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Golgins are coiled-coil proteins that have been implicated in the structure and function of the Golgi complex. Here, we identify and characterize a trypanosomal golgin, TbG63, showing that it has a C-terminal membrane anchor and an N-terminus that projects into the cytoplasm. TbG63 in procyclic parasites is localized to the Golgi and interacts with the active, GTP-form of TbRab1A. Overexpression of TbG63 has dramatic effects on Golgi architecture – effects that require the N-terminus – whereas depletion has little, if any, effect on the growth rate. By contrast, in the bloodstream form of the parasite, depletion of TbG63 slows growth, although it has no obvious effect on the transport of a variant surface glycoprotein (VSG) or on Golgi structure. TbG63 might be a useful tool to study the structure and functioning of the Golgi complex.

The homotypic fusion of yeast vacuoles requires Sec18p (NSF)-driven priming to allow vacuole docking, but the mechanism that links priming and docking is unknown. We find that a large multisubunit protein called the Vam2/6p complex is bound to cis-paired SNAP receptors (SNAREs) on isolated vacuoles. This association of the Vam2/6p complex with the cis-SNARE complex is disrupted during priming. The Vam2/6p complex then binds to Ypt7p, a guanosine triphosphate binding protein of the Rab family, to initiate productive contact between vacuoles. Thus, cis-SNARE complexes can contain Rab/Ypt effectors, and these effectors can be mobilized by NSF/Sec18p-driven priming, allowing their direct association with a Rab/Ypt protein to activate docking.

Vam2p/Vps41p is known to be required for transport vesicles with vacuolar cargo to bud from the Golgi. Like other VAM-encoded proteins, which are needed for homotypic vacuole fusion, we now report that Vam2p and its associated protein Vam6p/Vps39p are needed on each vacuole partner for homotypic fusion. In vitro vacuole fusion occurs in successive steps of priming, docking, and membrane fusion. While priming does not require Vam2p or Vam6p, the functions of these two proteins cannot be fulfilled until priming has occurred, and each is required for the docking reaction which culminates in trans-SNARE pairing. Consistent with their dual function in Golgi vesicle budding and homotypic fusion of vacuoles, approximately half of the Vam2p and Vam6p of the cell are recovered from cell lysates with purified vacuoles.

We thank Pamela Silver, Charles Barlowe, Carol Kumamoto, and (especially) Tony Pugsley for critical comments. Work in our laboratory has been supported by NIH grant GM23377. J. E. received fellowship support from the Human Frontier Science Program Organization and F. D. from the Association pour la Recherche sur le Cancer and the Institut de la Sante et de la Recherche Medicale.

The SecA subunit of preprotein translocase drives ATP-dependent translocation of preproteins across the bacterial inner membrane concomitant with cycles of membrane insertion and de-insertion (Economou, A., and Wickner, W. (1994) Cell 78, 835-843). We have identified the membrane-inserting region of SecA as a 30-kDa domain in the C-terminal third of the protein beginning at aminoacyl residue 610. Limited proteolysis in the absence of translocation ligands indicates that the SecA monomer is composed of two primary structural domains, the 30-kDa membrane-inserting domain and an N-terminal 65-kDa ATPase domain. This limited protease treatment of SecA results in constitutive ATPase activity, indicating that intramolecular constraints between the two domains may play a role in the regulation of ATP hydrolysis by SecA.

Yeast vacuoles undergo priming, docking, and homotypic fusion, although little has been known of the connections between these reactions. Vacuole-associated Vam2p and Vam6p (Vam2/6p) are components of a 65S complex containing SNARE proteins. Upon priming by Sec18p/NSF and ATP, Vam2/6p is released as a 38S subcomplex that binds Ypt7p to initiate docking. We now report that the 38S complex consists of both Vam2/6p and the class C Vps proteins [Reider, S. E. and Emr, S. D. (1997) Mol. Biol. Cell 8, 2307–2327]. This complex includes Vps33p, a member of the Sec1 family of proteins that bind t-SNAREs. We term this 38S complex HOPS, for ho motypic fusion and vacuole p rotein s orting. This unexpected finding explains how Vam2/6p associates with SNAREs and provides a mechanistic link of the class C Vps proteins to Ypt/Rab action. HOPS initially associates with vacuole SNAREs in “cis” and, after release by priming, hops to Ypt7p, activating this Ypt/Rab switch to initiate docking.

The homotypic fusion of yeast vacuoles occurs in an ordered cascade of priming, docking, and fusion. The linkage between these steps has so far remained unclear. We now report that Vam7p (the vacuolar SNAP-23/25 homolog) signals from the cis-SNARE complex to Ypt7p (the vacuolar Rab/Ypt) to initiate the docking process. After Vam7p has been released from the cis-SNARE complex by Sec18p-mediated priming, it is still required for Ypt7p-dependent docking and it needs Ypt7p to remain on the vacuole. Thus, after priming, Vam7p is released from the vacuole altogether if Ypt7p has been extracted by Gdi1p or inactivated by antibody but is not released if docking is blocked simply by vacuole dilution; it is therefore Ypt7p function, and not docking per se, that retains Vam7p. In accord with this finding, cells deleted for the gene encoding Ypt7 have normal amounts of Vam7p but have little Vam7p on their isolated vacuoles. Interaction of Vam7p and Ypt7p is further indicated by two-hybrid analysis [Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., et al. (2000) Nature (London) 403, 623–627] and by the effect of Vam7p on the association of the Rab/Ypt-effector HOPS complex ( ho motypic fusion and vacuole p rotein s orting; Vam2p and Vam6p plus four vacuole protein sorting class C proteins) with Ypt7p. Vam7p provides a functional link between the priming step, which releases it from the cis-SNARE complex, and docking.

During the last 30 years the osteopathic profession has undergone a remarkable transformation from osteopathy, characterized by manipulative therapy, to osteopathic medicine, characterized by full-service health care, and in the process it has won acceptance from the government, the military, and physicians. These changes in status have resulted in new problems for the profession, because D.O. graduates are turning increasingly toward M.D. programs for residency training, and osteopathic medicine's primary care orientation is being replaced by an emphasis on specialty training. The authors advocate that osteopathic medicine return to its original mission of primary care, abandon specialty training or restrict it to those who have completed primary care residencies, abolish its separate-but-equal posture, and establish lines of communication with allopathic medicine and the American Medical Association to facilitate the development of a rational national policy for primary care that considers the potential osteopathy has to offer in meeting the nation's primary care needs.

The linkage between innate and adaptive immunity is well established. The recognition of specific pathogen associated molecular patterns (PAMPs) by members of the Toll-like receptor (TLR) family plays a prominent role in the activation of adaptive immune responses. Previously, we have demonstrated that the linkage of specific PAMPs with defined antigens provides more potent and protective antigen-specific responses in the absence of adjuvants or complex formulations. Here we utilized this approach to develop a recombinant protein based influenza vaccine. The TLR5 ligand flagellin was linked to hemagglutinin (HA) to generate a single component vaccine. The recombinant E. coli expressed protein is recognized by influenza-convalescent antisera, indicating that the HA portion of the fusion protein is properly folded and presents conformational epitopes. In mice, the recombinant vaccine induces strong humoral and cellular responses that are quantitatively and qualitatively superior to those observed when the antigen is delivered with alum or co-delivered with flagellin unlinked. Analysis of serum antibody responses reveals strong reactivity with cells infected with influenza A and the ability to inhibit viral entry in vitro. Moreover, immunization with the fusion protein fully protects BALB/c mice from a lethal intranasal challenge with influenza A. These data demonstrate that linkage of the TLR5-ligand flagellin with HA provides significant promise as a non-egg based approach in the development of novel vaccines to combat seasonal and pandemic influenza.

During the past 40 years, the osteopathic medical profession has undergone a transformation from "osteopathy" to "osteopathic medicine." The former was characterized by manipulative treatment; the latter, by full-service healthcare. During this transformation, the profession has won acceptance from the government, the military, and MDs. These changes in status have resulted in new problems for the profession because DO graduates are increasingly turning toward allopathic programs for residency training. Thus, osteopathic medicine's primary care orientation is being replaced by an emphasis on specialty training. The authors propose that osteopathic medicine return to its original mission of primary care, abandon or restrict specialty training to those who have completed primary care residencies, and rethink its separate-but-equal posture. They also propose that osteopathic medicine establish lines of communication with allopathic medicine, the American Medical Association, and the government to facilitate the development of a rational national policy for primary care that considers the potential osteopathic medicine has to offer in meeting the nation's primary care needs.

The energy spectra of photoneutrons from the absorption of 22-Mev bremsstrahlung by silver and bismuth have been measured with nuclear emulsions. Comparisons are made between the observed photoneutron spectra and those predicted on the basis of a statistical nuclear model. Energy level densities are assumed proportional to $\mathrm{exp}[{(a\ensuremath{\epsilon})}^{\frac{1}{2}}]$ and $\mathrm{ln}(\frac{A\ensuremath{\epsilon}}{20}+1)$, where $\ensuremath{\epsilon}$ is the excitation energy of the residual nucleus. The exponential form of the energy level density is in better agreement with the data than is the logarithmic form. However, for neutron energies greater than 4 Mev there are more photoneutrons observed than predicted by the exponential energy level density scheme.The angular distributions of photoneutrons from several elements have been measured with a variety of neutron detectors. The moderated neutron detector, which is equally sensitive to neutrons of most energies, indicates the photoneutron flux to be predominately isotropic. The small anisotropic component has a maximum at 90 degrees to the x-ray beam. Photoneutrons from bismuth have a larger anisotropic component than photoneutrons from any other element which was tested. In that case 21 percent of the neutron flux is in the anisotropic component.The $\mathrm{Al}(n, p)$ and $\mathrm{Si}(n, p)$ reactions have been used to measure the angular distributions of photoneutrons from several elements. These reactions have thresholds at 1.95 Mev and 2.69 Mev, respectively and are primarily sensitive to neutrons with more than 4-Mev energy. The angular distributions thus observed have a maximum at 90 degrees to the x-ray beam. In general, photoneutrons from the heavy elements deviate more from isotropy than do the light elements. An epithermal neutron detector indicates that the low-energy photoneutrons are emitted isotropically.It is concluded that most photoneutrons are generated in a manner consistant with the statistical nuclear model. The high-energy photoneutrons, which constitute a small fraction of the total photoneutron flux, are ejected predominately at right angles to the x-ray beam as expected by dipole absorption of the photons.