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The bryozoans Bugula neritina and Bugula simplex harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy. In B. neritina, the bacterial symbiont has been characterized as a gamma-proteobacterium, "Candidatus Endobugula sertula." "Candidatus E. sertula" has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments. In this study, the bacterial symbiont in B. simplex larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with "Candidatus E. sertula." In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the B. simplex symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a B. simplex larva. The taxonomic status "Candidatus Endobugula glebosa" is proposed for the B. simplex larval symbiont. Degenerate polyketide synthase (PKS) primers amplified a gene fragment from B. simplex that closely matched a PKS gene fragment from the bryostatin PKS cluster. PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with B. simplex. Bryostatin activity assays and chemical analyses of B. simplex extracts reveal the presence of compounds similar to bryostatins. Taken together, these findings demonstrate a symbiosis in B. simplex that is similar and evolutionarily related to that in B. neritina.

Background: 1,1-Dichloro-2,2-bis(P-chlorophenyl)ethylene (P,P'-DDE, DDE), a metabolite of DDT, is a persistent hormonally active environmental toxicant which has been found in human serum and follicular fluid. The objective of this study was to investigate the interaction between FSH and ppDDE on aromatase activity in primary cultures of human granulosa cells., Methods: Granulosa cells were obtained at the time of oocyte retrieval for IVF procedures and cultured in defined medium containing FSH and environmentally relevant concentrations of DDE. Aromatase activity was measured by incubating the cells with 1beta-[(3)H]androstenedione and measuring the release of (3)H(2)O., Results: The granulosa cell response to FSH was highly dependent on the basal level of aromatase activity (r = -0.703, P = 0.001, n = 17) with the highest activity occurring at low basal levels of aromatase activity. Enzyme activity was significantly stimulated at 100 ng DDE/ml. A synergistic effect on aromatizing activity was observed when cells were co-cultured with DDE and FSH., Conclusions: Concentrations of DDE similar to those present in human follicular fluid enhance basal and FSH-stimulated granulosa cell aromatizing enzyme activity.

This review discusses approaches to identify, clone, and express bioactive metabolite genes from symbionts of marine invertebrates. Criteria for proving symbiotic origin of bioactive metabolites are presented, followed by a comprehensive, practically-oriented overview of techniques to be applied. The Bugula neritina/Endobugula sertula association is used as a primary example, but other symbioses are discussed. Thirty-six compounds are presented and 111 references are cited.

Recently, drug interactions with grapefruit juice (GFJ) have received considerable attention from basic scientists, physicians, industry and drug regulatory agencies. GFJ has been shown to inhibit cytochrome P-450 3A4 isoenzyme and P-glycoprotein transporters in the intestine and liver. The GFJ-induced inhibitory effects are considered to be responsible for alterations in drug bioavailability, and pharmacokinetic and pharmacodynamic changes when drugs are ingested concurrently with GFJ. However, little or no interaction is observed when GFJ is taken concomitantly with parentally administered drugs. It is well known that risk factors for cardiovascular disease increase with advancing age, while hepatic metabolic activity decreases in elderly individuals. It is, therefore, possible that the combination of GFJ and cardiovascular medications may pose a health risk, especially in elderly patients. A number of studies have shown interactions of GFJ with cardiovascular drugs such as calcium-channel blockers, angiotensin II receptor antagonists, beta-blockers, and statins. Such interactions are likely to change the pharmacokinetics and pharmacodynamics of these drugs, consequently causing undesirable health effects. Therefore, health care professionals and the public need to be advised of the potential risks associated with the concomitant use of GFJ and interacting medications, especially cardiovascular drugs and agents with a narrow therapeutic index. This review focuses on the adverse interactions of GFJ and cardiovascular medications, and the proposed underlying mechanisms of such interactions.

The marine bryozoan, Bugula neritina, is the source of the bryostatins, a family of macrocyclic lactones with anticancer activity. Bryostatins have long been suspected to be bacterial products. B. neritina harbors the uncultivated gamma proteobacterial symbiont "Candidatus Endobugula sertula." In this work several lines of evidence are presented that show that the symbiont is the most likely source of bryostatins. Bryostatins are complex polyketides similar to bacterial secondary metabolites synthesized by modular type I polyketide synthases (PKS-I). PKS-I gene fragments were cloned from DNA extracted from the B. neritina-"E. sertula" association, and then primers specific to one of these clones, KSa, were shown to amplify the KSa gene specifically and universally from total B. neritina DNA. In addition, a KSa RNA probe was shown to bind specifically to the symbiotic bacteria located in the pallial sinus of the larvae of B. neritina and not to B. neritina cells or to other bacteria. Finally, B. neritina colonies grown in the laboratory were treated with antibiotics to reduce the numbers of bacterial symbionts. Decreased symbiont levels resulted in the reduction of the KSa signal as well as the bryostatin content. These data provide evidence that the symbiont E. sertula has the genetic potential to make bryostatins and is necessary in full complement for the host bryozoan to produce normal levels of bryostatins. This study demonstrates that it may be possible to clone bryostatin genes from B. neritina directly and use these to produce bryostatins in heterologous host bacteria.

CD8 is a T cell surface glycoprotein that participates in recognition of peptide/MHC class I molecules by binding to their alpha 3 domains. In addition, the cytoplasmic domain of CD8 associates with the intracellular tyrosine kinase p56(lck) (lck) promoting recruitment of lck to the TCR signaling complex. Recent data have suggested also that CD8 may interact with the TCR to promote energetically favorable conformations which increase its ligand binding. We have used the techniques of co-capping and confocal microscopy to ask whether we can detect an association between CD8 and the TCR independently of their binding to MHC class I molecules. We show that capping CD8 heterodimers with antibodies to the CD8 beta polypeptide is significantly more efficient than antibodies to the CD8 alpha polypeptide at inducing co-localization of TCR molecules with CD8, suggesting that there may be preferred conformations of CD8 which stabilize interactions with the TCR. In addition, we show by microscopy that intracellular lck redistributes very efficiently to the area of a CD8 cap, suggesting that there is a stronger association between lck and CD8 than has been proposed from immunoprecipitation analyses.

CBA and BALB/c mice produced polyspecific and monospecific polyclonal antibody responses, respectively, following immunization with Wuchereria bancrofti stage-3 larvae. Two monoclonal antibodies (MAbs) were produced from the immunized BALB/c mouse. These MAbs (both isotype M) recognized a previously undescribed highly expressed W. bancrofti antigen present in stage-3 larvae. The epitopes bound by the MAbs appear to be species specific for W. bancrofti since the MAbs did not bind to antigens of either nine other nematode species or two vector species in Western blots (immunoblots). Phosphorylcholine epitopes, responsible for immunological cross-reactivity among nematodes, were identified only on a 200-kDa antigen and not on the 95-kDa molecule. The targets of these immunoglobulin M MAbs are not carbohydrate epitopes.

Cytotoxic T lymphocytes (CTL) are generally specific for class I MHC proteins plus antigen and express CD8 co-receptor molecules. The effector function of some CTL can be blocked by antibodies to CD8 (CD8 dependent CTL), whereas that of others is resistant to blocking (CD8 independent CTL). This difference in sensitivity to antibody-mediated inhibition is assumed to reflect variations in affinity of particular TCR for antigen. However, we have found that a major difference between CD8 independent and CD8 dependent T cells lies in their sensitivity to stimulation, the former responding to lower concentrations of anti-CD3 antibody than the latter. Thus the contribution to cell signalling provided by the co-association of p56lck and CD8 is particularly relevant for CD8 dependent cells. These data challenge the notion that the affinity of an individual TCR for antigen is related to the sensitivity of a cell to inhibition by anti-CD8 antibodies. Furthermore we show that antibodies to co-receptor molecules have multiple distinct effects on T cell activation, only some of which may be related to T cell affinity.

The filarial-specific humoral immune response of adult residents of two areas of Papua New Guinea, differing in transmission of Wuchereria bancrofti infection was compared. The majority of residents of the village of Bonahoi, in an area where transmission of filariasis had been interrupted by a 20-year insecticide spray program to control malaria, showed no parasitologic signs of active W. bancrofti infection and were negative for both circulating phosphorylcholine Ag and peripheral blood microfilariae. In contrast, adult residents of the village of Nanaha were in an area exposed to infection, and were phosphorylcholine-Ag- and microfilariae-positive. The antibody response of these two groups to both adult worm excretory/secretory (ES) Ag and somatic antigen extract was examined to determine which components of the filarial-specific immune response were dependent on active infection. Identification of these immune responses may point to immunologic methods to evaluate control programs for lymphatic filariasis. Adults from Bonahoi were found to have significant immune responses to [35S] methionine-labeled ES Ag by immunoprecipitation and to adult somatic antigen extracts by ELISA and by immunoblotting. This result is consistent with the fact that these individuals were previously exposed to and/or infected with W. bancrofti. Similarly, residents of the endemic village had detectable immune responses to these Ag irrespective of if they were microfilaremic. The most striking immunologic difference observed between the two groups was that residents of Bonahoi had a dramatically reduced filarial-specific IgG4 antibody response to both adult somatic Ag and adult ES Ag. These data suggest that longitudinal measurement of filarial-specific IgG4 levels may be a useful seroepidemiologic indicator of changes in W. bancrofti infection status.

We describe here the genetic control of humoral responses to filarial nematode Ag elicited by live adult Brugia malayi parasites in mice. Inbred and congenic mice of two different MHC haplotypes, H-2k and H-2d, were examined. Serologic analysis showed that the humoral responses to the major surface 29-kDa glycoprotein of adult parasites and a 40-kDa Ag from the surface of the microfilarial stage were restricted to mice with H-2k alleles (B10.BR, CBA/Ca, and CBA/N), whereas mice of the H-2d haplotype (B10.D2/n and BALB/c) were nonresponsive to these Ag. Conversely, internal adult Ag of molecular mass of 24 and 66 kDa were recognized only by animals with the H-2d haplotype. Apart from MHC-restricted recognition, the level of responses to phosphorylcholine and to a 15-kDa adult surface molecule were found to be influenced by non-MHC genes. A sharp restriction was also observed to an adult surface Ag complex of 17 to 200 kDa, which was recognized only by BALB/c mice. Thus, multiple examples of both H-2 and background genetic effects on the immune response to distinct filarial Ag can be found.

A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis.

We report here a broad analysis of the excretory/secretory (E/S) products of adult Brugia malayi, collected by in-vitro cultivation of the parasite. Culture media and conditions were optimized, and non-essential amino acids were found to be crucial for efficient protein synthesis under cell- and serum-free culture conditions. A close correlation was found between total protein secretion, phosphorylcholine-bearing antigen release and lactate production on each day of culture, indicating that E/S molecules are actively secreted. Parasites cultured in vitro take 2-3 days to adjust to the new environment, and show peak levels of secretion at days 3 and 4. The active secretion of phosphorylcholine by the parasite therefore justifies the measurement of this molecule as an indication of active infection, possibly reflecting total worm burdens. By comparing metabolically labelled E/S from male and female worms, several molecules of low mol. wt, namely 10,000, 13,000, 14,000 and 22,000, together with high mol. wt components of above 12,000 were found to be female specific. Tracing the origin of the E/S products, several molecules were also found to be associated with the surface. Among these, there are at least two glycoproteins, 29,000 and 51,000 of which the 29,000 molecule is a major surface protein. The immunogenicity of the E/S was examined and antigenic cross-reactivity was found with sera from most filarial infections but not with non-filarial nematodiases such as hookworm or Trichinella. However, two molecules of low mol. wt, 15,000 and 19,000, were not recognized by anti-Onchocerca sera and appeared to be potential Brugia-specific diagnostic molecules. Possible functional roles of the adult E/S products were examined but we could find no evidence of protease activity in the E/S or glutathione S-transferase activity in either the E/S or in whole somatic extract.

Adult Brugia malayi nematode parasites possess a range of cuticular and epicuticular molecules which may be defined by various surface-labelling techniques. We present here evidence that at least two distinct antigens are associated with the surface, a glycoprotein of 29 kDa, and a complex of twelve components forming a regular series or 'ladder' between 17 and 200 kDa (17/200 kDa) which have not previously been described. Each of these products is antigenic in infected hosts, although responses in infected humans to the 17/200 kDa are relatively weak. Digestion of the 29 kDa antigen with proteases and endoglycosidases indicates that it is closely conserved between B. malayi and B. pahangi, and that it carries at least two N-linked oligosaccharide chains each of 1.5-2 kDa. By contrast, a smaller surface-labelled antigen of 15 kDa shows no glycosylation by either lectin adherence or endoglycosidase digestion assays. Trypsin treatment of intact, labelled parasites results in cleavage of 29 kDa molecules isolated 17/200 kDa 'ladder' to trypsin abolishes all bands except the 17 kDa base unit. Both the 29 kDa and 17/200 kDa antigens can be recovered as water-soluble molecules by homogenisation of the parasite in the absence of detergent, or by disruption of the cuticle with reducing agents such as 2-mercaptoethanol. In the presence of such agents, both the 17/200 kDa series and the 29 kDa glycoprotein are shed rapidly from intact parasites. Finally, two-dimensional electrophoretic analysis shows that while the 29 kDa glycoprotein is strongly basic and the 15 kDa acidic, the 17/200 kDa antigens form a related series of neutral pI.