| Research Abstract |
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
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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
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