Azaspiracid shellfish poisoning: a review on the chemistry, ecology, and toxicology with an emphasis on human health impacts

Azaspiracids (AZA) are polyether marine toxins that accumulate in various shellfish species and have been associated with severe gastrointestinal human intoxications since 1995. This toxin class has since been reported from several countries, including Morocco and much of western Europe. A regulatory limit of 160 μg AZA/kg whole shellfish flesh was established by the EU in order to protect human health; however, in some cases, AZA concentrations far exceed the action level. Herein we discuss recent advances on the chemistry of various AZA analogs, review the ecology of AZAs, including the putative progenitor algal species, collectively interpret the in vitro and in vivo data on the toxicology of AZAs relating to human health issues, and outline the European legislature associated with AZAs.

Ciguatera Fish Poisoning: treatment, prevention and management

Ciguatera Fish Poisoning (CFP) is the most frequently reported seafood-toxin illness in the world, and it causes substantial physical and functional impact. It produces a myriad of gastrointestinal, neurologic and/or cardiovascular symptoms which last days to weeks, or even months. Although there are reports of symptom amelioration with some interventions (e.g. IV mannitol), the appropriate treatment for CFP remains unclear to many physicians. We review the literature on the treatments for CFP, including randomized controlled studies and anecdotal reports. The article is intended to clarify treatment options, and provide information about management and prevention of CFP, for emergency room physicians, poison control information providers, other health care providers, and patients.

Scientific assessment of marine harmful algal blooms

Algae are the most abundant photosynthetic organisms in marine ecosystems and are essential components of marine food webs. Harmful algal bloom or “HAB” species are a small subset of algal species that negatively impact humans or the environment. HABs can pose health hazards for humans or animals through the production of toxins or bioactive compounds. They also can cause deterioration of water quality through the buildup of high biomass, which degrades aesthetic, ecological, and recreational values. Humans and animals can be exposed to marine algal toxins through their food, the water in which they swim, or sea spray. Symptoms from toxin exposure range from neurological impairment to gastrointestinal upset to respiratory irritation, in some cases resulting in severe illness and even death. HABs can also result in lost revenue for coastal economies dependent on seafood harvest or tourism, disruption of subsistence activities, loss of community identity tied to coastal resource use, and disruption of social and cultural practices. Although economic impact assessments to date have been limited in scope, it has been estimated that the economic effects of marine HABs in U.S. communities amount to at least $82 million per year including lost income for fisheries, lost recreational opportunities, decreased business in tourism industries, public health costs of illness, and expenses for monitoring and management. As reviewed in the report, Harmful Algal Research and Response: A Human Dimensions Strategy1, the sociocultural impacts of HABs may be significant, but remain mostly undocumented.

Toxic effects of dietary seaweeds Sargassum bovianum, Caulera faridii and Gracilaria corticata on blood parameters of albino rats

The present communication deals with the feeding trials of brown (Sargassum bovianum), green (Caulerpa faridii) and red (Gracilaria corticola) seaweeds in albino rats for a period of thirty days in order to investigate their digestibility and acceptability as supplementary food for animals. The parameters used were: changes in blood hemoglobin, ESR, MCHC, PCV and plasma vitamin levels. The result revealed that all the three species of seaweeds had acceptability up to 5% level, as no ill effect was noted during the experiment. But at 10% and 20% levels, marked changes were observed in blood parameters with diarrhea, vomiting and convulsions indicating possibilities of either tissue and muscular dystrophy, gastrointestinal tract necrosis or functional disorder of central nervous system. A heavy mortality was noted due to excessive water loss through diarrhea and vomiting. However, no mortality was observed after 22nd day at both 10% and 20% levels with subsided clinical signs. The results suggest that these three seaweed species could be used safely as a supplementary food, in native form, in animals at low concentrations.

Isolation and cDNA cloning of cholecystokinin from the skin of Rana nigrovittata

Many neuroendocrine peptides that are distributed in amphibian gastrointestinal tract and central nervous system are also found in amphibian skins, and these peptides are classified into skin-gut-brain triangle peptides, such as bombesins, gastrin-releasi

Motilin and ghrelin gene experienced episodic evolution during primitive placental mammal evolution

Motilin and ghrelin, members of a structure-function-related hormone family, play important roles in gastrointestinal function, regulation of energy homeostasis and growth hormone secretion. We observed episodic evolution in both of their prehormone gene sequences during primitive placental mammal evolution, during which most of the nonsynonymous changes result in radical substitution. Of note, a functional obestatin hormone might have only originated after this episodic evolution event. Early in placental mammal evolution, a series of biology complexities evolved. At the same time the motilin and ghrelin prehormone genes, which play important roles in several of these processes, experienced episodic evolution with dramatic changes in their coding sequences. These observations suggest that some of the lineage-specific physiological adaptations are due to episodic evolution of the motilin and ghrelin genes.

Influence of a fish bacterium Lactobacillus sp. on the production of swordtail xiphophorus helleri (Heckel 1848)

The influence of a fish gut bacterium Lactobacillus sp on the production of swordtail Xiphophorus helleri was studied for a period of one year. The Lactobacillus sp P21 produced bacteriocin-like inhibitory substance and exhibited wide spectrum of action against Aeromonas hydrophila, Bacillus spp, Pseudomonas spp and Citrobacter freundi in vitro. The growth performance of X. helleri reared in the presence of Lactobacillus P21 at 106/ml rearing water was better than the control. The total plate counts, total MRS agar counts and the counts of motile aeromonads, presumptive pseudomonads, lactose fermenters and lactose non-fermenters in the gut of probiotic group were comparatively low than the control. On day 60 the count of Lactobacillus sp P21 was observed to be log 5.28/g in the gut of X. helleri indicating colonization of this bacterium in the gastrointestinal tract. The fecundity of X. helleri was in the range of 9-134. On average, it produced from 39.42±18.72 fry/female in control group to 53.00±23.57 fry/female in probiotic group. The increase in average fecundity in probiotic group over the control group was about 25%. There existed significant difference between probiotic group and control in respect of average fecundity/female (p<0.02), average number of fry survived /female (p<0.006) and average number of fry dead/female (p<0.029). The results of the present study demonstrated that the rearing of X. helleri in probiotic-enriched water have growth inducing ability and favourably influenced the reproductive performance in terms of high fecundity, high fry survival, reduced fry mortality and reduced fry deformity.

Study on effects of extracted probiotics from microbial flora of digestive system on growth indices in beluga (Huso huso) and Persian sturgeon (Acipenser persicus)

Isolation of Lactic Acid Bacteria (LAB) was carried out from gastro intestinal tract of beluga and Persian sturgeon at international sturgeon research institute and PCR has been used for bacteria Identification. Two species of LAB including Enterococcus seriolicida and Leuconostoc mesenteroides were isolated from Gastrointestinal tract (GI) of persian sturgeon in this study and the counts of Leu. mesenteroides (4.63×102 CFU/gr of GI) was significantly higher than other species. Lactobacillus curvatus, Lactococcus raffinolactis, Lactococcus lactis and Streptococcus sp. were also isolated from GI of beluga and maximum counts was belonged to Lb. curvatus (4.63×102 CFU/gr of GI) in this species. Dominant species were lyophilized and adding to the water since start of mix feeding of sturgeon with different counts including 2×109, 5×109 and 9×109 CFU/gr of live food, 4 times a day. The results revealed that the maximum and minimum growth rate and protease, amylase, and lipase activity in beluga was gained by using of Lb. curvatus with total viable count of 9×10 9 CFU/gr of live food and Leu. mesenteroides with total viable count of 9×109 CFU/gr of live food. According to the results of this study, the maximum and minimum growth rate and protease, amylase, and lipase activity in Persian sturgeon was gained by using of Leu. mesenteroides with total viable count of 2×10 9 CFU/gr of live food and Lb. curvatus with total viable count of 9×109 CFU/gr of live food. Histological study showed that gastrointestinal development was same during larva rearing in control and other treatments but the size of liver was bigger in treatments that received nonspecific LAB in both species. According to the results, positive effects of using dominant specific LAB bacteria for larviculture of sturgeon has been proved in this study.

New data on the morphology and systematic status of Spinitectus petrowi and Spinitectus gigi (Nematoda : Cystidicolidae) parasitic in catfishes in central China

Two little-known nematode species of the genus Spinitectus Fourment, 1883, S. petrowi Belous, 1965 (prevalence 25%, intensity 1-8) and S. gigi Fujita, 1927 (prevalence 10%, intensity 2-3), were collected from the gastrointestinal tract of the yellow catfish, Pelteobagrus fulvidraco (Richardson), from Liangzihu Lake, Hubei Province, central China, in September of 2002. The light and scanning electron microscopical examination of this material, supplemented by a few museum specimens of S. gigi collected from the catfish Clarias fuscus (Lacepede) in southern China, made it possible to study in detail the morphology of these parasite species and to redescribe them. The first species, whose correct name is S. petrowi Belous, 1965, exhibits some morphological features (e.g., unusually short vestibule, shape of pseudolabia and of the left spicule) not found in most other congeners; a unique feature is the presence of peculiar pairs of transversely oriented peg-like cuticular spines with rounded ends on the ventral surface of the female tail. Spinitectus gigi was found to have 28-31 cuticular spines in the first ring, relatively long distances between the 2nd-7th rings of spines, and anterior rings divided into 2 sectors; the excretory pore is located at the level of the 4th ring of cuticular spines; males posses 4 pairs of preanal- and 6 pairs of postanal caudal papillae and a pair of small phasmids. Spinitectus bagri Wang, Wu et Yu, 1993 and S. wulingensis Yu et Wang, 1997 are considered junior synonyms of S. petrowi, whereas S. clariasi Ky, 1971, S. ophicephali Ky, 1971 and S. yuanjiangensis Wang, Wit et Yu, 1997 are regarded to be junior synonyms of S. gigi. Spinitectus petrowi was not previously reported from China.

PAR4 和TFF2 在胃肠癌中的表达变异与临床关系以及 TFF2 的重组表达

胃癌和肠癌是常见的威胁人类健康的消化道恶性肿瘤，其发生发展涉及多因 子的作用及调控。其中，在胃肠道都有表达的蛋白酶激活受体（PARs）和三叶 因子蛋白（TFFs）家族都参与肿瘤发生发展的调控过程。正常生理条件下，PARs 的表达与胃肠道消化液的分泌和肌肉的收缩舒张相关。同时，在胃肠道肿瘤的发 生、浸润和转移过程中PARs和TFF2也发挥了作用。而PAR-4，除了具有凝血酶 激活后的血小板聚集功能外，还参与感染、细胞迁移和肿瘤的发生发展。在溃疡 性结肠炎，肠癌组织以及某些肠癌细胞系中都出现PAR4的异常表达，而这种异 常表达可能作为启动肠癌发生的重要环节。TFFs家族蛋白能够对抗粘膜损伤并 且参与修复以发挥保护胃肠道的功能。在肿瘤发生中，三叶因子既有报道作为肿 瘤抑制因子，又有报道作为潜在的肿瘤促进因子。含两个三叶因子结构域的 TFF2，主要表达在胃粘膜的颈细胞。在胃溃疡、慢性萎缩性胃炎及胃癌中，TFF2 的表达具有下降的趋势；而且分化程度越低的胃癌，TFF2的表达量越少，这是 因为TFF2的表达与胃粘膜细胞的增殖和恶性转移相关。在肠道，TFF2可以抑制 一氧化氮（NO）的生成以调节由NO引起的肠炎；在肠炎老鼠的模型中，TFF2 能减轻炎症和溃疡发生的程度，表明TFF2可能通过调节机体的免疫反应来抑制 肠道炎症的发生。 而本实验室前期对大蹼铃蟾皮肤分泌物中获得的新型血小板激动蛋白 -Bm-TFF2与PARs相互作用的实验，促使我们去研究人TFF2与PARs的关系。由 于免疫组化提示TFF2和PAR4在正常胃黏膜中都分布在从基底部到中间的位置， 而且TFF2第二个Loop区序列的保守性，以及和PAR4连接配体（tethered-ligand） 的高度相似性，促使我们推测PAR4和TFF2之间是否存在一种相互作用，或者 hTFF2是否能调节PAR4的生物学活性。所以该篇论文落脚于PAR4和hTFF2，着 重介绍PAR4和TFF2在胃肠道肿瘤中的表达变异以及TFF2对过表达PAR4的细胞 的趋化作用。 我们先用半定量PCR方法检测TFF2和PAR4在胃癌、肠癌及周围远癌部位组 织中mRNA的表达水平。结果提示两个基因在胃癌组织中的表达较周围远癌部位组织减弱，而在肠癌组织中的表达则较周围远癌部位组织增强。Western blotting 也得到相似的结果。为进一步明确PAR4和TFF2在胃癌和肠癌中表达的具体变化 情况，我们继而用实时荧光定量PCR对28例胃癌和38例肠癌及其周围远癌部位组 织中TFF2和PAR4的表达进行了研究。结果显示胃癌组织中两个基因mRNA的表 达都显著低于远癌部位组织（P<0.001），而肠癌组织中两个基因mRNA的表达 则显著高于远癌部位组织（P<0.001）。结合临床病理资料提示PAR4在淋巴结转 移的胃癌患者中的表达低于无淋巴结转移的患者（P<0.05），在胃窦癌中的表达 明显低于非胃窦癌（P<0.05）；而发生淋巴结转移的肠癌患者其TFF2和PAR4基 因的表达都显著高于无淋巴结转移的肠癌患者（P<0.05）；两个基因在中低分化 肠癌中的表达也显著高于高分化肠癌（P<0.001）。免疫组化结果也提示TFF2和 PAR4在胃癌中的表达显著低于周围远癌部位组织（P<0.001），而在肠癌中的表 达则显著高于周围远癌部位组织（P<0.001）。表明TFF2和PAR4在胃肠道肿瘤的 发生中可能受到某些因素的调节而协调性地一致性表达。 在细胞水平上，我们发现在同等浓度hTFF2的诱导下，过表达PAR4的Lovo 稳定株的细胞迁移能力较不表达者明显增强，并且hTFF2的促细胞迁移活性呈剂 量依赖性，同时伴随ERK1/2磷酸化的增强。同时，过表达PAR4的Lovo细胞增殖 能力强于无PAR4表达的细胞，但TFF2作用后其增强能力反而下降，表明TFF2 对过表达了PAR4的Lovo细胞具有抗增殖的能力。 总之这些结果提示PAR4和TFF2在胃肠道中协同表达的现实为两者之间产 生一定的作用提供了基础，而且这种共存为粘膜受损后的修复，组织自身平衡状 态的维持都发挥了一定的作用，同时也为临床相关疾病的诊断，治疗及预后提供 一个新的理论依据。当然，生理和病理情况下，存在于PAR4和TFF2之间的调控 和相互作用的分子机制仍不清楚，这也是进一步研究的关键所在。 为探讨其它动物体内三叶因子家族蛋白结构和功能的关系，我们进而利用原 核表达体系构建并表达纯化了Bm-TFF2以及它的两个单结构域。由于Bm-TFF2 分子中有三对二硫键，所以我们选用pET-32a表达体系表达融合的重组蛋白，并 利用融合蛋白N端引入的Xa因子酶切位点将融合蛋白中的硫氧还蛋白切除，亲和 柱及反向高压液相色谱纯化游离的重组蛋白。重组的Bm-TFF2全长具有血小板聚集活性，而第一个结构域只有诱导血小板变形的作用；三种重组蛋白都具有剂量 依赖性地诱导AGS细胞迁移的功能，但三种重组蛋白的细胞迁移活性无明显差 异。pET-32a表达体系成功表达Bm-TFF2的事实为我们研究人三叶因子家族蛋白 结构及功能关系提供一种方便而可靠的手段。

5-氟尿嘧啶-β-环糊精结肠靶向前体药物的研究

5-氟尿嘧啶(5-Fluorouracil， 5-FU)是一种抗代谢药物，广泛用于临床治疗结直肠癌、胃癌、乳腺癌等多种癌症，但其首过代谢显著、亲脂性较低，选择性差、毒副作用大。为克服这些缺点人们对5-FU进行了大量的修饰工作，包括小分子修饰以及与各种载体形成微球、微囊、纳米粒、共价前药等。 环糊精(Cyclodextrin，简称CD)，可被结肠中的糖苷酶特异性地降解成小分子糖，而胃和小肠中由于缺乏相应的酶而使环糊精不被降解，这一特性在结肠药物的靶向输送及释放中有重要应用价值。环糊精中含有丰富的羟基，易进行化学修饰，将药物与环糊精通过共价键结合制成前药，使其在胃和小肠中不降解，而在盲结肠中被特异性的酶降解释出药物，达到结肠靶向释药的目的。研究表明，环糊精作为一种前药载体为结肠靶向释药和缓释、控释系统提供了一种有效的手段。 本工作选择5-氟尿嘧啶为模型药物、β-环糊精作为载体，通过中间体5-FU羧酸衍生物的制备及其与β-环糊精的偶联，合成了系列5-FU-β-CD前体药物，并利用紫外、红外、质谱、核磁、元素分析、热分析等手段对其进行结构表征。同时，还研究了前体药物的体外释药性质。具体内容包括： 1. 含有羧基的5-FU衍生物中间体的合成：(5-氟尿嘧啶-1-基)-乙酸(FUAC)、3-(5-氟尿嘧啶-1-基)-丙酸(FUPC)、5-(5-氟尿嘧啶-1-基)-戊酸(FUVC)的合成。 2. 中间体5-FU的羧酸衍生物与β-CD的偶联：分别通过以6-OTs-β-CD为中间体的取代法和活化酯法，合成了第一面取代和第二面取代的5-FU-β-CD大分子前体药物。在二面取代的前体药物制备中，通过改变原料的比例，合成了系列不同取代度(DS)的2-[(5-氟尿嘧啶-1-基)-乙酰基] -β-环糊精结合物。 3. 对上述前体药物进行体外释放研究：分别考察了前体药物在不同pH缓冲溶液中的水解行为及其在小鼠胃肠道人工体液中的酶解行为，并通过UV-Vis及HPLC对前体药物释放情况进行检测分析。 5-Fluorouracil(5-Fu), commonly known as a broad-spectrum antineoplastic drug, has been widely used in the treatment of various kinds of cancer including colon cancer for 40 years. However, this antitumor agent exhibits serious adverse effects, such as their marrow toxicity, gastrointestinal reaction and low selectivity in their clinical use. In order to improve its antitumor activity and reduce its toxicity, the compound was modified in various ways, including the formation of conjugated prodrugs with kinds of carrier, microsphere and nanoparticles etc. Cyclodextrins(CDs) are known to be barely capable of being hydrolyzed and only slightly absorbed in passing through the stomach and small intestine; however they are fermented into small saccharides by colonic microflora and thus absorbed as small saccharides in the large intestine. This biodegradation property of CDs may be useful as a colon-targeting carrier, and thus CD prodrugs may serve as a source of site-specific delivery of drugs to colon. It was demonstrated that prodrugs of CDs can provide a versatile means for construction of not only colon targeted delivery systems, but also delayed release systems. 5-Fluorouracil was taken as a model drug and β-CD as the carrier in this study. Series prodrugs of 5-FU was prepared through the preparation of reactive 5-FU derivatives containing carboxyl group and coupling to hydroxyl groups of CD. The structures of the conjugates were charactered by using IR, UV–vis, ESI-MS, 1H, 13C-NMR spectra, elemental analyses, and thermal analysis. In vitro hydrolysis behavior in aqueous solution and in rat gastrointestinal tract contents of the conjugates were also investigated. The main content of this dissertation includes following aspects: 1. The preparation of 5-FU derivatives containing carboxyl group: 5-Fluorouracil- acetic acid(FUAC)、3-(5-FU-1)-propionic acid (FUPC)、and 5-(5-FU-1)-valeric acid(FUVC). 2. The coupling of 5-FU derivatives to β-CD: 5-FU was selectively conjugated onto the primary or secondary hydroxyl groups of β-CD through an ester linkage, by the substitution of 6-OTs-β-CD and the activated ester method respectively. For the secondary face conjugation, the degree of substitution(DS) can be controlled by changing the mole ratio of the starting materials(FUAC and β-CD). 3. In vitro release behavior of the conjugates in aqueous solution and in rat gastro- intestinal tract contents of the conjugates were investigated, and the reaction was monitored and analyzed by using UV-Vis and HPLC methods.

Studies on the Biotransformation of Arctigenin Using Electrospray Ionization Mass Spectrometry

To study the biotransformation of arctigenin, arctigenin was anaerobically incubated with Eubacterium sp. ARC-2 of human intestinal bacteria in vitro. Arctigenin formed a molecular ion [M-H](-) in negative ion mode. The arctigenin and its metabolites were investigated directly by the electrospray ionization tandem mass spectrometry ion trap and Fourier transform ion cyclotron resonance. Arctigenin was transformed to 4',4 ''-dihydroxylenterolactone by E sp. ARC-2 through 3 types of demethylation products.

Miméticos do “glucagon-like peptide-1” (GLP-1) e o seu potencial farmacêutico no controlo da diabetes tipo 2 e da obesidade

A diabetes mellitus do tipo 2 é caracterizada pela resistência à insulina e pela disfunção das células β do pâncreas. Os péptidos gastrintestinais, “gastric inhibitory polypeptide” (GIP) e “glucagon-like peptide-1” (GLP-1), são hormonas incretinas que estimulam, maioritariamente, a produção de insulina pós-prandial. Formulações contendo GLP-1 possuem um grande potencial no tratamento desta doença. Porém, o GLP-1 é eficaz apenas quando administrado por via parentérica. Para o tratamento da diabetes mellitus tipo 2 são usados análogos do GLP‑ 1 ou miméticos da incretina os quais são eficazes por via subcutânea. The pathogenesis of diabetes mellitus type 2 includes insulin resistance and progressive β-cell dysfunction. The gastrointestinal peptides, gastric inhibitory polypeptide (GIP) and glucagon‑like peptide-1 (GLP-1), are incretin hormones which are responsible for the major part of postprandial insulin secretion. Formulations containing GLP-1 have a great potential in the treatment of diabetes mellitus type 2. Nonetheless, GLP-1 is only efficient by continuous parenteral administration. GLP-1 analogues or incretin mimetics, exendine-4, are active after subcutaneous injection and can be used in the treatment of diabetes mellitus of type 2.

Talidomida

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

Relação entre a Infeção por Escherichia coli Aderente-Invasina e a Doença de Crohn

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