Science, Xining, China during 1996 - 1997 with three dry yak cows (initial body weight 163 - 197 kg, age 5 - 6 years) by using 3 x 3 Latin Square Design to determine the effect of levels of feed intake on digestion, nitrogen balance and purine derivative excretion in urine of yak cows. The animals were fed oat hay (nitrogen 13.5 g/kg dry matter (DM), metabolisable energy 8.3 MJ/kg DM), i.e., 0.3, 0.6 and 0.9 of voluntary intake (VI). Each intake treatment lasted for 17 days and the samples (feeds, faeces and urine) were collected during last 7 days of each period. The results indicate that digestibility of dietary DM, OM, NDF and ash declined when intake levels increased from 0.3 to 0.9 VI [DM, from 66.1% to 59.1% (P < 0.05); OM, from 68.1% to 59.9% (P < 0.05); NDF, from 62.1% to 54.3% (P < 0.05); and ash, from 33.9% to 11.8% (P < 0.05)]. Around 0.10 g N/kg W-0.75 was deficient daily in yak cows at 0.3 VI, and positive N balances were observed at 0.6 and 0.9 VI. Intake levels significantly (P < 0.05) affected total PD excretion in yak urine. The proportion of allantoin increased (P < 0.05) and uric acid decreased (P < 0.05) as intake level of feed increased. (C) 2004 Elsevier B.V. All rights reserved.

Rapid protein identification using monolithic enzymatic microreactor and LC-ESI-MS/MS

A monolithic enzymatic microreactor was prepared in a fused-silica capillary by in situ polymerization of acrylamide, glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, followed by ammonia solution treatment, glutaraldehyde activation and trypsin modification. The choice of acrylamide as co-monomer was found useful to improve the efficiency of trypsin modification, thus, to increase the enzyme activity. The optimized microreactor offered very low back pressure, enabling the fast digestion of proteins flowing through the reactor. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at high flow rate. The digests were then characterized by CE and HPLC-MS/MS with the sequence coverage of 57.7%. The digestion efficiency was found over 230 times as high as that of the conventional method. in addition, for the first time, protein digestion carried out in a mixture of water and ACN was compared with the conventional aqueous reaction using MS/MS detection, and the former solution was found more compatible and more efficient for protein digestion.

Tratamento de dejetos no âmbito do programa do ABC.

2015

Caratterizzazione proteomica di fluidi e tessuti in diverse condizioni fisio-patologiche

This thesis has been focused on the proteomic characterization of human saliva from donors of different ages, starting from birth up to adult age, and pediatric brain tumor tissues. The first study has been performed in order to compare the acid-insoluble fraction of saliva from preterm with at-term newborns and adults and establish if differences exist. In the second study medulloblastoma and pilocytic astrocytoma pediatric brain tumor extracts have been compared. In both studies 2- DE analysis was coupled with high resolution tandem mass spectrometry (MS/MS). The proteomic characterization of the acid-insoluble fractions of saliva from preterm newborns allowed to integrate data previously obtained on the acid-soluble fraction by HPLC-electrospray ionization (ESI)-mass spectrometry (MS), and to evidence several differences between preterm newborns, at-term newborns and adults. Spots differentially expressed between the three groups, according to image analysis of the gels, were submitted to in-gel tryptic digestion and the peptide mixture analyzed by high performance HPLC-ESI-MS/MS for their characterization. By this strategy, we identified three over-expressed proteins in atterm newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, two proteoforms of annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100A6, S100A7, two proteoforms of S100A9, several proteoforms of prolactin-inducible protein, Ig kappa chain, two proteoforms of cystatin SN, one proteoform of cystatin S and several proteoforms of α-amylase 1). Moreover, for the first time, it was possible to assign by MS/MS four spots of human saliva 2-DE, already detected by other authors, to different proteoforms of S100A9. The strategy applied used a sequential staining protocol to the 2-DE gels, first with Pro-Q Diamond, that allows specific detection of phosphoproteins, and successively with total protein SYPRO Ruby stain. In the second study, proteomic analysis of two pediatric brain tumor tissues pointed out differences between medulloblastoma, the prevalent malignant tumor in childhood, and pilocytic astrocytoma, the most common, that only rarely shows a malignant progression. Due to the limited availability of bioptic tissue, the study was performed on pooled tumor tissues, and was focused on acid-insoluble fraction to integrate the characterization performed by a group of colleagues in Rome on the acid-soluble fraction by high performance HPLC-ESI-MS/MS. The results indicated that the two tumors exhibit different proteomic profiles and evidenced interesting differential expression of several proteins. Among them, peroxiredoxin- 1, peptidyl-prolyl cis–trans isomerase A, heterogeneous nuclear ribonucleoproteins A2/B1, mitochondrial isoform of malate dehydrogenase, nucleoside diphosphate kinase A, glutathione S-transferase P and fructose bisphosphate aldolase A resulted significantly over-expressed in medulloblastoma while glial fibrillary acidic protein, serotransferrin, α crystallin B chain, ferritin light chain, annexin A5, fatty acid-binding protein (brain), sorcin and apolipoprotein A-I resulted significantly over-expressed in pilocytic astrocytoma. In conclusion, the work done allowed to evidence the usefulness of using an integrated bottom-up/top-down approach, based on 2-DE-MS analysis and high performance MS in order to obtain a complete characterization of the proteome under investigation, revealing and identifying, not only peptides and small proteins, but also proteins with higher MW, that often it is not possible to identify by using exclusively a top-down ESI-MS approach.

Dynamic effects of cofactors and DNA on the oligomeric state of human mitochondrial DNA helicase

We examined the effects of cofactors and DNA on the stability, oligomeric state and conformation of the human mitochondrial DNA helicase. We demonstrate that low salt conditions result in protein aggregation that may cause dissociation of oligomeric structure. The low salt sensitivity of the mitochondrial DNA helicase is mitigated by the presence of magnesium, nucleotide, and increased temperature. Electron microscopic and glutaraldehyde cross-linking analyses provide the first evidence of a heptameric oligomer and its interconversion from a hexameric form. Limited proteolysis by trypsin shows that binding of nucleoside triphosphate produces a conformational change that is distinct from the conformation observed in the presence of nucleoside diphosphate. We find that single-stranded DNA binding occurs in the absence of cofactors and renders the mitochondrial DNA helicase more susceptible to proteolytic digestion. Our studies indicate that the human mitochondrial DNA helicase shares basic properties with the SF4 replicative helicases, but also identify common features with helicases outside the superfamily, including dynamic conformations similar to other AAA⁺ ATPases.

Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome

Matthew J. Nicholson, Michael K. Theodorou and Jayne L. Brookman. (2005). Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome. Microbiology, 151 (1), 121-133. Sponsorship: BBSRC RAE2008

A Doença Celíaca

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Branqueamento Dentário

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas