Role of the C2A domain of synaptotagmin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal.

Squid synaptotagmin (Syt) cDNA, including its open reading frame, was cloned and polyclonal antibodies were obtained in rabbits immunized with glutathione S-transferase (GST)-Syt-C2A. Binding assays indicated that the antibody, anti-Syt-C2A, recognized squid Syt and inhibited the Ca(2+)-dependent phospholipid binding to the C2A domain. This antibody, when injected into the preterminal at the squid giant synapse, blocked transmitter release in a manner similar to that previously reported for the presynaptic injection of members of the inositol high-polyphosphate series. The block was not accompanied by any change in the presynaptic action potential or the amplitude or voltage dependence of the presynaptic Ca2+ current. The postsynaptic potential was rather insensitive to repetitive presynaptic stimulation, indicating a direct effect of the antibody on the transmitter release system. Following block of transmitter release, confocal microscopical analysis of the preterminal junction injected with rhodamine-conjugated anti-Syt-C2A demonstrated fluorescent spots at the inner surface of the presynaptic plasmalemma next to the active zones. Structural analysis of the same preparations demonstrated an accumulation of synaptic vesicles corresponding in size and distribution to the fluorescent spots demonstrated confocally. Together with the finding that such antibody prevents Ca2+ binding to a specific receptor in the C2A domain, these results indicate that Ca2+ triggers transmitter release by activating the C2A domain of Syt. We conclude that the C2A domain is directly related to the fusion of synaptic vesicles that results in transmitter release.

Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal.

Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles.

Intratumoral injection of an adenovirus expressing interleukin 2 induces regression and immunity in a murine breast cancer model.

Rodent tumor cells engineered to secrete cytokines such as interleukin 2 (IL-2) or IL-4 are rejected by syngeneic recipients due to an enhanced antitumor host immune response. An adenovirus vector (AdCAIL-2) containing the human IL-2 gene has been constructed and shown to direct secretion of high levels of human IL-2 in infected tumor cells. AdCAIL-2 induces regression of tumors in a transgenic mouse model of mammary adenocarcinoma following intratumoral injection. Elimination of existing tumors in this way results in immunity against a second challenge with tumor cells. These findings suggest that adenovirus vectors expressing cytokines may form the basis for highly effective immunotherapies of human cancers.

Increase of skeletal muscle relaxation speed by direct injection of parvalbumin cDNA.

Parvalbumin (PV) is a high affinity Ca(2+)-binding protein found at high concentration in fast-contracting/relaxing skeletal muscle fibers of vertebrates. It has been proposed that PV acts in the process of muscle relaxation by facilitating Ca2+ transport from the myofibrils to the sarcoplasmic reticulum. However, on the basis of metal-binding kinetics of PV in vitro, this hypothesis has been challenged. To investigate the function of PV in skeletal muscle fibers, direct gene transfer was applied in normal and regenerating rat soleus muscles which do not synthesize detectable amounts of PV. Two weeks after in vivo transfection with PV cDNA, considerable levels of PV mRNA and protein were detected in normal muscle, and even higher amounts were detected in regenerating muscle. Twitch half-relaxation time was significantly shortened in a dose-dependent way in transfected muscles, while contraction time remained unaltered. The observed shortening of half-relaxation time is due to PV and its ability to bind Ca2+, because a mutant protein lacking Ca(2+)-binding capacity did not promote any change in physiology. These results directly demonstrate the physiological function of PV as a relaxing factor in mammalian skeletal muscle.