Lovchik, C. about the signaling pathways involved Ac-Lys-AMC in spore germination (28, 32). The first step in the germination process is most commonly the binding of metabolites by Ac-Lys-AMC germination (Ger) receptors (8, 23, 38). These receptors are membrane proteins mostly encoded by tricistronic operons. Up to seven Ger receptors have been characterized in (13). Combinations of Ger receptors may be involved in different interacting pathways for germination (13, 30). Usually a purine and an amino acid are required for the efficient germination of spores (2, 23, 37). Once germination is usually activated, a series of degradative events break up spore-specific structures and proteins (24, 29, 34). Germination is usually followed by a period of outgrowth, during which actively dividing cells are regenerated (19, 20, 22). It has been observed that and spore germination can be blocked by alcohols (11, 36), ion channel blockers (26), protease inhibitors (9), sulfhydryl reagents (14), and other miscellaneous compounds (10). Most of these studies targeted specific germination pathways in different organisms and are not directly comparable. A more recent Ac-Lys-AMC study tested the activities of subsets of the different types of compounds against and germination (10). Research from many groups, including ours, has shown that nucleoside and amino acid analogues act as competitive inhibitors of spore germination (2, 21, 25). Of these inhibitors, d-alanine (d-Ala) and d-histidine (amino acid analogues) and 6-thioguanosine (6-TG; a nucleoside analogue) were shown to also safeguard macrophages from spore germination and in macrophage cultures. Structure-activity relationship analysis allowed identification of epitopes necessary for nucleoside acknowledgement by spores. However, we found no correlation between germination inhibition and the ability of nucleosides to protect macrophages from cytotoxicity. We also showed that a nucleoside analogue (6-TG) and an amino acid analogue (d-Ala) combined to increase macrophage protection from cytotoxicity. MATERIALS AND METHODS Cell lines, reagents, and Rabbit Polyclonal to ATG16L2 gear. Murine macrophage J774A.1 cells were a nice gift from Jrgen Brojatsch (Albert Einstein College of Medicine, NY). Sterne 34F2 strain was a nice gift from Arturo Casadevall (Albert Einstein College of Medicine, NY). Immunicillin H (IH; compound XXXVIII) was a nice gift from Vern Schramm (Albert Einstein College of Medicine, NY). Nucleoside analogues of 6-benzylthioinosine (6-BTI; compound XVII), 6-spore germination and macrophage viability were monitored in a Tecan Infinite M200 multimode microplate reader. Open in a separate windows FIG. 1. Compounds tested as spore germination inhibitors and in cell culture (with the compound number shown in roman numerals in parentheses): INO (I), 6-TG (II), 2-mercaptopyrimidine (III), 2-thiouracil (2-TU; IV), trithiocyanuric acid (TTCA; V), 2,4-diamino-6-mercaptopyrimidine (DAMPy; VI), 2-mercaptopyridine (VII), 4-mercaptopyridine (VIII), 2-mercaptobenzimidazole (2-MBI; IX), 2-methylmercaptobenzimidazole (2-MMBI; X), 6-TI (XI), ADE (XII), GUA (XIII), 6-CPR (XIV), 2-APR (XV), 6-MMPR (XVI), 6-BTI (XVII), 6-methylaminopurine riboside (6-MAPR; XVIII), 6-spore preparation. cells were plated in nutrient agar (EMD Chemicals Inc.) and incubated at 37C to yield single cell clones. Individual colonies were produced in nutrient broth and replated to obtain bacterial lawns. Plates were incubated for 5 days at 37C. The producing bacterial lawns were collected by flooding with ice-cold deionized water. Spores were pelleted by centrifugation and resuspended in new deionized water. After two washing steps, spores were separated from vegetative and partially sporulated cells by centrifugation through a 20%-to-50% HistoDenz gradient (1). Spores were resuspended in water and washed three times before storage at 4C. Spores in all preparations were more than 95% real as determined by microscopic observation of Schaeffer-Fulton-stained aliquots. Spore viability was assessed by heat treatment followed by serial dilution plating in nutrient agar. Spore viability was retested every 15 to 20 days. Activation of spore germination. spore germination was monitored spectrophotometrically whereby the loss in light diffraction following addition of a germinant was reflected by a decreased optical density at 580 nm (OD580). All germination experiments were carried out in a Tecan Infinite M200 multimode microplate reader in UV-visible mode with its monochromator set at 580 nm. The final volume of each reaction combination was 0.2 ml. Experiments were carried out in triplicate on.