Zebrafish and medaka as models for bone research including implications regarding space-related issues

Summary. Teleost fish develop bones directly from mesenchymal condensations and from cartilage precursors. At the cellular level, the involved cell populations share many features with their mammalian counterparts. In addition, several genes are already described in fish showing high homology in amino acid sequence and expression with the corresponding genes of tetrapods that are involved in bone metabolism. Therefore, analysis of the underlying molecular mechanism in fish, in particular zebrafish and medaka, will increase the knowledge in teleosts. Furthermore, it will help to identify novel genes and regulatory pathways of bone homeostasis and skeletal disorders also in higher vertebrates, including disorders caused by altered gravity. Correspondence and reprints (present address): Department of Physiological Chemistry I, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Federal Republic of Germany.     

Physicochemical and structural characterization of a polyionic matrix of interest in biotechnology, in the pharmaceutical and biomedical fields

This paper reports on the swelling degree and the rheological and structural characteristics of a hydrogel composed by chitosan and xanthan. The latter is a polyionic hydrogel obtained by complexation between the both polysaccharides. The swelling degree has been found to be influenced by the time of coacervation, the pH of the solution of chitosan used to form the hydrogel and the pH of the swelling solution. The molecular weight and the degree of acetylation of the chitosan also influence the swelling degree of this matrix. The connectivity between chitosan and xanthan affects the swelling degree of this matrix. A rheological study has been carried out in order to understand the formation of the coacervate and of the subsequent hydrogel. The evolution of the storage modulus with time during the coacervation has allowed to optimize the time of coacervation required for a subsequently hydrogel, with desirable swelling degree. The kinetics has shown that (a) the coacervate is formed in two distinct steps and (b) the storage modulus of the hydrogel reaches a stable plateau. The values of the storage modulus have been correlated with the swelling degree. The microscopic characterization has shown the presence of a porous network with a fibrillar structure. To complete the characterization studies fine powder of this hydrogel has been used to determine the surface, perimeter, Feret diameter and sphericity factor distribution of dry and hydrated (swollen) particles.     

Zebrafish (Danio rerio) androgen receptor: Sequence homology and up-regulation by the fungicide vinclozolin

Steroid hormones regulate gene expression in organisms by binding to receptor proteins. These hormones include the androgens, which signal through androgen receptors (ARs). Endocrine disrupters (EDCs) are chemicals in the environment that adversely affect organisms by binding to nuclear receptors, including ARs. Vinclozolin, a fungicide used on fruit and vegetable crops, is a known anti-androgen, a type of EDC that blocks signals from testosterone and its derivatives. In order to better understand the effects of EDCs, further research on androgen receptors and other hormone signaling pathways is necessary. In this study, we demonstrate the evolutionary conservation between the genomic structure of the human and zebrafish ar genes and find that ar mRNA expression increases in zebrafish embryos exposed to vinclozolin, which may be evolutionarily conserved as well. At 48 and 72 h post-fertilization, vinclozolin-treated embryos express ar mRNA 8-fold higher than the control level. These findings suggest that zebrafish embryos attempt to compensate for the presence of an anti-androgen by increasing the number of androgen receptors available.     

TCDD诱发斑马鱼胚胎shh基因表达降低及其对下颌发育的影响

2,3,7,8-四氯-二苯基-对-二恶英(2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD)为毒性 很强的环境污染物质。用O-1．0μg／L的TCDD处理受精后24 h(24 hpf)的斑马鱼胚至观察时为止,进行了形 态学观察、基因阻断、原位杂交、TUNEL染色以及免疫学染色等实验。结果表明,TCDD处理后会引起斑马鱼 下颌短小,Shh基因在下颌部表达降低;而在芳烃基受体AhR功能阻断的胚胎中,TCDD并没有引起斑马鱼下 颌短小,同时也没有观察到Shh基因表达缺失;免疫学染色表明TCDD和Shh阻断物质Cyclopamine均可引起 斑马鱼下颌区域增殖细胞的减少。TCDD引起的下颌短小可能是首先引起以AhR为媒介的Shh表达缺失,进而 造成下颌部增殖细胞减少,最终引起下颌短小的发生     

Liquid storage of Asian elephant (Elephas maximus) sperm at 4 degrees C

The Asian elephant (Elephas maximus) population in the wild has been in decline for several decades and breeding in captivity has not been self-sustaining. The use of artificial insemination (AI) can help overcome many of the difficulties associated with breeding elephants in captivity; however, the ability to store semen for extended periods of time is critical to the successful application of AI to elephants. The objective of the present study was to assess the effects of four different semen extenders and the presence of egg yolk on the viability and motility of Asian elephant semen stored at 4 °C. High quality ejaculates (n=4) were collected from two Asian elephant bulls by rectal massage. Aliquots of each ejaculate were extended in four different diluents (Beltsville thawing solution (BTS); Tris–citric acid (TCA)/fructose-based; Beltsville F5 (BF5); dextrose-supplemented phosphate-buffered saline (PBS)) with or without egg yolk then cooled and stored at 4 °C. The percentages of viable (viability) and motile (motility) sperm were evaluated at 8, 24 and 48 h following collection. The addition of egg yolk significantly reduced the percentage loss in viability from initial collection to 48 h compared to extenders without egg yolk (17.0±8.2 versus 32.6±8.9 decline in percent viable sperm in the population, respectively; P<0.05). Extender and egg yolk affected (P<0.005) total motility and percent progressively motile sperm at all evaluation times during incubation. TCA + egg yolk maintained higher (P<0.05) levels of progressive motility compared to other extenders supplemented with egg yolk. These results indicate that Asian elephant semen extended in TCA diluent supplemented with egg yolk can maintain at least 50% viability and motility when stored at 4 °C for 48 h.     

马兜铃酸对斑马鱼胚胎肾毒性作用

选用肾发育完成(受精后3天)的斑马鱼(Danio rerio)胚胎,用马兜铃酸进行染毒处理,观察胚胎的表型变化及死亡情况,分析马兜铃酸对胚胎的毒性作用及规律;利用肾荧光观察及肾组织切片,观察马兜铃酸处理后胚胎肾形态和肾组织结构的改变情况;利用qPCR检测马兜铃酸处理前后nephrin的表达变化,初步探讨足细胞在马兜铃酸毒性作用中的功能状态。20μmol/L马兜铃酸处理24 h后,胚胎出现明显眼周水肿表现;在马兜铃酸高浓度组(40~80μmol/L),除眼周水肿外,胚胎血循环系统功能出现异常,表现为心率降低、血流缓慢甚至停滞;荧光显微镜下观察发现,马兜铃酸处理组胚胎肾出现肾小球囊性膨胀、前肾管囊性扩张和形态异常;切片显示马兜铃酸处理组胚胎肾组织结构受到损害,表现为肾小球结构疏松、囊性扩张,前肾管上皮细胞细胞排列松散、紊乱及管腔扩张样改变;qPCR结果,马兜铃酸处理组斑马鱼胚胎nephrin的表达水平比对照组显著降低(P0.01)。研究表明,马兜铃酸能损害斑马鱼胚胎肾结构和功能,其毒性作用与肾小球足细胞的功能改变有关。     

Cytological study of Rubenstrema exasperatum (Trematoda: Omphalometridae)

Cytological studies on Rubenstrensa exasperatum were carried out. The number of chromosomes in diploid cells (2n=16), and the morphological characteristics of the karyotype were determined. All the chromosomes (from Nos 2 to 8) had a well-defined short arm and, in accordance with the I^c values, were determined to be meta-submetacentric. Polymorphism was established in the length of the 2 bomologues of the 1st chromosome. This pair is probably linked with sex determination.     

Cryoenzymology: How to practice kinetic and structural studies

For a full understanding of an enzyme reaction pathway, one must identify the reaction intermediates and obtain their structures and rates of interconversion. It is impossible to obtain all this information under normal conditions. An approach is to work suboptimally, in particular at subzero temperatures. This is cryoenzymology, an approach that implies both kinetic and structural measurements on enzyme systems below 0°C. To work below 0°C one must add an antifreeze so cryoenzymology means perturbation by two agents: temperature and antifreeze, usually an organic solvent. Certain precautions are needed with these agents, which we will discuss here. In particular, we discuss the importance of choosing the right solvent: this requires extensive exploratory studies but it is the key for the successful practice of cryoenzymology. Each system has its particularities and its own ‘good’ solvent. Cryoenzymology is not only a way of reducing reaction rates, it is also a way of perturbing one's system. Thus, it is a method that allows for the accumulation of intermediates that cannot be observed under normal conditions by slowing down their kinetics of formation, by changes in rate limiting steps or by shifts in equilibria. We illustrate the usefulness of cryoenzymology by myosin and actomyosin ATPases and by creatine, arginine and 3-phosphoglycerate kinases. We also discuss recent results obtained by X-ray crystallography.     

Transcriptional and Translational Regulation of Zebrafish Connexin 55.5 (zf.Cx.55.5) and Connexin 52.6 (zf.Cx52.6)

Zebrafish connexin 55.5 (zf.Cx55.5) and connexin 52.6 (zf.Cx52.6) show highly restricted expression patterns in the nervous system. Both connexins are confined to subsets of neurons in the fish retina. In order to get initial answers to the questions of pattern definition in neuronal subsets, we elucidated molecular mechanisms responsible for their expression. Different upstream DNA fragments were subcloned into a pGL3-basic vector and transiently transfected in HeLa and N2A cells. Luciferase activity showed the presence of two putative promoter elements in zfCx55.5 and a promoter element in zfCx52.6 that showed different promoter activities in HeLa and N2A cells. Moreover, fusion constructs of zfCx55.5 with EGFP revealed the presence of a new isoform with an additional short exon I.     

Transcriptional and Translational Regulation of Zebrafish Connexin 55.5 (zf.Cx.55.5) and Connexin 52.6 (zf.Cx52.6)

Zebrafish connexin 55.5 (zf.Cx55.5) and connexin 52.6 (zf.Cx52.6) show highly restricted expression patterns in the nervous system. Both connexins are confined to subsets of neurons in the fish retina. In order to get initial answers to the questions of pattern definition in neuronal subsets, we elucidated molecular mechanisms responsible for their expression. Different upstream DNA fragments were subcloned into a pGL3-basic vector and transiently transfected in HeLa and N2A cells. Luciferase activity showed the presence of two putative promoter elements in zfCx55.5 and a promoter element in zfCx52.6 that showed different promoter activities in HeLa and N2A cells. Moreover, fusion constructs of zfCx55.5 with EGFP revealed the presence of a new isoform with an additional short exon I.     

The distribution and abundance of Lernaeocera lusci (Copepoda) on hake (Merluccius merluccius) and bid (Trisopterus luscus) (Teleostei)

Distribution patterns, mean intensity and prevalence of Lernaeocera lusci (Copepoda, Pennellidae) in its 2 hosts Trisopterus luscus (Teleostei, Gadidae) and Merluccius merluccius (Teleostei, Merlucciidae) were examined, together with parasitic abundance and aggregation in relation to the size of the host. The mean parasite abundance and the variance to mean abundance ratio increased with host size, suggesting that the accumulation of this parasite had no noticeable impact on the structure of either host population. Merluccius merluccius being a newly colonized host in the Mediterranean, these results are discussed in relation to current ideas on the evolution of pathogenicity in heterospecific associations.     

Isolation and Characterization of a Carbonic Anhydrase Homologue from the Zebrafish (Danio rerio)

We have isolated a 29,000-Da carbonic anhydrase (CA) protein from the zebrafish, Danio rerio, sequenced two peptide fragments, and tentatively identified it as a high-activity CA by inhibition kinetics. We have also characterized a 1,537-bp message whose deduced sequence of 260 amino acids matches that of the isolated protein. This CA is clearly an α-CA based on the similarity of its sequence to that of other members of the α-CA gene family. A phylogenetic analysis suggested CAH-Z diverged after the branching of the CA-V and CA-VII genes and prior to the duplications that generated the CA-I, CA-II, and CA-III genes of amniotes. This marks the first characterization of the mRNA and its protein product from the CA gene of a teleost. Received: 31 March 1996 / Accepted: 8 September 1996     

用细胞色素b部分序列研究斑马鱼的分子分类与系统发育

用细胞色素b特异性引物,对长尾斑马鱼、短尾斑马鱼、豹纹斑马鱼、食蚊鱼和青石斑鱼的总DNA进行PCR扩增、克隆、测序后与Genbank数据库的团头鲂、青鲻鱼、草鱼、金桔鱼的细胞色素6基因同源序列进行序列比较。结果显示,长度为426bp的细胞色素b同源序列的平均碱基组成是：26．7％A,26．1％c．16．6％G．30．5％T,密码子第三位点的碱基组成存在较大的偏倚,G的含量仅占6．4％。长尾斑马鱼、短尾斑马鱼和豹纹斑马鱼之间细胞色素b DNA序列变异很小,相似性高于99%。NJ法构建的分子系统树表明：豹纹斑马鱼先与长尾斑马鱼聚在一起,再与短尾斑马鱼聚为一组。本研究结果支持Frankel和David等人认为豹纹斑马鱼应该是Danio rerio的一个亚种,而不是单独的一个种的观点。     

斑马鱼鳃的光镜和透射电镜观察

应用光学显微镜和透射电镜对斑马鱼(Danio rerio)鳃的组织结构及鳃丝、鳃小片超微结构进行了观察。结果表明,斑马鱼有4对全鳃,鳃耙呈长锥状,鳃丝呈梳状排列在鳃弓上,鳃小片均匀排列在鳃丝两侧。鳃小片由上皮细胞、柱细胞、内皮细胞和毛细血管网组成,鳃小片基部和血管周围分布有泌氯细胞,胞内有丰富的线粒体和排泄小泡,根据线粒体形态特征和细胞质电子密度可将其分为两个亚型。黏液细胞通常与泌氯细胞对生存在,并且有通外的开口。斑马鱼鳃组织结构与其他硬骨鱼鳃结构相似,其结构和功能有密切的关系。     

N-(4-hydroxyphenyl)retinamide (Fenretinide) and nephrectomy alter normal plasma retinol-binding protein metabolism

We have reported previously that injecting vitamin A-deficient rats with N-(4-hydroxyphenyl)retinamide causes a significant reduction in the liver retinol-binding protein concentration and a 2 fold rise in the kidney retinol-binding protein concentration. This presumably reflects a rapid translocation of retinol-binding protein from the liver to the kidney through the plasma, although no rise in plasma retinol-binding protein is detected. In the present studies, nephrectomized rats were used to determine if retinol-binding protein accumulating in kidneys passes through the plasma. Bilateral nephrectomy in control rats caused the plasma retinol-binding protein concentration to approximately double by 5 hr postsurgery. However, nephrectomy plus N-(4-hydroxyphenyl)retinamide treatment did not result in an increase in the plasma retinol-binding protein concentration. Therefore, the lowering of liver retinol-binding protein concentration in response to N-(4-hydroxyphenyl)retinamide treatment was not accounted for by an accumulation of retinol-binding protein in the plasma compartment. Interestingly, the muscle retinol-binding protein concentration increased with nephrectomy plus N-(4-hydroxyphenyl)retinamide treatment. The ratio of muscle retinol-binding protein:plasma retinol-binding protein in vitamin A-deficient nephrectomized rats treated with N-(4-hydroxyphenyl)retinamide was significantly higher than in comparable rats treated with either carrier or retinol. We conclude that in vivo N-(4-hydroxyphenyl)retinamide induces the secretion of retinol-binding protein from the liver. Since the N-(4-hydroxyphenyl)retinamide-retinol-binding protein complex does not bind with transthyretin it rapidly leaves the plasma. In non-nephrectomized rats this complex is rapidly filtered by the kidney. Nephrectomizing rats causes the retinol-binding protein secreted in response to N-(4-hydroxyphenyl)retinamide to diffuse into interstitial fluid.     

Mutation scanning methods for the analysis of parasite genes

Molecular variation is widespread in parasite populations, and its analysis has important implications for studying aspects relating to the function and organisation of genes, and the taxonomy, phylogeny and population genetics of parasites. This article reviews some PCR-based mutation scanning techniques that have advantages over currently used DNA methods for the analysis of genetic variation in parasites. The review is technical and describes briefly the principles of relevant techniques, examines some of their advantages and disadvantages and gives several examples for possible applications.     

Basiepidermal nervous system in Nemertoderma westbladi (Nemertodermatida): GYIRFamide immunoreactivity

The Nemertodermatida are a small group of microscopic marine worms. Recent molecular studies have demonstrated that they are likely to be the earliest extant bilaterian animals. What was the nervous system (NS) of a bilaterian ancestor like? In order to answer that question, the NS of Nemertoderma westbladi was investigated by means of indirect immunofluorescence technique and confocal scanning laser microscopy. The antibodies to a flatworm neuropeptide GYIRFamide were used in combination with anti-serotonin antibodies and phalloidin-TRITC staining. The immunostaining revealed an entirely basiepidermal NS. A ring lying outside the body wall musculature at the level of the statocyst forms the only centralisation, the "brain". No stomatogastric NS has been observed. The GYIRFamide immunoreactive part of the "brain" is formed of loosely packed nerve fibres with multiple small neurones and a few large ones. The peptidergic and aminergic patterns of the NS do not correspond to each other: the former is more developed on the ventral side, the latter is more pronounced on the dorsal side. A pair of GYIRFamide immunoreactive nerve cords innervates the ventral side of the animal, the mouth and the male genital opening. The nemertodermatids studied to-date display no common NS pattern. Possible synapomorphies of the Acoelomorpha are discussed. The study demonstrates that the nemertodermatid NS possesses a number of plesiomorphic features and appears more primitive than the NS in other worms, except the Xenoturbellida. The bilaterian ancestor supposedly possessed only a basiepidermal nerve net and had no centralised brain-like structures and no stomatogastric NS.