Abstract. Purified Golgi membranes of the human intestinal adenocarcinoma cell line Caco-2 were used as an antigen to produce a monoclonal antibody, G1/93, which specifically labels a tubulovesicular compartment near the cis side of the Golgi apparatus, including the first cis-cisterna itself, as visualized by single and double immunoelectron microscopy with antibodies against galactosyltransferase. The antigen recognized by G1/93 was identified as a protein with a subunit size of 53 kD. Pulse-chase experiments revealed that the 53-kD protein dimerizes immediately after synthesis followed by formation of oligomers of ~ 310 kD, probably homohexamers. The protein has a transmembrane topology with only a short cytoplasmic segment as assessed by protease protection experiments.

Surface display of heterologous proteins on bacterial cells is an important objective for many applications in microbiology and molecular biology. In gram-negative bacteria, different types of surface proteins have been exploited for this purpose. These include the following: (i) outer membrane proteins LamB (4), PhoE (1), and OmpA (35); (ii) lipoproteins TraT (19) and the peptidoglycan-associated lipoprotein (10); (iii) the fimbria protein fimbrillin (20, 21); and (iv) the flagellar protein flagellin (32). The different surface display systems have been used extensively to express heterologous antigenic determinants on the bacterial cells for the purpose of developing live bacterial vaccine vehicles. Enteric bacteria, such as Escherichia coli and Salmonella typhimurium, have been studied in this context, and the cell surface presentation has been considered advantageous to induce an antibody response to the exposed antigens with live cells for immunization (11,28,41).The expression of functional single-chain antibodies on the surface of E. coli cells (7, 10) has opened the discussion of whether this strategy would be an alternative to the rapidly developing phage technology for the selection of peptides or recombinant antibody fragments from large libraries (31). An interesting application for bacterial cells exposing heterologous proteins might be the development of whole-cell adsorbents by the surface expression of suitable protein ligands. Immobilized recombinant bacteria cannot be considered for separation processes in pharmaceutical industries but might because of their low cost constitute competitive adsorbents for certain separations (3, 11). The use of enzyme-coated bacteria as novel biocatalysts has also been envisioned, because enzymes with retained activity have been surface displayed on E. coli cells (8, 9).Investigations with gram-positive bacteria for cell surface display of heterologous proteins have recently been initiated. Expression systems for the mouth commensal bacterium Streptococcus gordinii (37) and the nonpathogenic bacterium Staphylococcus xylosus (18) have been developed on the basis of the fibrillar M6 protein from Streptococcus pyogenes and protein A from Staphylococcus aureus, respectively. These cell surface display systems have been used for the surface expression of several antigenic determinants, and immunization with live recombinant bacteria induces both local and systemic antibody responses to the hybrid receptors (33, 37), suggesting that gram-positive bacteria might constitute potential live bacterial vaccine delivery systems. The surface receptors of gram-positive bacteria seem to be more permissive for the insertion of extended sequences of foreign proteins (6) than the different gram-negative systems, in which both translocation through the cytoplasmic membrane and correct integration into the outer membrane are required for proper surface exposure of the heterologous polypeptide. Considering the development of whole-cell adsorbents or bacterial biocatalysts by surface ...

The extraordinary structural diversity of subunits forming type A y-aminobutyric acid (GABAA) (al-6, 1-4, y1-3, 8, p) (1-5) provides the basis for an extraordinary structural diversity of GABAA receptors. The heterogeneity of GABAA receptors is expected to provide a hitherto unexplored diversity in the functions of receptor subtypes affecting their sensitivity to GABA, modulation by allosteric effectors, adaptation to stimulus conditions, distribution within a neuron, ontogenetic development, or alterations in disease states (1).As a prerequisite for a functional analysis of receptor subtypes, elucidation of their cellular location and subunit composition is essential. So far, the distribution of subunits has largely been mapped at the mRNA level (6-12). However, the coassembly of subunits and the presence of multiple receptors in a single neuron can be demonstrated only by detailed immunohistochemical analysis. In this study, the possible colocalization ofthe al, a3, P2,3, and Y2 subunits was assessed in rat brain neurons by double and triple immunofluorescence staining. The pattern of subunit immunoreactivity (IR) was analyzed at the cellular and subcellular levels by confocal laser microscopy. To visualize the al, a3, and 'Y2 subunits, polyclonal antisera were used whose specificity had been assessed by Western blotting, immunoprecipitation, and immunohistochemistry (13-15). Since antibodies for single P subunits are not available, the monoclonal antibody bd-17 (16, 17), which recognizes both the P2 and the f3 subunit (18), was used to represent the /-subunit family. Subunits not included in the present study are not of major preponderance (a2, a4, a5, 81, yi, )y3, p) (5,8,9,11,(19)(20)(21) or are largely restricted to a particular population of neuronse.g., cerebellar granule cells (a6, 8) (7,22,23). The present analysis was restricted to the olfactory bulb, basal forebrain, basal ganglia, brainstem, and cerebellum, where the pattern of subunit distribution was most distinctive. Five distinct combinations of subunits were detected denoting different GABAA receptor subtypes. Some of the receptors could be allocated to defined neurons by double staining with neurotransmitter markers. MATERIALS AND METHODSFollowing anesthesia with chloral hydrate, adult male Wistar rats were perfused transcardially with paraformaldehyde/ picric acid (24). Brains were removed, postfixed for 2 hr, and stored overnight in phosphate-buffered saline containing 10%6 dimethyl sulfoxide for cryoprotection. Sections were cut from frozen blocks with a sliding microtome and processed free-floating for double and triple immunofluorescence staining (25). Polyclonal antisera directed against subunit-specific peptides (a,, residues 1-16; a3, 1-15; Y2, 1-15) raised in rabbits (al, a3, Y2) (13-15) and guinea pigs (a3, V2), as well as the mouse monoclonal antibody bd-17 (16, 18) (#2 and 183 subunits), were used as primary antibodies. After overnight incubation of sections with antibodies diluted in phosphatebuffered saline containi...

Virus-like particles (VLPs) are known to induce strong Ab responses in the absence of adjuvants. In addition, VLPs are able to prime CTL responses in vivo. To study the efficiency of this latter process, we fused peptide p33 derived from lymphocytic choriomeningitis virus to the hepatitis B core Ag, which spontaneously assembles into VLPs (p33-VLPs). These p33-VLPs were efficiently processed in vitro and in vivo for MHC class I presentation. Nevertheless, p33-VLPs induced weak CTL responses that failed to mediate effective protection from viral challenge. However, if APCs were activated concomitantly in vivo using either anti-CD40 Abs or CpG oligonucleotides, the CTL responses induced were fully protective against infection with lymphocytic choriomeningitis virus or recombinant vaccinia virus. Moreover, these CTL responses were comparable to responses generally induced by live vaccines, because they could be measured in primary ex vivo 51Cr release assays. Thus, while VLPs alone are inefficient at inducing CTL responses, they become very powerful vaccines if applied together with substances that activate APCs.

Jaw1 is an endoplasmic reticulum (ER) resident protein representative of a class of proteins post translationally inserted into membranes via a type II membrane anchor (cytosolic NH2 domain, lumenal COOH domain) in a translocon-independent manner. We found that Jaw1 can efficiently deliver a COOH-terminal antigenic peptide to class I molecules in transporter associated with antigen processing (TAP)-deficient cells or cells in which TAP is inactivated by the ICP47 protein. Peptide delivery mediated by Jaw1 to class I molecules was equal or better than that mediated by the adenovirus E3/19K glycoprotein signal sequence, and was sufficient to enable cytofluorographic detection of newly recruited thermostabile class I molecules at the surface of TAP-deficient cells. Deletion of the transmembrane region retargeted Jaw1 from the ER to the cytosol, and severely, although incompletely, abrogated its TAP-independent peptide carrier activity. Use of different protease inhibitors revealed the involvement of a nonproteasomal protease in the TAP-independent activity of cytosolic Jaw1. These findings demonstrate two novel TAP-independent routes of antigen processing; one based on highly efficient peptide liberation from the COOH terminus of membrane proteins in the ER, the other on delivery of a cytosolic protein to the ER by an unknown route.

A novel process for the preparation of water-borne biomaterials for hard tissue repair from injectable precursors is described, where the precursors form crosslinked materials in situ under physiological conditions. The precursors react by means of a Michael-type addition reaction that makes use of addition donors such as pentaerythritol tetrakis 3'-mercaptopropionate (QT) and addition acceptors such as poly(ethylene glycol) diacrylate 570 MW (PEGDA), pentaerythritol triacrylate (TA), and poly(propylene glycol) diacrylate 900 MW (PPODA). These crosslinked materials (at 75 wt% solid), prepared from water dispersions or reverse emulsions, showed ultimate strengths in compression of 1.8 +/- 0.2 and 6.7 +/- 0.5 MPa and ultimate deformations of 35 +/- 2+/- and 37 +/- 2%, respectively. Scanning electron microscopy (SEM) shows that the morphology of the precursors templated the morphology of the final materials. The current study indicates that it is possible to obtain injectable high-modulus materials that have appropriate mechanical properties and gelation kinetics for tissue augmentation and stabilization applications such as mechanical stabilization of the intervertebral disc annulus.

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