Genetic relationships among actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin

Summary A polyphasic taxonomic study was undertaken to establish the genetic and phenotypic relationships among six actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin. Chemotaxonomic studies reveal that all have Type I cell walls. Gas chromatography (GC) of fatty acid methyl esters revealed patterns consistent for strains ofStreptomyces with 160 and 150anteiso predominating. Principal component analysis of GC data revealed distinct profiles for each culture. Reciprocal DNA homology studies atT _m -25 showed the rapamycin-producing strain and one FK506-producing strain to have 38–50% homology with the type strain ofStreptomyces hygroscopicus (ATCC 27438). The remaining strains exhibited 6–17% homology. To further explore the relationships among these strains all were probed for the presence of anO-methyltransferase gene specific to this biosynthetic pathway. Among the strains of interest, onlyStreptomyces hygroscopicus subsp.yakushimaensis, the patent strain for FK520/FK523, failed to hybridize with the probes.     

Mass mortality of Diadema antillarum on the Caribbean coast of Panama

The ecologically important sea urchin Diadema antillarum suffered mass mortalities in 1983, first noted in Panama and then reported from the rest of the Caribbean. We documented the effects of this mortality at two localities on the Atlantic coast of Panama, Punta Galeta and the San Blas Archipelago. At Punta Galeta, affected by the mortality in January 1983, the numbers of D. antillarum changed from an estimated 14,000 per ha in June 1982 to 0.5 per ha in May 1983; by February 1984 they had increased to 38 per ha. In the San Blas, where mass mortality started in April 1983, the number of D. antillarum in permanent quadrats on 8 reefs was reduced by an average of 94.2%. The average reduction in population density measured in transects on nine reefs was 98.9%. Data taken in permanent quadrats on four reefs in 1978, 1979 and 1980 indicate that population fluctuations of D. antillarum are normally much smaller, justifying the labeling of the 1983 event as mass mortality. Size structure of the San Blas populations was also affected; mean test diameter of D. antillarum on four reefs was reduced from 48.6 mm to 25.0 mm. Other echinoids (Echinometra viridis, E. lucunter, Lytechinus variegatus, L. williamsi, Eucidaris tribuloides, Tripneustes ventricosus, Clypeaster rosaceus and Echinoneus cyclostomus) suffered no ill effects at either Galeta or the San Blas; their population densities remained stable or increased. Density determinations of Diadema mexicanum at the island of Taboguilla on the Pacific side of Panama indicate that Diadema mass mortality did not extend to the eastern Pacific. Sea surface temperatures, tidal levels, rainfall and salinity showed no abnormal fluctuations during the time of D. antillarum mass mortality at Galeta, suggesting that mortality was not due to physical stress. The wide geographical spread and species-specificity of the mortality suggest a water-borne pathogen as the most likely causative agent. Recovery of D. antillarum populations is likely to be slow because there are few, if any, unaffected populations in the Caribbean to contribute larvae for the recolonization of depleted areas. The absence of D. antillarum will probably be reflected by changes in the algal, coral and echinoid communities, and by altered patterns of bioerosion.     

Isolation of Pittsburgh pneumonia agent from a hospital shower.

Tatlockia (Legionella) micdadei, the Pittsburgh pneumonia agent, was isolated from a hospital shower. Although it was not possible, at the current time, to establish an epidemiological link to disease acquisition, this information may be significant because it provides further evidence that a water-associated reservoir of this organism exists within the hospital.     

Discharge of nematocysts isolated from aeolid nudibranchs

Nematocysts were extracted from 3 nudibranch species and one sea anemone species, and the ability of several test fluids to promote discharge was examined. Except when isolated in sodium citrate, nudibranch nematocysts did not discharge in response to any test fluids. Nudibranch nematocysts isolated in sodium citrate discharged when tested with EGTA, distilled water, and calcium-free artificial seawater, but there were differences among the 3 nudibranch species. Nematocysts isolated from one nudibranch species and nematocysts isolated from that nudibranch's sea anemone prey differed in the percentage that discharged in response to EGTA and distilled water. These results suggest that nematocysts stored by nudibranchs are altered in some way, resulting in the different discharge responses.     

Biological nitrogen fixation for sustainable agriculture: A perspective

The economic and environmental costs of the heavy use of chemical N fertilizers in agriculture are a global concern. Sustainability considerations mandate that alternatives to N fertilizers must be urgently sought. Biological nitrogen fixation (BNF), a microbiological process which converts atmospheric nitrogen into a plant-usable form, offers this alternative. Nitrogen-fixing systems offer an economically attractive and ecologically sound means of reducing external inputs and improving internal resources. Symbiotic systems such as that of legumes and Rhizobium can be a major source of N in most cropping systems and that of Azolla and Anabaena can be of particular value to flooded rice crop. Nitrogen fixation by associative and free-living microorganisms can also be important. However, scientific and socio-cultural constraints limit the utilization of BNF systems in agriculture. While several environmental factors that affect BNF have been studied, uncertainties still remain on how organisms respond to a given situation. In the case of legumes, ecological models that predict the likelihood and the magnitude of response to rhizobial inoculation are now becoming available. Molecular biology has made it possible to introduce choice attributes into nitrogen-fixing organisms but limited knowledge on how they interact with the environment makes it difficult to tailor organisms to order. The difficulty in detecting introduced organisms in the field is still a major obstacle to assessing the success or failure of inoculation. Production-level problems and socio-cultural factors also limit the integration of BNF systems into actual farming situations. Maximum benefit can be realized only through analysis and resolution of major constraints to BNF performance in the field and adoption and use of the technology by farmers.     

Detergent induced inhibition of eukaryotic RNA polymerase B activity and amanitin binding

We studied the effect of detergents on the binding of amanitin to RNA polymerase and on enzymatic activity. SDoS, Sarkosyl and deoxycholate were most inhibitory. Cholate and non-ionic detergents were less inhibitory. Evidence is presented that Sarkosyl inhibits chain elongation. The inhibition of amanitin binding was most influenced by the hydrophilicity of the detergent.     

Valveless pumping behavior of the simulated embryonic heart tube as a function of contractile patterns and myocardial stiffness

Biomechanics and Modeling in Mechanobiology - During development, the heart begins pumping as a valveless multilayered tube capable of driving blood flow throughout the embryonic vasculature. The...     

Isolation of genes mediating resistance to inhibitors of nucleoside and ergosterol metabolism in Leishmania by overexpression/selection

We tested a general method for the identification of drug resistance loci in the trypanosomatid protozoan parasite Leishmania major. Genomic libraries in a multicopy episomal cosmid vector were transfected into susceptible parasites, and drug selections of these transfectant libraries yielded parasites bearing cosmids mediating resistance. Tests with two antifolates led to the recovery of cosmids encoding DHFR-TS or PTR1, two known resistance genes. Overexpression/selection using the toxic nucleoside tubercidin similarly yielded the TOR (toxic nucleoside resistance) locus, as well as a new locus (TUB2) conferring collateral hypersensitivity to allopurinol. Leishmania synthesize ergosterol rather than cholesterol, making this pathway attractive as a chemotherapeutic target. Overexpression/selection using the sterol synthesis inhibitors terbinafine (TBF, targeting squalene epoxidase) and itraconazole (ITZ, targeting lanosterol C(14)-demethylase) yielded nine new resistance loci. Several conferred resistance to both drugs; several were drug-specific, and two TBF-resistant cosmids induced hypersensitivity to ITZ. One TBF-resistant cosmid encoded squalene synthase (SQS1), which is located upstream of the sites of TBF and ITZ action in the ergosterol biosynthetic pathway. This suggests that resistance to "downstream" inhibitors can be mediated by increased expression of ergosterol biosynthetic intermediates. Our studies establish the feasibility of overexpression/selection in parasites and suggest that many Leishmania drug resistance loci are amenable to identification in this manner.     

Compartmentalization of visual centers in the Drosophila brain requires Slit and Robo proteins

Brain morphogenesis depends on the maintenance of boundaries between populations of non-intermingling cells. We used molecular markers to characterize a boundary within the optic lobe of the Drosophila brain and found that Slit and the Robo family of receptors, well-known regulators of axon guidance and neuronal migration, inhibit the mixing of adjacent cell populations in the developing optic lobe. Our data suggest that Slit is needed in the lamina to prevent inappropriate invasion of Robo-expressing neurons from the lobula cortex. We show that Slit protein surrounds lamina glia, while the distal cell neurons in the lobula cortex express all three Drosophila Robos. We examine the function of these proteins in the visual system by isolating a novel allele of slit that preferentially disrupts visual system expression of Slit and by creating transgenic RNA interference flies to inhibit the function of each Drosophila Robo in a tissue-specific fashion. We find that loss of Slit or simultaneous knockdown of Robo, Robo2 and Robo3 causes distal cell neurons to invade the lamina, resulting in cell mixing across the lamina/lobula cortex boundary. This boundary disruption appears to lead to alterations in patterns of axon navigation in the visual system. We propose that Slit and Robo-family proteins act to maintain the distinct cellular composition of the lamina and the lobula cortex.