MOLECULAR BIOLOGICAL AND CELL BIOLOGICAL STUDIES ON CYPRIS CEMENTATION OF BARNACLES

藤壶苏铁的永久附着是由特殊的粘连物质，即所谓的粘合剂释放出来的。这种水泥是由一对水泥腺分泌出来的。我们研究的最终目的是了解赛普里斯水泥是如何分泌的，以及赛普里斯水泥(S)是如何硬化的。在这里，我们致力于在分子和细胞水平上研究骨水泥释放和骨水泥物质的技术。我们开发的离体骨水泥腺体制剂对于研究藤壶鱼骨化的生理机制是非常有用的。从藤壶、大玫瑰、加勒比半条鱼和长嘴小鱼中分离出鲤鱼水泥腺，并对候选神经递质的有效性进行了测试。Cariosus和B.rostratus的骨水泥腺体对亚微摩尔多巴胺有反应，并表现出许多胞吐融合事件，这与M.rosa(1996年)的观察结果相似，表明多巴胺有可能成为…属。除藤壶外，还有更多的L神经递质参与骨水泥的分泌。分离的玫瑰花水泥腺在十二烷基硫酸钠裂解缓冲液中裂解，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分析，发现有4个主要组分，分别为36K、57K、110K和一个极高分子量组分。Cariosus水泥腺的格局基本相同，而长吻水泥腺没有36k的迹象，上移了57k和110k。我们还没有成功地确定这些是什么。100个分离的水泥腺的积累也使我们能够提取总RNA，这足以用于RT-PCR或其他分子研究。研究了鲤鱼水泥腺细胞膜上电压依赖性钙通道(VGCC)的电生理、药理和分子生物学特性。膜片钳全细胞构型显示骨水泥腺体上的VGCC具有高阈值、无失活特性和部分DHP敏感性。利用简并的引物(Jeziorski等人，1998年)，使用月见草鱼和月季水泥腺的总RNA进行RT-PCRi，随后的测序为VGCCα_1亚基的存在提供了证据，两种类型的VGCC是L型和P/Q型。这两个VGCC可能是玫瑰根茎永久性附着过程中钙离子诱导的粘固剂释放的原因。抑制消减杂交技术(SSH)用于检测玫瑰根茎幼虫阶段特异性表达序列标签(EST)。由于我们对鲤鱼骨水泥腺体的发育和骨水泥颗粒的增大很感兴趣，所以我们选择了两个特定的阶段--阶段6的无节幼虫和无节幼虫-鲤鱼蜕皮后的阶段作为试验者，而阶段4-5和阶段2的无节幼体分别被选为驱动者。从消减文库中进一步通过差异筛选筛选克隆，然后测序获得阶段特异性EST。到目前为止，阳性克隆包括BCS-1的同源物，藤壶鲤幼虫特异性基因-1，来自Amphitite的Balanus Amphitite(Okazaki&Shizuri，2000)。较少

The permanent attachment of the barnacle cyprids is initiated by the release of special adhesive substances, the so-called cement. The cement is secreted from a pair of cement glands. The ultimate goal of our studies is to understand how the cypris cement is secreted and how the cypris cement(s) harden. Here, we have concentrated on developing the techniques to study cement release and cement substances at molecular and cellular levels.The isolated cement gland preparation we have developed is very useful to study physiological mechanisms underlying barnacle cypris cementation. Cypris cement glands from the barnacles, Megabalanus rosa, Semibalanus cariosus, and Balanus rostratus were isolated and tested for the efficacy of neurotransmitter candidates. Cement glands from S. cariosus and B. rostratus responded at sub-micro molar dopamine and exhibited many exocytotic fusion events, similar to that observed in that of M. rosa (1996), suggesting that dopamine has a potential to be a genera … More l neurotransmitter for cement secretion beyond barnacle species.Accumulation of isolated cement glands allowed us to investigate the protein composition of the cement glands. Isolated M. rosa cement glands were lysed in SDS-lysis buffer and subjected to SDS-PAGE, which revealed 4 major components, 36K, 57k, 110k and a very high molecular one. The pattern of S. cariosus cement glands was basically the same, while B. rostratus cement glands showed no sign of 36k and upper shifts of 57 and 110k. We have not succeeded to identify what these are. Accumulation of one hundred of isolated cement glands also allowed us to extract total RNA, which was adequate for RT-PCR or other molecular studies.Electrophysiological, pharmacological and molecular biological properties of voltage-dependent Ca^<2+> channels (VGCC) on the membrane of cement gland cells of the cypris larvae of M. rosa were studied. Whole cell configuration of patch-clamp was used to reveal that VGCC on the cement glands had high threshold, non-inactivating characteristics with partial DHP sensitivity. RT-PCR with degenerate primer sets (Jeziorski et al., 1998) using mRNA from M. rosa cyprids or total RNA from M. rosa cement glands, and subsequent sequencing provided evidence for the presence of two types of VGCC α_1 subunits, L-type and P/Q type. These two VGCCs are presumably responsible for Ca^<2+> -induced cement release during permanent attachment of the cyprids of M. rosa.Suppression subtractive hybridization (SSH) was applied to detect larvel stage-specific expressed sequence tags (ESTs) of M. rosa. Because we were interested in the development of cypris cement glands and the enlargement of cement granules, two specific stages, stage 6 nauplius larvae and cypris larvae just after nauplius-cypid molt, were selected as testers, while stage 4-5 and stage 2 nauplii were selected as drivers, respectively. Clones from subtracted cDNA libraries were further selected by differential screening, and then sequenced to obtain stage-specific ESTs. So far, positive clones includes the homologue of bcs-1, barnacle cypris larva-specific gene-1, from Balanus amphitrite (Okazaki & Shizuri, 2000). Less