Lactobacillus reuteri is a prominent member of the LactobaciUus population in the gastrointestinal ecosystem of humans, poultry, swine, and other animals. Reuterin is a newly discovered, broad-spectrum antimicrobial substance produced by this species during fermentation of glycerol. In this report, we describe procedures for (i) producing reuterin in sufficient amounts to isolate from a fermentation mixture and (ii) isolating this substance by high-performance liquid chromatography. By using uniformly labeled [14C]glycerol, reuterin was identified as a product of glycerol fermentation associated with the production of 0-hydroxypropionic acid and trimethylene glycol.Axelsson and co-workers (L. Axelsson, T. C. Chung, W. J. Dobrogosz, and S. E. Lindgren, submitted for publication) and Chung and colleagues (T. C. Chung, L. Axelsson, S. E. Lindgren, and W. J. Dobrogosz, submitted for publication) reported the discovery of a broad-spectrum antimicrobial substance termed reuterin produced by Lactobacillus reuteri. L. reuteri resides in the gastrointestinal tract of healthy humans and animals (2, 5) and is believed to function as a symbiont in the enteric ecosystem. Synthesis of such an antimicrobial substance by an enteric resident raises a number of interesting questions and possibilities as to the role these residents may play in the health of the host. Yet little is known about L. reuteri and less about reuterin except that it is produced specifically from glycerol by anaerobic resting cells under physiological conditions of temperature and pH. Preliminary investigations indicate that it is a lowmolecular-weight, neutral, water-soluble, nonprotein material that has antibacterial, antimycotic, and antiprotozoal activity.The

Lactobacillus reuteri converts glycerol into a potent cell growth inhibitor. This substance, termed reuterin, inlhibits the growth of gram-positive and gram-negative bacteria as well as yeasts, fungi, and protozoa. Semipreparative chromatography was used to purify reuterin, and Fourier transform infrared spectroscopy and liquid chromatography-mass spectrometry were used to establish the molecular weight as well as the molecular functionality of the reuterin molecule. Nuclear magnetic resonance studies of purified reuterin carried out with deuterium oxide confirmed the presence of two three-carbon compounds, I8-hydroxypropionaldehyde and the corresponding hydrated acetal, and a six-carbon cyclic dimer of the aldehyde. Further nuclear magnetic resonance studies with deuterated methanol revealed that in this solvent the compound existed as a three-carbon compound in a methoxy form. Trimethylsilyl derivatives of reuterin were analyzed by gas chromatography-mass spectrometry, and a molecule was identified which had a molecular weight corresponding to a disilylated dimeric structure. On the basis of the above information, reuterin was determined to be an equilibrium mixture of monomeric, hydrated monomeric, and cyclic dimeric forms of l-hydroxypropionaldehyde. This was subsequently confirmed by chemical synthesis.

Lactobacillus reuteri utilizes exogenously added glycerol as a hydrogen acceptor during carbohydrate fermentations, resulting in higher growth rates and cell yields than those obtained during growth on carbohydrates alone. Glycerol is first converted to 3-hydroxypropionaldehyde by a coenzyme B12-dependent glycerol dehydratase and then reduced to 1,3-propanediol by an NAD+-dependent oxidoreductase. The latter enzyme was purified and determined to have a molecular weight of 180,000; it is predicted to exist as a tetramer of identical 42,000-molecular-weight subunits.

A coenzyme B12-dependent glycerol dehydratase from Lactobacillus reuteri has been purified and characterized. The dehydratase has a molecular weight of approximately 200,000, and sodium dodecyl sulfatepolyacrylamide gel electrophoresis yielded a single major band with a molecular weight of 52,000. Km values for substrates and coenzyme B12 were in the millimolar and the submicromolar range, respectively.

Purpose: Prostate-specific membrane antigen (PSMA) is an attractive target for active immunotherapy. Alphavirus vaccines have shown promise in eliciting immunity to tumor antigens. This study investigated the immunogenicity of alphavirus vaccine replicon particles (VRP) that encode PSMA (PSMA-VRP). Experimental Design: Cells were infected with PSMA-VRP and evaluated for PSMA expression and folate hydrolase activity. Mice were immunized s.c. with PSMA-VRP or purified PSMA protein. Sera, splenocytes, and purified T cells were evaluated for the magnitude, durability, and epitope specificity of the anti-PSMA response. Antibodies were measured by flow cytometry, and cellular responses were measured by IFN-g enzyme-linked immunospot and chromium release assays. Cellular responses in BALB/c and C57BL/6 mice were mapped using overlapping 15-mer PSMA peptides. A Good Laboratory Practice^compliant toxicology study was conducted in rabbits. Results: PSMA-VRP directed high-level expression of active PSMA. Robust T-cell and B-cell responses were elicited by a single injection of 2 Â 10 5 infectious units, and responses were boosted following repeat immunizations. Anti-PSMA responses were detected following three immunizations with 10 2 infectious units and increased with increasing dose. PSMA-VRP was more immunogenic than adjuvanted PSMA protein. Responses to PSMA-VRP were characterized by Th-1cytokines, potent CTL activity, and IgG2a/IgG2b antibodies. T-cell responses in BALB/c and C57BL/6 mice were directed toward different PSMA peptides. Immunogenic doses of PSMA-VRP were well tolerated in mice and rabbits. Conclusions: PSMA-VRP elicited potent cellular and humoral immunity in mice, and specific anti-PSMA responses were boosted on repeat dosing. PSMA-VRP represents a promising approach for immunotherapy of prostate cancer.