MOLECULAR BIOLOGICAL AND CELL BIOLOGICAL STUDIES ON CYPRIS CEMENTATION OF BARNACLES

藤壶类的永久附着是通过释放特殊的粘合剂物质，即所谓的水泥开始的。骨水泥由一对骨水泥腺分泌。我们研究的最终目标是了解塞浦路斯水泥是如何分泌的，以及塞浦路斯水泥是如何硬化的。在此，我们致力于发展从分子和细胞水平研究骨水泥释放和骨水泥物质的技术，我们所开发的分离的骨水泥腺制备物对于研究藤壶cypris骨水泥形成的生理机制非常有用。从藤壶，Megabalanus rosa，Semibalanus cariosus和Balanus rostratus中分离Cypris水泥腺，并测试神经递质候选物的功效。来自S. Cariosus和B. rostratus响应于亚微摩尔多巴胺，并表现出许多胞吐融合事件，类似于在M. Rosa（1996），表明多巴胺有可能成为一种 关于我们 l神经递质的水泥分泌超出藤壶物种。积累孤立的水泥腺允许我们调查的蛋白质组成的水泥腺。孤立的M。用SDS-裂解液裂解玫瑰花粘固腺，经SDS-PAGE电泳，显示4种主要组分，36 K、57 K、110 K和一种非常高分子量组分。S.龋齿骨水泥腺基本相同，而B.喙状骨水泥腺无36 K的迹象，57和110 K的上移。我们还没有成功地确定这些是什么。100个离体牙骨质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。玫瑰花研究全细胞膜片钳技术显示，牙骨质腺体上的VGCC具有高阈值、非失活特性，对DHP部分敏感。使用简并引物组的RT-PCR（Jeziorski等人，1998）使用来自M. rosa cyprids或M.结果表明，VGCC α_1亚基存在L型和P/Q型两种类型。这两个VGCC可能是在M的Cyprids永久附着过程中Ca^<2+>诱导的骨水泥释放的原因。应用抑制性消减杂交（SSH）技术检测家蚕幼虫期特异性表达序列标签（ESTs）。罗莎由于我们感兴趣的cypris水泥腺的发展和水泥颗粒的扩大，两个特定的阶段，阶段6无节幼体和cypris幼虫后无节幼体-cypid蜕皮，被选为测试者，而阶段4-5和阶段2无节幼体被选为驱动器，分别。从消减cDNA文库中进一步通过差异筛选选择克隆，然后测序以获得阶段特异性EST。到目前为止，阳性克隆包括bcs-1的同源物，藤壶cypris幼虫特异性基因-1，来自藤壶amphitrite（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