Studies on the mechanism of T cell inhibition by the Pseudomonas aeruginosa phenazine pigment pyocyanine

Pseudomonas aeruginosa and its products have been shown to inhibit mitogen-induced human lymphocyte blastogenesis as measured by [3H]TdR uptake. The phenazine pigment pyocyanine has been identified as one of the inhibitors present in cellfree culture supernatants. To determine the mechanism of the inhibitory action of pyocyanine, we studied its effect on the early stages of T cell activation. Pyocyanine inhibited lymphocyte stimulation induced by specific antigens, the lectin concanavalin A and the calcium ionophore, ionomycin, suggesting that its inhibitory effect is not dependent on interference with the T cell antigen receptor complex itself. Using quin-2, we showed that pyocyanine did not interfere with the mitogen-induced increase in cytosolic-free Ca2+. We also showed that pyocyanine did not interfere with the function of calmodulin stimulated Ca2+-Mg2+ ATPase activity, indicating that the mechanism of action of pyocyanine differs from that of the structurally related phenothiazine compounds. Analysis of IL 2 production and IL 2 receptor expression clearly showed that pyocyanine inhibits the production of this essential lymphokine as well as the expression of IL 2 receptors on the T cell membrane. This inhibition is dose dependent and not due to cellular toxicity. There was parallel inhibition of growth in cell volume as well as [3H]TdR uptake. Thus, our results demonstrate that pyocyanine inhibits T cell proliferation by decreasing the production of the critical lymphokine IL 2 and by decreasing the expression of the IL 2 receptor. Local suppression of lymphocyte stimulation by phenazine pigments such as pyocyanine may interfere with cellular immune responses that may be necessary for eradication of chronic infection with P. aeruginosa.     

F prostaglandins function as potent olfactory stimulants that comprise the postovulatory female sex pheromone in goldfish

This study establishes that ovulated female goldfish release F type prostaglandins (PGFs) to the water where they stimulate male spawning behavior and comprise the goldfish postovulatory pheromone. We first demonstrated that ovulated and prostaglandin-injected female goldfish release immunoreactive PGFs to the water. Next, using electro-olfactogram recording (EOG), we determined that waterborne prostaglandins function as potent olfactory stimulants for mature male goldfish. Prostaglandin F2 alpha (PGF2 alpha) and its metabolite 15-keto-prostaglandin F2 alpha (15K-PGF2 alpha) were the most potent prostaglandins; the former had a detection threshold of 10(-10) M and the latter a detection threshold of 10(-12) M. Studies of prostaglandin-injected fish indicated that PGF metabolites are an important component of the pheromone. Cross-adaptation experiments using the EOG demonstrated that goldfish have separate olfactory receptor sites for PGF2 alpha and 15K-PGF2 alpha that are independent from those that detect other olfactory stimulants. Finally, we established that male goldfish exposed to low concentrations of waterborne PGFs exhibit reproductive behaviors similar to those elicited by exposure to the odor of ovulated fish. Together with our recent discovery that a steroidal maturational hormone functions as a preovulatory "priming" pheromone for goldfish, these findings suggest that hormones and their metabolites may commonly serve as reproductive pheromones in fish.     

Molecular mechanics studies of dermorphin

Molecular mechanical simulations have been carried out on dermorphin. Presence of D-Ala2 at the N-terminus and L-Pro6 residue at the C-terminus indicated the probability of beta-turns. From the stereochemical considerations, three types- II', III' and V' - for the beta-turn at the N-terminus of the peptide and two types-I and III- for the C-terminus side of the peptide are possible. In our molecular mechanics calculations, we considered six folded and one extended conformations for dermorphin to asses the relative stabilities. Three of the six folded conformations are lower in energy and have the following general feature-similar in energy, three hydrogen bonds, semirigid beta-sheet segment and favorable Tyr1-Tyr5 interaction. The presence of beta-sheet structure might play a role in mu-receptor selective interaction of dermorphin.     

Cellular interactions of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)-specific suppressor factors. I. Inhibition of the activity of GAT-specific helper T cell clones by monoclonal GAT-specific suppressor T cell factors

Considerable information concerning the serology and biochemistry of antigen-specific, T cell-derived suppressor factors has been obtained with the use of T cell hybridomas as a source of homogeneous material. Similarly, knowledge of helper T cell products and receptors is accumulating from studies of helper T cell clones and hybridomas. Our strategy for studying the mechanisms by which suppressor factors inhibit responses was to determine whether monoclonal suppressor factors could inhibit antibody responses specific for L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in cultures containing unprimed splenic B cells, macrophages, and GAT-specific T cell clones as a source of helper activity. The MHC-restricted, two chain suppressor factors, GAT-TsF2, inhibited these responses if the helper T cell clones and suppressor factor were derived from H-2-compatible mice. Furthermore, responses were inhibited by briefly pulsing T cell clones with GAT-TsF2 in the presence of GAT, indicating that suppressor factors need not be present continuously. In addition, helper T cell clones adsorbed syngeneic, but not allogeneic, GAT-TsF2 in the presence of GAT. Adsorption also requires a shared antigenic specificity between the H-2b-derived helper T cells and TsF2 factor. Thus, helper T cells can serve as the cellular target of antigen-specific, MHC-restricted GAT-TsF2, and cloned helper T cells can be used as a homogeneous target population for analysis of the molecular mechanisms of T cell suppression.     

Identification and characterization of a suppressor T cell hybridoma specifically inducible by L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)

In vitro activation of naive spleen cells from C57BL/10 mice with GAT and the monoclonal GAT-TsF1, 372B3.5, followed by fusion with BW5147 resulted in generation of a hybridoma that fails to produce GAT-TsF constitutively, but upon reexposure to GAT and 372B3.5 is induced to secrete GAT-TsF2. The induction is GAT specific and requires de novo RNA, protein synthesis, and DNA synthesis. Although both GAT and 372B3.5 are required for induction, they may be added sequentially, provided the GAT is added first. The GAT-TsF produced by the induced cell is antigen specific and composed of two polypeptide chains: one capable of binding antigen, the other bearing determinants encoded by the I-J region of the MHC. The utility of this inducible GAT-TsF2 cell line for molecular biology and other studies is discussed.     

Techniques for studying the execution of foreign queens by temporal subcastes in fire ants (Solenopsis invicta)

Using monogynous and polygynous colonies of the fire ant, Solenopsis invicta Buren, we developed methodology to determine which behavioural subcaste of workers was responsible for executing unfamiliar queens that were introduced to them. Separation of subcastes and introduction of queens to each of these separately was partly effective, but more accurate results were obtained by removing workers that were in the act of executing queens, marking them individually, and returning them to the colony. Their subsequent behaviour was then recorded to determine whether they behaved like nurses, reserves, or foragers. The results showed that foragers are dominant in execution behaviour and that all morphological subcastes (minors, medias, and majors) are involved. This methodology may be applied to other social insect species.

---

Résumé A l'aide de colonies monogynes de S. invicta Buren, nous avons testé la sensibilité de deux méthodes: Premièrement, nous avons isolé les trois sous-castes temporelles des ouvrières (nurses, réserves et fourrageuses) auxquelles nous avons introduit des reines étrangères. Nous avons trouvé que les fourrageuses étaient le groupe le plus agressif, exécutant un pourcentage élevé des reines qui leur étaient présentées. Le degré de physogastrie de la reine étrangère, sa colonie d'origine et la présence de couvain n'avaient pas d'effet visible sur sa destinée. La sensibilité de cette méthode était limitée par la difficulté d'une séparation complète des souscastes temporelles.Deuxièmement, nous avons capturé des ouvrières exécutant activement des reines étrangères, après marquage nous les avons remises dans leur colonie pour observations ultérieures. Nous avons remarqué que leur localisation dans le nid et leur comportement correspondaient aux séquences trouvées pour les fourrageuses bien plus qu'à celles des ouvrières réserves ou nurses. Cette méthode est plus sensible. Des mesures de la largeur de la capsule céphalique de ces ouvrières ont montré qu'aucune des sous-caste morphologiques (mineurs, moyennes et majeurs) étaient dominantes dans le comportement d'exécution. Nos résultats indiquent que la sensibilité des ouvrières à certaines phéromones de la reine et odeur de la colonie augmente avec leur âge.

     

Cell-mediated immune responses to Chlamydia trachomatis in mothers and infants

Cell-mediated immunity to Chlamydia trachomatis was studied in pregnant women with chlamydial infection of the cervix, in infants born vaginally to these women, and in infants presenting with chlamydial conjunctivitis. Uninfected pregnant women and their infants were studied as controls. McCoy cell cultures were used to isolate C. trachomatis from clinical specimens. Cell-mediated immunity was measured by lymphocyte proliferative responses in vitro to stimulation by chlamydial antigens. Chlamydial IgG antibody in serum specimens was detected by a microenzyme-linked immunosorbent assay technique. The mean lymphocyte proliferative responses to chlamydial antigens were greater in infected women than in uninfected women both during pregnancy and in the postpartum period. Lymphocyte responsiveness in infected pregnant women, however, was less than in postpartum women. Despite failure to detect chlamydial infection in exposed infants, lymphocyte proliferative responses were greater in umbilical cord blood and later in peripheral blood samples from neonates born to infected mothers than in infants born to uninfected mothers. These responses were also greater in infants with chlamydial conjunctivitis than in infants of uninfected mothers. These data suggest that cellular immune responses to chlamydial antigens are increased in infected mothers and infants and that infants may acquire chlamydial cell-mediated immunity transplacentally.     

Origins of the freshwater attractant(s) of migrating elvers of the American eel,Anguilla rostrata

Synopsis Origins of the freshwater attractant(s) of migrating elvers of the American eel were investigated by assaying elvers' responses to rinses of plants, animals, and inanimate objects collected from a Rhode Island (U.S.A.) brook with a sizable elver run. Odor rinses were tested in a Y-maze at naturally occurring concentrations against both blank and brook water. Many items were attractive, several were repulsive, and some caused a reduction in elvers' rheotactic behavior, suggesting that elvers respond to a bouquet of odors. The odor of abundant decaying leaf detritus was highly attractive as were odors of the surfaces of aquatic plants, submerged stones, and migrating alewives. Conspecific odor was only weakly attractive. Because unattractive leaves became attractive when cultured with stream water, microorganisms responsible for detrital decomposition and present in/on most stream objects are thought to be the major source of the attractant(s). Decaying detritus and its associated microorganisms are abundant in most freshwater streams, where they often constitute the ecosystem's primary energy source; their odor could serve as an index of environmental suitability for migrating eels.