MOLECULAR BIOLOGICAL AND CELL BIOLOGICAL STUDIES ON CYPRIS CEMENTATION OF BARNACLES

藤壶塞的永久附着是由特殊粘合物质（所谓的水泥）的释放引起的。水泥是由一对水泥腺分泌的。我们研究的最终目的是了解骨水泥是如何分泌的以及骨水泥是如何硬化的。在这里，我们专注于开发在分子和细胞水平上研究水泥释放和水泥物质的技术。本研究开发的离体骨水泥腺对研究塞浦路斯藤壶骨水泥的生理机制具有重要意义。从藤壶、蔷花大虾、齿状虾和喙状虾中分离出塞氏水泥腺，并对候选神经递质进行了有效性测试。来自S. cariosus和B. rostratus的水泥腺体对亚微摩尔多巴胺有反应，并表现出许多胞外融合事件，与M. rosa（1996）的观察结果相似，这表明多巴胺有可能是除藤壶物种外的一种更广泛的水泥分泌神经递质。孤立水泥腺的积累使我们能够研究水泥腺的蛋白质组成。分离的玫瑰水泥腺体在sds -裂解缓冲液中裂解，并进行SDS-PAGE分析，结果显示有4种主要成分：36K、57k、110k和一个非常高分子的成分。齿状螺杆鱼骨水泥腺的形态基本一致，而喙状螺杆鱼骨水泥腺在36k和57k、110k的上移处无明显变化。我们还没有成功地确定它们是什么。积累100个分离的水泥腺也使我们能够提取总RNA，这足以用于RT-PCR或其他分子研究。本文研究了月牙蛾水泥腺细胞膜上电压依赖性Ca^<2+>通道（VGCC）的电生理、药理学和分子生物学特性。膜片钳的全细胞结构揭示了VGCC在水泥腺体上具有高阈值、非失活特性和部分DHP敏感性。Jeziorski等人，1998年使用退化引物集（Jeziorski et al., 1998）对玫瑰玫瑰水泥腺体的mRNA或总RNA进行RT-PCR，随后的测序证实了VGCC α_1亚基的两种类型，l型和P/Q型。这两种vgc可能是在玫瑰草的永久附着过程中Ca^<2+>诱导水泥释放的原因。采用抑制减法杂交（SSH）技术检测了玫蔷薇幼虫期特异性表达序列标签（ESTs）。由于我们对赛鱼水泥腺体的发育和水泥颗粒的增大感兴趣，我们选择了两个特定的阶段，即6期nauplius幼虫和刚换壳后的赛鱼幼虫作为测试者，而4-5期和2期nauplii分别作为驱动者。从缺失的cDNA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ESTs。到目前为止，阳性克隆包括来自Balanus amphitrite的bcs-1同源物，即塞浦路斯藤壶幼虫特异性基因-1 （Okazaki & Shizuri, 2000）。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