Purification and characterization of a matrix shell protein from the shell of scallop <Emphasis Type="Italic">Patinopecten yessoensis</Emphasis>

ABSTRACT:   A new protein named MSP-SC (matrix shell protein from scallop) with a molecular weight of 14 kDa was isolated from the shell of a scallop, Patinopecten yessoensis , using gel filtration column chromatography, ion exchange column chromatography, and reverse phase C4 column chromatography. A comparison of the known protein sequences with the N-terminal sequence of MSP-SC showed that the protein sequence of MSP-SC was novel. Immunohistochemistry using a polyclonal antibody against MSP-SC showed that MSP-SC is expressed in the mantle pallial cell layer but not in the muscle tissue, and showed a punctate distribution along the horizontal calcified layer in the shell. The isolated MSP-SC inhibited the formation of calcium carbonate (CaCO₃) crystals in a dose-dependent manner. The CaCO₃ crystals grown in the presence of a lower concentration of MSP-SC were much larger and aggregated when compared with those formed in the absence of MSP-SC. In addition, the crystal had a radial and not cubical morphology. These results suggest that MSP-SC regulates the formation and the morphology of CaCO₃ crystals in the shell. Moreover, its ability to aggregate CaCO₃ crystals and its localization along the horizontal calcified layer in the shell suggest that MSP-SC may serve to connect the CaCO₃ layers in the scallop shell.     

Properties of scallop mantle collagen: its content,tissue distribution and thermal behavior

ABSTRACT:   To obtain fundamental information for the effective use of scallop Patinopecten yessoensis mantle, which is one of the underutilized marine resources. Some properties of collagen contained in the mantle were examined by chemical and histochemical techniques. Collagen content in the mantle varied annually, ranging from 0.98 to 1.72% of wet tissue, 7.7 to 12.6% of dry tissue and 13.5 to 26.5% of total protein, being relatively in high level of collagen content of invertebrate muscles. Collagen fiber was densely distributed in the inner connective tissue matrix of the mantle pallial, in contrast to the inner fold part which was rich in muscle fibers. The collagen contained in the crude collagen fraction (residue after alkali extraction), prepared from the mantle, was revealed to have considerably low solubility on hot-water extraction, constantly less than 20% of the total collagen at the temperatures in the range of 20–90°C.     

三角帆蚌HcCUBDC基因cDNA的全长克隆与表达分析

利用cDNA末端快速扩增（Rapid Amplification of cDNA Ends,RACE）方法,获得了三角帆蚌HcCUBDC基因的全长cDNA序列,共5158bp,开放阅读框（ORF）1920bp,编码639个氨基酸,5′端非编码区576bp,3′端非编码区2662bp,GeneBank登陆号为KP067952。生物信息学分析表明,三角帆蚌HcCUBDC蛋白含有一个由19个氨基酸组成的信号肽和4个CUB结构域,但未能比对上已知的蛋白。经荧光定量PCR检测,HcCUBDC基因在紫色和白色蚌前端缘膜、后端缘膜、中央膜、鳃、斧足、肝胰腺、肠和肾8个组织中均有表达,并且都是肝胰腺表达量最低。在紫色蚌中,后端缘膜表达量极显著高于前端缘膜;在白色蚌中,前端和后端缘膜之间表达差异不显著。HcCUBDC基因在紫色蚌后端缘膜中的表达量极显著高于白色蚌,在紫色和白色蚌前端缘膜中的表达量差异不显著。外套膜原位杂交结果显示,HcCUBDC基因主要在缘膜外上皮细胞中表达。研究表明,HcCUBDC基因在三角帆蚌内壳色形成中发挥作用,可为进一步深入研究该基因在珍珠颜色形成过程中的功能及其调控机理提供基础资料。     

体外培养三角帆蚌外套膜细胞的生物学特性及有核珍珠培育

通过光学显微镜、透射电镜、流式细胞仪等方法对体外培养的三角帆蚌(Hyriopsis cumingiiLea)外套膜细胞进行观察和检测,证明其具有与蚌体内细胞相同的生物学结构、分裂增殖能力和分泌珍珠质的生物活性。在此基础上,对三角帆蚌有核珍珠培育技术进行探讨。将培养细胞黏附在表面经过促黏壁物质处理的珠核表面,再插入育珠蚌体内培育,120 d后,蚌体内可形成完备的珍珠囊,并在珠核表面有明显的珍珠质沉积;6个月后,珍珠质可将整个珠核包裹起来形成有核珍珠。本研究初步证明了细胞法培育有核珍珠的可行性。[中国水产科学,2007,14(1):149-154]     

福寿螺足组织和外套膜组织细胞培养的初步研究

采用改良的M199培养基,对福寿螺(Ampullaria gigas Spix)足组织和外套膜组织细胞进行了体外培养研究。结果显示:接种6 h后,细胞开始从组织块中迁出,培养至第3天,迁出细胞在组织块周围形成生长晕,第21~28天,细胞覆盖大部分培养瓶底壁。足组织和外套膜组织细胞分别被传至8代和9代。足组织和外套膜组织细胞在原代培养物中主要有两类,一为上皮样小细胞,形态为圆形、椭圆形或多边形,直径7~15μm;另一类为上皮样中型和大型细胞,形态椭圆形或多边形,直径15~30μm。上皮样小细胞数量多,增殖速度较快,在原代和传代培养的中后期皆可形成细胞单层。外套膜组织细胞的生长和增殖能力高于足组织细胞,其贴壁性也好于足组织细胞,Hoechst荧光染色显示外套膜组织细胞的细胞凋亡指数明显低于足组织细胞。结果表明,贝类外套膜组织有潜力成为建立连续细胞系的组织来源。     

Pearl formation in the Japanese pearl oyster (Pinctada fucata) by CaCO3 polymorphs: Pearl quality‐specific biomineralization processes and their similarity to shell regeneration

Pearl oyster shell consists of two layers: a calcite prismatic layer (outer layer) and an aragonite nacreous layer (inner layer). Calcite and aragonite are CaCO₃ polymorphs, and their formations are controlled by shell‐forming tissue called mantle. Pearl sacs originating in the mantle form cultured pearls. Therefore, it has been widely accepted that pearl and shell are produced by the same process. However, this idea has been called into question by some recent mineralogical studies indicating microstructural and crystal‐polymorphic diversity in pearls. The pearl biomineralization process is still not fully understood in detail. Thus, in this study, we focused on the diversity of CaCO₃ polymorphism of non‐nacreous structures (NNSs) underlying the nacreous layer in pearl and regenerated shell, to reveal the biomineralization process of the Japanese pearl oyster (Pinctada fucata). Using Meigen's stain and scanning electron microscope‐energy dispersive X‐ray (SEM‐EDX), NNSs polymorphs in valuable and valueless pearls, in addition to regenerated shell, were compared. Aragonite was exclusively observed in the NNSs of valuable pearls, whereas calcite was dominant in those of valueless pearls. The same analysis of NNSs of regenerated shells was carried out. As in valueless pearls, almost all regenerated shell NNSs consisted of calcite, but one NNS was composed of aragonite. Accordingly, it seems that pearls are formed by the same biomineralization process as shell regeneration rather than shell formation.     

三角帆蚌贝壳基质蛋白研究进展

贝壳和珍珠是碳酸钙在有机基质调控下形成的结构高度有序的生物矿化物。贝壳有机基质包括贝壳基质蛋白、糖和少量的脂类,其中贝壳基质蛋白对贝壳和珍珠的形成过程具有重要的调控作用。三角帆蚌是我国重要的淡水育珠蚌,三角帆蚌贝壳基质蛋白的研究对于揭示淡水珍珠形成机理和培育高品质淡水珍珠具有重要意义。本文介绍了已发现的与三角帆蚌棱柱层和珍珠层形成相关的26个基质蛋白,包括氨基酸组成、一级结构、高级结构等结构特征,以及参与贝壳碳酸钙沉积、贝壳有机框架形成、晶体形貌调控、贝壳着色调控及与珍珠重量性状的关联性等生物矿化功能方面的最新研究进展,为进一步提高珍珠质量提供参考。     

In vivo adherence of Flavobacterium psychrophilum to mucosal external surfaces of rainbow trout (Oncorhynchus mykiss) fry

The adherence of Flavobacterium psychrophilum to surfaces of epithelial tissues has been inconclusively suggested as a mechanism, which enables the bacterium to invade the host. Hence, the present study aimed to examine the adherence of the cells of two colony phenotypes, smooth and rough, of F. psychrophilum to mucosal tissues of rainbow trout fry and to test the skin mucus as a nutrient for the growth of F. psychrophilum. Fish were immersed in water containing 10⁶ CFU mL^?1 F. psychrophilum for each colony phenotype. Mucosal tissue samples from fins, gills, skin and eyes, and swab samples from spleen and kidney were taken and inoculated onto TYES agar plates. Colony phenotypes of F. psychrophilum were identified and number of colonies counted. The results showed that cells of both phenotypes initially (0 h) adhered to all mucosal surfaces, but only the rough cells were still present on tissues 1 h post‐immersion. Both phenotypes showed a tissue tropism with the fin tissue being the most adhered. Furthermore, skin mucus promoted the growth of both colony phenotypes. We suggest that the growth of F. psychrophilum cells in skin mucus apparently facilitates the bacterial adherence to mucosal surfaces, and the subsequent invasion into the host.     

How cultured pearls acquire their colour

Round nucleated pearls are produced through a surgical operation, where a round nucleus and a mantle tissue ‘saibo’ from donor oyster are inserted into the gonad of the host oyster. The epithelial cells in the mantle tissue proliferate around the nucleus, and thus, the pearl sac is formed. Pearl sac secrets nacre and forms a pearl. The quality and economic value of pearls are assessed by pearl features such as colour, brightness, lustre and shape. Among all these features, colour has been reported as an important economic indicator and has been widely studied by researchers. Generally, pearl colour is affected by the donor oyster which is determined genetically and biological pigments (melanin and carotenoid). Organic matrices, metal ions and other factors have also been reported to influence the colour of a cultured pearl. Recently, multi‐omics methods have been used to study the colour formation of pearl, and some key genes and signal pathways related to the colour formation of pearls have been identified. Nevertheless, the specific mechanism of pearl formation needs further research. The review combines both fresh and sea water pearls focusing on Hyriopsis cumingii and pearl oysters to provide a general overview and understanding for pearl colour formation.     

育珠对合浦珠母贝N19和Prismalin-14基因表达水平的影响

为探讨育珠对壳基质蛋白基因表达的影响,开展了合浦珠母贝（Pinctada fucata）N19和Prismalin-14基因在育珠贝和非育珠贝中的荧光定量表达研究。结果显示,N19和Prismalin-14在育珠贝与非育珠贝的闭壳肌、肝胰脏、性腺、肠、鳃等组织中均无表达,在外套膜均有表达;N19在珍珠囊表达,而Prismalin-14则不表达。N19勺Prismalin．14在育珠贝外套膜的表达量均大于非育珠贝,表明育珠可能对某些壳基质蛋白的表达具有一定的促进作用。其中Prismalin-14在育珠贝外套膜的表达量最高（P〈0．01）,是非育珠贝外套膜Prismalin-14的1．23倍,是育珠贝外套膜N19的25．04倍,是非育珠贝外套膜N19的41．04倍,推测在珍珠贝生长过程中,对Prismalil7-14的需求量可能比N19大。N19在育珠贝珍珠囊中表达量最高（P〈0．01）,是育珠贝N19外套膜的11．58倍,是非育珠贝外套膜的18．97倍,推测在珍珠形成的过程中,对N19的需求量可能比贝壳自身生长对N19的需求量大。     

The effects of scallop shell extract on collagen synthesis

ABSTRACT:   Previous observations showed that scallop shells contain organic components that have various useful applications for skin. In this study, the effect of the organic components of scallop shell (scallop shell extract) on collagen metabolism is investigated. Collagen metabolism is tightly controlled by the collagen degrading matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP). Treatment of human skin fibroblast cells with the scallop shell extract increased the mRNA expression levels of type I collagen, MMP-1 and TIMP-1, suggesting that the scallop shell extract may activate collagen metabolism in skin fibroblast cells. Sirius red staining and the colorimetric quantification of collagen in fibroblast cells demonstrated that the scallop shell extract increased collagen content by approximately 1.3-fold. In vivo studies also revealed that the topical application of the scallop shell extract to rat dorsal skin increased the collagen content in the skin tissue section. These results suggest that the scallop shell extract may be effective for the treatment of photoaged and aging skin, which undergo collagen loss.     

Invasion and survival of Photobacterium damselae subsp. piscicida in non-phagocytic cells of gilthead sea bream, Sparus aurata L.

Fluorescence microscopy and gentamicin protection assays were used to investigate the ability of four Photobacterium damselae subsp. pisicida (Phdp) strains to adhere to and to invade the fish epithelial cell line, SAF-1, derived from Sparus aurata . All strains tested were detected intracellularly using both techniques, although internalization levels varied among strains. Treatment with cytochalasin D and experiments carried out at 4 °C demonstrated that a functional host cell cytoskeleton and active cell metabolism are necessary for bacterial internalization. Intracellular bacteria were detected for up to 7 days with a round morphology and were stained with DAPI, indicating that some bacterial cells may remain viable inside SAF-1 cells. Our in vitro findings indicate that Phdp are capable of adhering, entering and surviving within the non-phagocytic epithelial cell line SAF-1, which may be important for persistence and establishment of a carrier state in S. aurata .     

海湾扇贝一种球形病毒的形态发生及细胞病理学观察

2001年3-5月,青岛海区养殖海湾扇贝发生外套膜"糜烂病"并导致约50%亲贝死亡,主要表现为外套膜糜烂;严重者,约2/3的外套膜溃烂呈胶水状.电镜检测发现病贝体内感染有病毒等病原微生物.本文报道了该病毒粒子的形态、发生及宿主细胞由此所产生的细胞病理学变化.成熟的病毒粒子近球形,直径150～180nm,具囊膜,在细胞核附近的溶酶体内发生和增殖.发生初期溶酶体内形成板层髓样结构,随后形成块状、泡状、絮状等多形态的病毒发生基质及具有正方形花样的蛋白质晶格结构.最后,大量的病毒粒子装配形成,填充在溶酶体内.该病毒粒子主要存在于消化盲囊上皮细胞及结缔组织细胞的胞质中.受感染的宿主细胞线粒体肿胀、嵴溶解,内质网肿胀、核糖体脱落,溶酶体数量增多,核膜膨胀、溶解等,大部分细胞器受损.     

鲁西黄牛耳缘组织成纤维细胞的体外培养

实验以鲁西黄牛耳缘组织为研究材料,采用组织块贴壁培养法对其进行了成纤维细胞的体外培养。通过细胞的传代培养、形态学和动力学观察与分析,结果表明,所建立的鲁西黄牛耳缘组织成纤维细胞系具有典型的成纤维细胞形态,胞体均为梭形和不规则三角形,且细胞丰满;细胞核形态为卵圆形,并位于胞质中央,核仁清晰。在细胞生长于繁殖过程中亦经历了潜伏期、指数增生、停滞期和衰退死亡期等4个时期。细胞群体倍增时期为72h。实验结果表明,已成功地培养了鲁西黄牛耳缘组织成纤维细胞。     

Identification of overwintering vegetative cells of the bivalve-killing dinoflagellate <Emphasis Type="Italic">Heterocapsa circularisquama</Emphasis> in Uranouchi Inlet,Kochi Prefecture,Japan

ABSTRACT:   Red tides of Heterocapsa circularisquama have led to serious damage of bivalve aquacultures in western coastal areas of Japan. To understand the whole picture regarding the ecology of this species, it is essential to clarify its overwintering mechanisms. In this study, the population dynamics of H. circularisquama were investigated from February 2004 to November 2005, and overwintering cells were identified for the first time in water columns of Uranouchi Inlet, Kochi Prefecture, Japan. Heterocapsa circularisquama cells were detected by the indirect fluorescent antibody technique using monoclonal antibodies that specifically recognize and react to this species. Vegetative cells were almost always detected from the first observation in February 2004 to November 2005 with temperatures of 10.5–30.6°C. During the period from winter to spring, this species survived in areas with a temperature higher than 10°C. The overwintering cells of H. circularisquama were isolated in March 2004, and identification was made via observation of the morphology and body scales of the cultured cells. These overwintering cells were identified as H. circularisquama and reacted to the monoclonal antibody. These results indicate that H. circularisquama can overwinter and survive throughout the year in a vegetative cell state in Uranouchi Inlet.     

Association of zinc with connective tissue in the digestive tract of common carp

ABSTRACT:   Zinc (Zn) concentration in the digestive tract of common carp is always >10 times higher than most animal tissues. In a previous paper, it was reported that this high Zn came from a 43 kDa Zn-binding membrane protein. In this present study it was further found that in the digestive tract of common carp, Zn content was closely associated with the amounts of extracellular macromolecules. The higher the Zn content, the more there is of collagen and glycosaminoglycans. An indirect immunoperoxidase staining method using antibody against the 43 kDa Zn-binding protein, or the fibroblast marker (Thy 1.1 protein) was applied to the sections of digestive tract of fish. It was found that the Zn-binding protein in the digestive tract of common carp is mainly located in the connective tissue of its lamina propria and submucosal layer. Connective tissue cells, probably fibroblasts, hold the 43 kDa Zn-binding protein. In the common carp Zn might be bound to the external side of the fibroblast. The present finding may have a significant meaning on extending the studies of Zn in biology to the field of Zn with extracellular matrix.     

Fine structure and differentiation of the alimentary canal in captive-bred Japanese eel Anguilla japonica preleptocephali

ABSTRACT:   The fine structure of the alimentary canal in preleptocephali produced by artificially matured Japanese eel was examined. At 1 day posthatch (dph), the alimentary canal was found only above the dorsal side of the yolk mass, and the epithelium was composed of a single layer of epithelial cells. By 5 dph, the alimentary canal was divided into three segments based on the structure of the epithelial cells: foregut, midgut and hindgut, corresponding to the future esophagus, intestine and rectum, respectively. After 7 dph, the epithelium in the foregut was surrounded by a circular muscle layer, suggesting a role in the transportation of food materials. The epithelial cells of the midgut exhibited well-developed membranous structures, which are deduced to be invaginations of the cytoplasmic membrane. Pinocytotic invaginations and vacuoles were observed in the epithelial cells of the hindgut; this observation suggests that this region is involved in the uptake of food. Significant changes in morphological features of the epithelial cells in each segment were observed until 7 dph; however, these were not evident between 7 dph and 13 dph. Consequently, the differentiation of the alimentary canal was completed by 7 dph, and preleptocephalus had developed the ability to absorb food by 7 dph.     

Photoprotective activity of scallop shell water-extract in keratinocyte cells

ABSTRACT:   The photoprotective activity of the scallop shell water-extract was investigated using cultured rat skin keratinocyte cells. It was found that the scallop shell water-extract protected keratinocyte cells against ultraviolet (UV)-B-induced damage. The scallop shell water-extract had protective effects against hydrogen peroxide (H₂O₂) in skin keratinocyte cells and also showed 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and lipid peroxidation inhibitory activity. In addition, the scallop shell water-extract promoted proliferation of keratinocyte cells. The scallop shell water-extract may protect skin keratinocyte cells against UV-B-induced damage through two factors; antioxidant activity and growth-promoting activity in skin keratinocyte cells. These results suggest utilization of the scallop shell water-extract as materials for protecting skin.     

A STUDY ON THE MASS MORTALITY OF MUSSELS IN THE LAGUNA VENETA

A digenetic trematode, Cercaria tenuans, was the probable causative agent of an extensive mortality of cultured mussels (Mytilus edulis = gallo-provincialis) in the southern part of the Laguna Veneta, Italy. Sporocysts of C. tenuans occurred in the mantle tissue, the visceral mass and in the extra-pallial fluid space between the mantle and the shell. Germballs and apparently viable cercaria, or daughter sporocysts, were observed in the sporocysts. Other microscopic parasites were found in low numbers.