Four monoclonal antibodies against the calcium ATPase in sarcoplasmic reticulum (SR) of rabbit fast-twitch skeletal muscle were characterized using SDS-PAGE, Western blots and immunofluorescence. The ultrastructural distribution of the antigens was determined using post-embedding immunolabeling. The antibodies recognized the calcium ATPase in the SR but not in transverse (T-) tubule or plasma membranes. The antibody, D12, had the same binding affinity for the calcium ATPase from fast-twitch (rabbit sternomastoid) and slow-twitch (rabbit soleus) fibers and the affinity fell by 30% after fixation for electron microscopy in both types of muscle fiber. Ultrastructural studies revealed that the density of D12 antibody binding to the terminal cisternae membrane of extensor digitorum longus (edl) and sternomastoid fibers was on average seven times greater than in the slow-twitch soleus and semimembranosus fibers. Since the affinity of the ATPase for the antibody was the same in SR from fast- and slow-twitch muscles, the concentration of calcium ATPase in the terminal cisternae membrane of fast-twitch fibers was seven times greater than in slow-twitch fibers. This conclusion was supported by the fact that the concentration of calcium ATPase in light SR membranes was six times greater in SR from fast-twitch fibers than in SR from slow-twitch fibers. The results provide strong evidence that the different calcium accumulation rates in mammalian fast- and slow-twitch muscles are due to different concentrations of calcium ATPase molecules in the SR membrane.

Induction of HIV-specific T-cell responses by vaccines may facilitate efficient control of HIV replication. Plasmid DNA vaccines and recombinant fowlpox virus (rFPV) vaccines are promising HIV-1 vaccine candidates, although delivering either vaccine alone may be insufficient to induce sufficient T-cell responses. A consecutive immunization strategy, known as "prime-boost," involving priming with DNA and boosting with rFPV vaccines encoding multiple common HIV antigens, is used to induce broad and high-level T-cell immunity and ameliorate AIDS in macaques. This vaccine strategy is proceeding to clinical trials. This chapter describes the use of prime-boost vaccines to induce T-cell responses against HIV-1 and protective immunity against AIDS in macaques. Methods for the construction of the vaccines, the use of animal models, and the detection of immune responses are described.

Cajal (1911) noted that bistratified amacrine cells were common in non mammalian species and extremely rare in the mammalian retina. An examination of the marsupial retina of the tammar wallaby, stained with a modified Golgi procedure, revealed that a particular type of bistratified amacrine was frequently impregnated with the silver stain. Flat mount and transverse sections showed that the morphology of this cell did not correspond with any of the species-dependent bistratified amacrines reproduced in Cajal's drawings. Instead, the cell appeared to be almost identical to the AII or rod amacrine that has been observed in a number of mammalian retinas. The relative frequency with which the cell appears in our material, and its confirmed rod input in other species, are both consistent with the grazing habits of the tammar wallaby which is a crepuscular animal that does most of its feeding at dusk and after dark.

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To investigate the role of nitric oxide during vaccinia virus (VV) infection of mice, a recombinant VV encoding the inducible nitric oxide synthase (iNOS) gene (VV-HA-iNOS) was constructed. Following infection of immunocompromised or immunocompetent mice, the virus was highly attenuated compared with a control recombinant VV. Athymic and sublethally irradiated mice survived infection with 10 7 PFU of VV-HA-iNOS, a dose that resulted in uniform mortality in mice infected with the control recombinant VV. Attenuated virus growth was evident as early as 24 h following infection, suggesting that NO had direct antiviral activity. We have previously shown that treatment of mice with the inhibitor of NO production N G-methyl-L-arginine did not influence the course of VV infection in mice. The present study has indicated that NO can potentially exert an antiviral effect during murine VV infection. We propose that during VV infection, nitric oxide production contributes to the control of virus growth, but that in its absence, other antiviral mechanisms are sufficient to mediate fully effective virus clearance. MATERIALS AND METHODS Mice. CBA/H and Swiss athymic nude mice were obtained from the Animal Breeding Establishment of the John Curtin School of Medical Research. The mice were bred under specific-pathogen-free conditions and were used at 6 to 8 weeks of age. Materials. The human osteosarcoma cell line 143B (48), the monkey kidney cell line CV-1 (24), the murine fibroblast line L929 (50), and the Moloney leukemia virus-induced murine lymphoma line YAC-1 (29) were grown in Eagle's minimum essential medium (GIBCO, Grand Island, N.Y.) supplemented with 5% fetal calf serum, 10 mM HEPES (N-2-hydroxyethylpiperazine-NЈ-2ethanesulfonic acid), 1 mM glutamine, and antibiotics. N G-methyl-L-arginine (NMA) was synthesized as previously described (46). Purity was assessed by thin-layer chromatography and microanalysis (percentages of C, H, and N within 0.2% of required values). NMA dose-dependently inhibited NO production (see Fig. 4B). Construction of VV-HA-iNOS. Murine macrophage iNOS cDNA in plasmid CL-BS-mac-NOS (38) was provided by Charles Lowenstein (Johns Hopkins University, Baltimore, Md.). A 4,100-bp fragment containing the iNOS cDNA was excised from CL-BS-mac-NOS by a NotI digest, and this fragment was subjected to a further HincII digest to remove 136 bp from the 5Ј end. The protruding 5Ј ends of the DNA were filled by using the Klenow fragment of Escherichia coli DNA polymerase I, and the iNOS cDNA was then cloned into the HincII-digested vector pPS7.5A (14) under the control of the early-late P7.5 VV promoter. Restriction analysis with BamHI and ClaI was used to confirm correct orientation of the iNOS gene. The resulting plasmid was designated pPS7.5A-iNOS (Fig. 1). An rVV expressing iNOS was constructed from the thymidine kinase (TK)negative virus VV-HA-PR8 (13) by using marker rescue of the TK gene (8) and methotrexate selection (45). The presence of the iNOS gene was determined by dot blot hybridization with 32 P-l...