Culture of mantle epithelial cells expressing shell matrix proteins from scallop Patinopecten yessoensis

ABSTRACT:   In molluscs, mantle epithelial cells secrete organic matrix proteins to form shells. In this study, we established a culture of mantle epithelial cells by using the mantle pallial layer of scallops. We aimed to identify the mantle epithelial cells expressing scallop shell matrix proteins and establish a culture system of epithelial cells. After the mantle pallial layer was carefully isolated from the mantle tissue, explant culture was performed at 4°C. Most cells that migrated from the explant tissue were round cells. Most of the adhered cells retained round morphology, while some of the cells adhered to the dish and showed morphology similar to that of epithelial-like and fibroblast-like cells. When the cultured cells were immunostained with a polyclonal antibody against the shell matrix protein, the antibody recognized many of the adhered cells. An estimation of the number of epithelial cells revealed that approximately 70% of the adhered cells were epithelial cells. This is the first report to describe epithelial cells in cultured mantle cells, which express shell matrix proteins. This culture system may be a useful method for characterization of the mantle epithelial cells.     

Complete nucleotide sequence of a cDNA encoding a myosin heavy chain from mantle tissue of scallop Patinopecten yessoensis

ABSTRACT: It has been reported that the amino acid sequences of striated and catch muscle myosin heavy chains from two scallop species ( Argopecten irradians and Placopecten magellanicus ) are almost identical, but that the ATPase activities between these myosins vary several-fold. These myosin sequences have been useful for identifying the region that modulates the ATPase activity of scallop myosin. In the present study, a cDNA encoding a myosin heavy chain was isolated from the mantle tissue of scallop Patinopecten yessoensis . The cDNA is composed of 6067 base pairs (bp) including an open-reading frame of 5841 p, which encodes an amino acid sequence of 1947 residues. The deduced amino acid sequence of P. yessoensis mantle myosin had a high identity of 90%, 92%, and 91% to P. magellanicus , A. irradians , and Pecten maximus striated muscle myosins, respectively. Interestingly, while the deduced amino acid sequences of around adenosine triphosphate-binding and actin-binding sites of the mantle myosin are homologous to those of A. irradians striated muscle myosin, the subfragment 2 hinge region and the non-helical tail region are similar to those of catch muscle myosin.     

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.     

三角帆蚌类丝状基质蛋白基因silkmaxin的克隆及其在贝壳和珍珠生物矿化中的作用

贝壳基质蛋白指导了珍珠形成过程中碳酸钙的成核、晶体生长和晶型选择等关键过程。为进一步研究珍珠形成的分子机理,本实验使用RACE-PCR技术克隆得到一个新的贝壳基质蛋白基因,并命名为silkmaxin。组织表达分析和原位杂交分析表明,该蛋白于外套膜缘膜部外上表皮组织特异性表达,证明silkmaxin基因所编码的蛋白属于珍珠层基质蛋白。silkmaxin基质蛋白氨基酸序列富含甘氨酸(Gly,33.0%)和丝氨酸(Ser,10.4%),蛋白结构由β-折叠构成,类似丝状蛋白结构。分析珍珠形成早期珍珠囊中该蛋白基因的表达发现,silkmaxin基因在珍珠囊内碳酸钙沉积物从无序到有序的转变过程中起了重要作用。通过RNA干扰实验可知,贝壳基质蛋白是珍珠层文石小片正常生长不可缺少的因子,当silkmaxin基因表达被抑制,文石小片的成核、大小和形状均发生了改变。     

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.     

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.     

Inheritance of shell colours in the noble scallop Chlamys nobilis (Bivalve: Pectinidae)

The noble scallop Chlamys nobilis is polymorphic in its shell colour, which includes orange, orange‐purple, purple and brown. In this study, the inheritance of shell colours of this species was investigated by analysing shell colour segregation in juvenile scallops produced from cross‐fertilization. Totally, twenty two families derived from four types of parental crosses were produced. The results clearly demonstrated that shell colour in this species was consistently inherited but insensitive to the environmental factors given that each family was maintained in a common environment. The pattern of the four superficial shell colours in C. nobilis appears to be genetically controlled by the interaction of one pair of nonallelic genes, with one locus showing dominance epistasis to the other one. One‐locus‐three‐allele model was proposed to explain the genetic relationship of the orange‐purple colour dominant to the purple and brown colours, and the purple colour dominant to the brown colour. While the phenotypic ratio of 12:3:1 detected in one specific family showed that the dominant allele of the other locus controlling orange colour may mask the effect of the locus controlling orange‐purple, purple and brown colours. The present results provide a genetic basis for selective breeding in the noble scallop in terms of shell colour.     

Thermally induced gelation of paramyosin from scallop adductor muscle

ABSTRACT:   Thermally induced gelation of paramyosin from scallop smooth adductor muscle was investigated by dynamic rheological measurements under various conditions. The paramyosin thermal gel was produced at pH 6.5 and 7.2 at temperatures above 30°C through a two-step increase in storage (G') and loss (G') moduli; these values were higher than in gels produced from actomyosin at a high temperature. The thermal gel properties were very firm and brittle. In contrast, one main peak of G' was observed during gelation at pH 8.0. The gel produced at pH 8.0 was more transparent and less soluble in a 6 M urea−0.5 M NaCl solution than those formed either at pH 6.5 or 7.2. These differences in the thermal gel properties are presumed to derive from the pH dependence of the gel matrix-forming process, such as oxidative cross-linking between cysteine residues, rather than from the thermal unfolding of the paramyosin molecules. The thermal gelation profile of chymotrypsin-digested paramyosin showed marked depression of G' at high temperature.     

栉孔扇贝wnt4基因cDNA克隆及表达分析

由栉孔扇贝(Chlamys farreri)转录组数据库获得wnt4基因的一段表达序列标签(EST)序列,利用SMART-RACE技术克隆了wnt4基因cDNA全长序列,该序列长1 239 bp,其中开放阅读框为1 068 bp,可以编码355个氨基酸,推导的氨基酸序列含有WNT家族特有序列,且与沙蚕(Platynereis dumerilii)、海胆(Heliocidariserythrogramma)、人(Homo sapiens)等物种WNT4同源性都在60%以上。半定量RT-PCR结果显示,除肾脏外,wnt4基因在栉孔扇贝的精巢、卵巢、闭壳肌、肝胰腺、鳃、外套膜组织中均有表达,但表达量较低。定量RT-PCR结果表明:wnt4基因在成熟期的精卵巢中表达量最高,增殖期和休止期表达量次之,生长期表达量最低;整个生殖周期中精巢表达量高于卵巢。该基因在栉孔扇贝多个组织中的表达特性,暗示其参与了多样的生物学过程;在性腺中的表达特征表明其可能参与两性性腺的发育并在生殖细胞成熟过程中发挥作用。     

花刺参钙调蛋白cDNA克隆及组织分布

钙调蛋白（ calmodulin,CaM）是一种钙离子（ Ca2＋）结合蛋白,在钙信号传导过程中起重要作用。文章扩增到花刺参（Stichopus monotuberculatus）钙调蛋白（命名为StmCaM）cDNA全长序列。StmCaM cDNA全长1394 bp,其中5＇UTR长138 bp,3＇UTR长806 bp,ORF为450 bp。ORF推导出的StmCaM含149个氨基酸（包含起始蛋白酸）,其预测分子量约为16.7 kDa,理论等电点为4.1。StmCaM氨基酸序列与其他物种的钙调蛋白高度相似,相似度百分比为95.9%~98.0%。采用realtime PCR分析StmCaM基因在花刺参不同组织中的表达,结果显示其在呼吸树中表达量最高,体腔细胞、肠次之,而体壁中表达量最低,几乎检测不到。     

Gelation of low salt soluble proteins from scallop adductor muscle in relation to its freshness

ABSTRACT: The low salt extract from scallop striated adductor muscle contained extra proteins in addition to sarcoplasmic proteins and was turned into a gel upon standing or immediately by the addition of Ca²⁺. The amount of extra protein, which was composed of a large amount of actin and a small amount of myosin, decreased with a decrease in adenosine triphosphate (ATP) content in the muscle during storage at 5°C. Actin was easily extracted from scallop myofibrils in low salt solution with ATP at 1 mM or above. Furthermore, ATP was required to induce the gelation of the low salt extract. A visual observation of the gelation of low salt extract is therefore a simple and easy method to inspect prerigor state of scallop adductor muscle.     

Scallop shell extract promotes recovery from UV-B-induced damage in rat skin epidermal layer

ABSTRACT:   It has been previously reported that scallop shell extract can protect keratinocyte cells against UV-B-induced damage in vitro by acting as an antioxidant and a growth promoter. To establish the growth promoting activity of scallop shell extract, the effect was investigated in vivo. Exposure of rat dorsal skin to UV-B caused the formation of erythema and eschar. Either the scallop shell extract or vehicle (water) was applied once a day for 5 days to the injured dorsal area and the area of the erythema and eschar was estimated using National Institute of Health (NIH) image software. Wound healing of the erythema and eschar was clearly promoted after application of scallop shell extract when compared to application of the vehicle control. Histological studies indicated that scallop shell extract promoted the recovery of the epidermal layer. These results suggest that scallop shell extract activates rat keratinocyte cells and promotes the turnover of skin stratum corneum. This conclusion was also supported by measurement of the turnover rate of the stratum corneum. Therefore, scallop shell extract may be effective in protecting skin against UV-B.     

条斑紫菜细胞质甘油醛-3-磷酸脱氢酶的cDNA克隆和序列分析

利用已知甘油醛-3-磷酸脱氢酶(GAPDH)的特征性保守区域获得的探针筛选条斑紫菜cDNA文库,获得了1个全长的cDNA克隆GAP2,编码细胞质甘油醛-3-磷酸脱氢酶(GAPC)。其中GAP2 cDNA插入片段为1596bp,包括一个1008bp的开放阅读框编码336个氨基酸的细胞质甘油醛-3-磷酸脱氢酶(GAPC)蛋白。将获得的GAPC蛋白的氨基酸序列与其它物种的GAPDH序列进行多重序列比较后,发现条斑紫菜的GAPC氨基酸序列包含了4个高度保守的结构域。条斑紫菜GAPC的cDNA序列中GC含量很高,为64．2％,与紫菜EST分析中得到的结果(65．2％)近似。通过GAPDH的分子发育进化分析,对红藻的分类地位进行了初步探讨,条斑紫菜的细胞质甘油醛-3-磷酸脱氢酶的cDNA序列已登记到GenBank数据库,序列号分别为：AY273820。     

海湾扇贝外套眼结构的初步观察

海湾扇贝外套眼由短柄与外套膜边缘相连,是由一个皮下眼囊构成。最上面是单层立方上皮细胞构成的“外角膜”,胞质内无色素。眼囊内具有由细胞构成的“晶体”以及网膜层和色素层。“晶体”内细胞特别大,胞膜厚,胞质对ＰＡＳ反应呈强阳性,说明其内含有丰富的糖类,胞质对茚三酮－雪夫反应显示弱阳性,说明存在少量的蛋白成分。     

Crosses between two subspecies of bay scallop Argopecten irradians and heterosis for yield traits at harvest

In order to improve the yield of bay scallop Argopecten irradians by exploiting geographic subspecies heterosis, crosses between and within two subspecies, the northern A. i. irradians (Lamarck) and the southern A. i. concentricus (Say), were created and the offspring were cultured under the same environmental conditions. The two parental populations were collected from Bohai Bay in northern China and Beibu Bay in southern China, which were distinct in particular shell colours. The results indicated that the shell colour of bay scallop was inheritable and could be used as a morphological mark to distinguish hybrid and purebred individuals. At harvest, hybrid offspring always produced a significantly higher yield than purebred offspring. Positive heterosis was achieved for shell length, shell height, shell width, total weight and adductor weight, which was 5.02, 5.22, 3.88, 7.53 and 9.47 respectively. Hybrid gain was 10.86% for shell length, 10.48% for shell height, 9.77% for shell width, 34.90% for total weight and 41.69% for adductor weight, respectively, and all were significantly >5%. The present study reveals that it is effective for improving yield by hybridization between different geographic subspecies in the bay scallop.