Antifungal activity of modified hederagenin glycosides from the leaves of Kalopanax pictum var. chinense.

Monodesmosides which were obtained from the partial degradation of hederagenin bisdesmosides exhibited significant antifungal effect against Microsporum canis, Coccidioides immitis, Trichophyton mentagrophytes, Cryptococcus neoformans, and Candida albicans at the minimal inhibitory concentrations of 6.25-25 microg/ml. The hederagenin glycosides were isolated from the leaves of Kalopanax pictum var. chinense.

Diarylheptanoids with in vitro inducible nitric oxide synthesis inhibitory activity from Alnus hirsuta.

Four diarylheptanoids were isolated from the leaf of Alnus hirsuta (Betulaceae) and have been assessed for nitric oxide (NO) production inhibitory effects in vitro. Oregonin (1) and hirsutanonol (2) were found to be potent inducible nitric oxide synthase (iNOS) inhibitors. Compounds 1 and 2 showed inhibition of NO synthesis in dose-dependent manners by murine macrophage-like RAW 264.7 cells stimulated with interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). Their 50% inhibitory concentrations (IC50) were 3.8 and 14.3 microM, respectively. The inhibitory effects of these compounds on NO synthesis were due to suppression of iNOS mRNA expression as determined by Northern blotting.

Producer-decomposer co-dependency influences biodiversity effects.

Producers, such as plants and algae, acquire nutrients from inorganic sources that are supplied primarily by decomposers whereas decomposers, mostly fungi and bacteria, acquire carbon from organic sources that are supplied primarily by producers. This producer-decomposer co-dependency is important in governing ecosystem processes, which implies that the impacts of declining biodiversity on ecosystem functioning should be strongly influenced by this process. Here we show, by simultaneously manipulating producer (green algal) and decomposer (heterotrophic bacterial) diversity in freshwater microcosms, that algal biomass production varies considerably among microcosms (0.0-0.67 mg ml(-1)), but that neither algal nor bacterial diversity by itself can explain this variation. Instead, production is a joint function of both algal and bacterial diversity. Furthermore, the range in algal production in microscosms in which bacterial diversity was manipulated was nearly double (1.82 times) that of microcosms in which bacterial diversity was not manipulated. Measures of organic carbon use by bacteria in these microcosms indicate that carbon usage is the mechanism responsible for these results. Because both producer and microbial diversity respond to disturbance and habitat modification, the main causes of biodiversity loss, these results suggest that ecosystem response to changing biodiversity is likely to be more complex than other studies have shown.

Hospital trauma registries linked with population-based data.

BACKGROUND: We sought to obtain more reliable population-based data for injury epidemiology and trauma system evaluation by linking several sources. METHODS: In the state of Maine, probabilistic computer methods were used to link data from hospitals contained in a trauma registry for 1995 to 1996 to data from the same years contained in death certificates, ambulance run reports, and hospital discharge abstracts. The most reliable data available from each source were merged to form a standard record for each identifiable case of acute trauma. RESULTS: A total of 8,924 cases of serious injury were identified that either were in the registry, had a death certificate, or had both an ambulance run report and a hospital discharge abstract. Only 74% of the Trauma Center cases and 33% of the cases overall were contained in the registry. Only 84% of fatal hospitalized cases matched to a death certificate. Incompleteness of the registry and occasional failures to match records from one source to another were attributable to intentional omissions and a variety of human data management problems. CONCLUSIONS: Combining sources of data for injury victims can produce a resource more descriptive than any single source alone. However, computer-assisted record linkage still requires human review and corrections. Feedback of discrepancies to the individual data sources should further improve the quality of data available for linkage.

Comparison of probabilistic and deterministic record linkage in the development of a statewide trauma registry.

We have been working to develop a statewide injury surveillance system using not only hospital-based trauma registries but also other sources of data (including ambulance run reports, hospital discharge abstracts, and death certificates). For this purpose, a commercially available probabilistic matching program was compared to the deterministic program described previously. Using the same data preprocessing and linkage strategy, we programmed the probabilistic software to perform the matching step and compared the results with those obtained from the previously tested program. The outcomes using our data were similar, but we expect the probabilistic program to be more adaptable for general use, especially if large amounts of data must be linked.

Optimal location for a helicopter in a rural trauma system: prediction using discrete-event computer simulation.

A discrete-event computer simulation was developed using the C programming language to determine the optimal base location for a trauma system helicopter in Maine, a rural area with unevenly distributed population. Ambulance run reports from a one-year period provided input data on the times and places where major injuries occurred. Data from a statewide trauma registry were used to estimate the percentage of cases which would require trauma center care and the locations of functional trauma centers. Climatic data for this region were used to estimate the likelihood that a helicopter could not fly due to bad weather. The incidence of trauma events was modeled as a nonstationary Poisson process, and location of the events by an empirical distribution. For each simulated event, if the injuries were sufficiently severe, if weather permitted flying, if the occurrence were not within 20 miles of a center or outside the range of the helicopter, and if the helicopter were not already in service, then it was used for transportation. 35 simulated years were run for each of 4 proposed locations for the helicopter base. One of the geographically intermediate locations was shown to produce the most frequent utilization of the helicopter. Discrete-event simulation is a potentially useful tool in planning for emergency medical services systems. Further refinements and validation of predictions may lead to wider utilization.

Transposition of Tn5096 and related transposons in Streptomyces species.

IS493 is an insertion sequence isolated from Streptomyces lividans by a method designed to 'trap' transposable elements. IS493 was converted to functional transposons by cloning antibiotic-resistance-encoding genes between ORF-A and ORF-B of IS493 or near the left-end inverted repeat of the element. Tn5096 transposed relatively randomly in several Streptomyces species. Tn5096 can be introduced into streptomycetes on temperature-sensitive vectors by protoplast transformation, FP43-mediated transduction, or by conjugation from Escherichia coli. We have shown that additional genes can be inserted in Tn5096 without disrupting transposition, and that Tn5096 insertions in a tylosin (Ty)-producing strain of Streptomyces fradiae frequently cause no deleterious effects on Ty production. A promoter probe transposon, Tn5099, containing a promoterless xylE gene, transposed in Streptomyces griseofuscus and S. fradiae, and transcriptional fusions were readily identified.

Tn5099, a xylE promoter probe transposon for Streptomyces spp.

Tn5099, a promoter probe transposon for Streptomyces spp., was constructed by inserting a promoterless xylE gene and a hygromycin resistance gene into IS493. Tn5099 transposed into different sites in the Streptomyces griseofuscus genome, and the xylE reporter gene was expressed in some of the transposition mutants. Strains containing Tn5099 insertions that gave regulated expression of the xylE gene were identified.

Properties of the streptomycete temperate bacteriophage FP43.

FP43 is a temperate bacteriophage for Streptomyces griseofuscus that forms plaques on many Streptomyces species. FP43 virions contain 56 kb of double-strand DNA that is circularly permuted and terminally redundant, and contains 65% G + C. A physical map of the FP43 genome was constructed, and the origin for headful packaging (pac) was localized to an 8.8-kb region of the genome (hft) that mediates high-frequency transduction by FP43 of plasmid pRHB101. The phage attachment site (attP), a replication origin (rep), a region that inhibits plaque formation (pin), and a 3-kb deletion (rpt) that caused a 100-fold reduction in plasmid transduction were mapped.

Transposition and transduction of plasmid DNA in Streptomyces spp.

To expand the application of molecular genetics to many different streptomycete species, we have been developing two potentially widely applicable methodologies: transposon mutagenesis and plasmid transduction. We constructed three transposons from the Streptomyces lividans insertion sequence IS493. Tn5096 and Tn5097 contain an apramycin resistance gene inserted in different orientations between the two open reading frames of IS493. These transposons transpose from different plasmids into many different sites in the Streptomyces griseofuscus chromosome and into its resident linear plasmids. Tn5099 contains a promoterless xylE gene and a hygromycin-resistance gene inserted in IS493 close to one end. Tn5099 transposes in S. griseofuscus giving operon fusions in some cases that drive expression of the xylE gene product, catechol deoxygenase, giving yellow colonies in the presence of catechol. We have also developed plasmid vectors that can be transduced into many streptomycete species by bacteriophage FP43. We describe the characterization of FP43 and mapping of several bacteriophage functions. The region of cloned FP43 DNA essential for plasmid transduction includes the origin for headful packaging.

Saponins from the stem bark of Kalopanax pictum var. magnificum (I).

Three triterpenoid saponins were isolated from the methanol extract of the stem bark of Kalopanax pictum Nakai var. magnificum (Araliaceae). The structures of these saponins were identified as hederagenin 3-O-alpha-L-arabinopyranoside, hederagenin-3-O-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinop yranoside and 3-O-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranosyl hederagenin 28-O-alpha-L-rhamnopyranosyl(1-->4)-beta-D- glucopyranosyl(1-->6)-beta-D-glucopyranosyl ester.

Triterpenoidal saponins from the bark of Kalopanax pictum var. typicum.

One new triterpenoidal saponin, saponin F(2) has been isolated from the bark of Kalopanax pictum Nakai var. typicum (Araliaceae), together with one known saponin, kizuta saponin K12 (1). On the basis of chemico-spectral evidences, the structure of 2 has been elucidated to be 3-O-beta-D-xylopyranosyl(1-->3)-alpha- L-rhamnopyranosyl(1-->2)-alpha- L-arabinopyranosyl-23-hydroxyolean-12-en-28-O-alpha-L- rhamnopyranosyl(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranosy l ester.

Characterization of FP22, a large streptomycete bacteriophage with DNA insensitive to cleavage by many restriction enzymes.

Bacteriophage FP22 has a very broad host range within streptomycetes and appeared to form lysogens of Streptomyces ambofaciens ATCC 15154. FP22 shared strong cross-immunity and antibody cross-reactivity with bacteriophage P23, but not with seven other streptomycete bacteriophages. FP22 particles had a head diameter of 71 nm and a tail length of 307 nm. The FP22 genome was 131 kb, which is the largest bacteriophage genome reported for streptomycetes. The G + C content of the genome was 46 mol% and restriction mapping indicated that FP22 DNA had discrete ends. NaCl- and pyrophosphate-resistant deletion mutants were readily isolated and the extent of the deletions defined at least 23 kb of dispensable DNA in two regions of the genome. The DNA was not cleaved by most restriction endonucleases (or isoschizomers) which have been identified in the streptomycetes, including the tetranucleotide cutter MboI (GATC).

Saponins from leaves of Kalopanax pictum var. maximowiczii, a Korean medicinal plant.

From leaves of Kalopanax pictum var. maximowiczii, a Korean medicinal plant, six known saponins of hederagenin were isolated. One of the monodesmosides was identified as sapindoside A, previously isolated from Sapindus spp. Another monodesmoside and four bisdesmosides were proved to be identical with saponins-K3, -K10 and -K12 and Kizuta saponins-K8 and -K11, respectively, all of which have been isolated from Hedera rhombea. It was observed that the water solubilities of these monodesmosides were increased in the presence of the co-occurring bisdesmosides. The relationship between structure and solubilizing effect is reported.

Regulation of proline utilization in Salmonella typhimurium: molecular characterization of the put operon, and DNA sequence of the put control region.

The two genes required for proline utilization (put) in Salmonella typhimurium form a divergent operon. Extensive genetic evidence suggests that transcription of the put operon is autoregulated by the putA gene product, a membrane-associated dehydrogenase. In order to understand the mechanism of regulation, we characterized plasmid clones of the put operon. A 7.5 kb clone contains both of the put structural genes and regulatory sites. This clone only expressed two unique proteins corresponding to the putA and putP gene products. By comparing the physical and genetic maps of the put operon, the position of the put regulatory region was defined and the DNA sequence of this region was determined. Analysis of the DNA sequence indicated several potential regulatory sites for the put genes. Based on genetic and physical mapping studies, the most likely regulatory sites are two convergent promoters approximately 30 bp apart. A 27 bp palindrome located between the two promoters may be the operator for autoregulation by the PutA protein. The putA translational start site is 40 bp downstream of its putative mRNA start site. The putP promoter and its translational start site are separated by a 400 bp untranslated region.

Regulation of the put operon in Salmonella typhimurium: characterization of promoter and operator mutations.

The two genes required for proline utilization by S. typhimurium form a divergent operon. Expression of the put operon is induced by proline and subject to catabolite repression. Genetic evidence suggests that putA protein autogenously represses transcription of the putA and putP genes. In order to establish the molecular mechanism of put operon regulation we isolated regulatory mutations in the put control region. These mutants were selected using two phenotypes: the ability to degrade a toxic proline analogue, dehydroproline, due to overexpression of putA enzyme activity, or overexpression of lacZ from put::Mud operon fusions. The effect of each mutation on transcription in both directions was determined by measuring lacZ expression from putA and putP operon fusions. These regulatory mutations were cis-dominant when the putA protein was provided in trans, and they map in a region between the two genes. The phenotypes of the mutants suggest that the put regulatory region has a single operator site where the putA protein binds to repress transcription in both directions, and the putA and putP promoters overlap.