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SUMMARYThe dense granule antigen 4 (GRA4) is known as an immundominant antigen of Toxoplasma gondii and, therefore, is considered as a vaccine candidate. For further evaluation of its vaccine effect, a recombinant plasmid and vaccinia virus, both expressing GRA4, were constructed, and a heterologous prime-boost vaccination regime was performed in a mouse model. The mice immunized with the heterologous prime-boost vaccination regime showed a high level of specific antibody response against GRA4 and a significantly high level of gamma interferon (IFN-gamma) production and survived completely against a subsequent challenge infection with a lethal dose of T. gondii. In addition, the formation of cysts was inhibited in the mice vaccinated with the heterologous prime-boost vaccination regime. These results demonstrate that the heterologous prime-boost vaccination regime using DNA and a vaccinia virus, both expressing GRA4, could induce both humoral and cellular immune responses and provide effective protection against lethal acute and chronic T. gondii infections in mice.

Asterosap, a group of equally active isoforms of sperm-activating peptides from the egg jelly of the starfish Asterias amurensis, functions as a chemotactic factor for sperm. It transiently increases the intracellular cGMP level of sperm, which in turn induces a transient elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)). Using a fluorescent Ca(2+)-sensitive dye, Fluo-4 AM, we measured the changes in sperm [Ca(2+)](i) in response to asterosap. KB-R7943 (KB), a selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), significantly inhibited the asterosap-induced transient elevation of [Ca(2+)](i), suggesting that asterosap influences [Ca(2+)](i) through activation of a K+-dependent NCX (NCKX). An NCKX activity of starfish sperm also shows K(+) dependency like other NCKXs. Therefore, we cloned an NCKX from the starfish testes and predicted that it codes for a 616 amino acid protein that is a member of the NCKX family. Pharmacological evidence suggests that this exchanger participates in the asterosap-induced Ca(2+) entry into sperm.

In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, namely ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for the induction of acrosome reaction. For the induction, ARIS alone is enough in high-Ca2+ or high-pH seawater, but, besides ARIS, the addition of either Co-ARIS or asterosap is required in normal seawater. Asterosap transiently increased both the intracellular pH (pHi) and Ca2+ ([Ca2+]i), while ARIS slightly elevated the basal level of [Ca2+]i. However, a sustained elevation of [Ca2+]i and acrosome reaction occurred if sperm were simultaneously treated with ARIS and asterosap. EGTA inhibited the sustained [Ca2+]i elevation and acrosome reaction. The sustained [Ca2+]i elevation and acrosome reaction were highly susceptible to SKF96365 and Ni2+, specific blockers of the store-operated Ca2+ channel (SOC). These results suggest that sustained [Ca2+]i elevation, mediated by the SOC-like channel, is a prerequisite for the acrosome reaction. In high-pH seawater, ARIS alone induced a prominent [Ca2+]i increase and acrosome reaction, which were similarly sensitive to SKF96365. The acrosome reaction was effectively induced by ARIS alone when pHi was artificially increased to more than 7.7. Asterosap increased pHi from 7.6 +/- 0.1 to 7.7 +/- 0.1. Furthermore, the sustained [Ca2+]i elevation and acrosome reaction, induced by a combination of ARIS and asterosap, were drastically inhibited by a slight reduction in pHi. Taking these results into account, we suggest that an asterosap-induced pHi elevation is required for triggering the ARIS-induced sustained [Ca2+]i elevation and consequent acrosome reaction.

In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, i.e. ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for inducing the acrosome reaction. Experimentally, ARIS and asterosap are sufficient for the induction. However, when sperm are treated only with asterosap, they become unresponsive to the egg jelly to undergo the reaction. In this study, we analysed the mechanism of the acrosome reaction, using sperm inactivation by asterosap as a clue. Asterosap causes a rapid and transient increase in intracellular cGMP through the activation of the asterosap receptor, a guanylyl cyclase, and causes an increase in intracellular Ca(2+). When sperm were pretreated with asterosap, the guanylyl cyclase seemed to be inactivated irreversibly by dephosphorylation. They were still responsive to ARIS but no longer to asterosap. However, in the presence of IBMX or zaprinast, inhibitors against phosphodiesterases (PDEs), they retained their capacity to undergo the acrosome reaction in response to the egg jelly or ARIS alone. IBMX and zaprinast suppressed the intracellular catabolism of cGMP, but not of cAMP. These results suggest that guanylyl cyclase and cGMP-specific, IBMX- and zaprinast-susceptible PDEs are involved in the regulation of the acrosome reaction.