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Biomedical subjects

D L Crawford

Publications and source records attributed to D L Crawford.

At least 19 recordsLinked to original sources

Characterization of streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots.

The actinomycete Streptomyces lydicus WYEC108 showed strong in vitro antagonism against various fungal plant pathogens in plate assays by producing extracellular antifungal metabolites. When Pythium ultimum or Rhizoctonia solani was grown in liquid medium with S. lydicus WYEC108, inhibition of growth of the fungi was observed. When WYEC108 spores or mycelia were used to coat pea seeds, the seeds were protected from invasion by P. ultimum in an oospore-enriched soil. While 100% of uncoated control seeds were infected by P. ultimum within 48 h after planting, less than 40% of coated seeds were infected. When the coated seeds were planted in soil 24 h prior to introduction of the pathogen, 96 h later, less than 30% of the germinating seeds were infected. Plant growth chamber studies were also carried out to test for plant growth effects and for suppression by S. lydicus WYEC108 of Pythium seed rot and root rot. When WYEC108 was applied as a spore-peat moss-sand formulation (10(8) CFU/g) to P. ultimum-infested sterile or nonsterile soil planted with pea and cotton seeds, significant increases in average plant stand, plant length, and plant weight were observed in both cases compared with untreated control plants grown in similar soils. WYEC108 hyphae colonized and were able to migrate downward with the root as it elongated. Over a period of 30 days, the population of WYEC108 colonized emerging roots of germinating seeds and remained stable (10(5) CFU/g) in the rhizosphere, whereas the nonrhizosphere population of WYEC108 declined at least 100-fold (from 10(5) to 10(3) or fewer CFU/g). The stability of the WYEC108 population incubated at 25 degrees C in the formulation, in sterile soil, and in nonsterile soil was also evaluated. In all three environments, the population of WYEC108 maintained its size for 90 days or more. When pea, cotton, and sweet corn seeds were placed into sterile and nonsterile soils containing 10(6) or more CFU of WYEC108 per g, it colonized the emerging roots. After a 1-week growing period, WYEC108 populations of 10(5) CFU/g (wet weight) of root were found on pea roots in the amended sterile soil environment versus 10(4) CFU/g in amended nonsterile soil. To further study the in vitro interaction between the streptomycete and P. ultimum, mycelia of WYEC108 were mixed with oospores of P. ultimum in agar, which was then used as a film to coat slide coverslips.(ABSTRACT TRUNCATED AT 400 WORDS)

Antifungal Agents

A Cryptosporidium parvum genomic region encoding hemolytic activity.

Successful parasitization by Cryptosporidium parvum requires multiple disruptions in both host and protozoan cell membranes as cryptosporidial sporozoites invade intestinal epithelial cells and subsequently develop into asexual and sexual life stages. To identify cryptosporidial proteins which may play a role in these membrane alterations, hemolytic activity was used as a marker to screen a C. parvum genomic expression library. A stable hemolytic clone (H4) containing a 5.5-kb cryptosporidial genomic fragment was identified. The hemolytic activity encoded on H4 was mapped to a 1-kb region that contained a complete 690-bp open reading frame (hemA) ending in a common stop codon. A 21-kDa plasmid-encoded recombinant protein was expressed in maxicells containing H4. Subclones of H4 which contained only a portion of hemA did not induce hemolysis on blood agar or promote expression of the recombinant protein in maxicells. Reverse transcriptase-mediated PCR analysis of total RNA isolated from excysted sporozoites and the intestines of infected adult mice with severe combined immunodeficiency demonstrated that hemA is actively transcribed during the cryptosporidial life cycle.

Amino Acid Sequence

Enteral human serum immunoglobulin treatment of cryptosporidiosis in mice with severe combined immunodeficiency.

The anti-cryptosporidial immunoglobulin G antibodies in two commercially available human serum immunoglobulin (HSIG) products were quantified and characterized. The mean level of Cryptosporidium parvum-specific immunoglobulin G in HSIG was eightfold higher than the antibody level found in the sera of three immunocompetent individuals convalescing from cryptosporidiosis. However, HSIG products displayed no reactivity to cryptosporidial antigens in immunoblot analyses, while convalescent-phase sera demonstrated characteristic banding patterns. When HSIG was given to newborn severe combined immunodeficiency (scid) mice before and shortly after experimental infection, a decreased intensity of infection was observed in the intestines of the mice compared with that of control mice. However, there was no difference in mortality or histopathologic findings in the intestines of HSIG-treated and control mice when treatment was not started until 22 days of age. These results indicate that HSIG may be beneficial when given prophylactically; however, HSIG cannot eradicate cryptosporidia from mucosal surfaces in an established infection.

Adult

Rapid in vivo footprinting method for the detection of DNA-protein interactions in isolated nuclei.

Here we report a simple method of in vivo footprinting for the detection of DNA-protein interactions in the liver of a small teleost fish, Fundulus heteroclitus. This method allows the determination of these interactions in nuclei isolated from intact liver, obviating the need for cell culture. Cells in culture often do not respond to environmental cues in the same way as do tissues within the intact organism and therefore may be inappropriate for the study of certain adaptive responses. Furthermore, cell lines are available for only a small number of marine organisms. This technique may therefore be of general utility for the study of gene regulation in a wide variety of marine organisms.

Animals

Nuclear genes from the copepod Calanus finmarchicus.

For biological oceanography it is important to understand the coupling between physical and biological processes in pelagic systems. The calanoid copepod Calanus finmarchicus dominates the zoo-plankton biomass and is an important link between primary producers and higher trophic levels in the northern Atlantic. Thus understanding how the physical environment affects gene expression or population genetics in this species is important. However, very few nuclear genes have been characterized from this species, making it difficult to perform these types of studies. Four cDNAs encoding actin, hexokinase, phosphoglucose isomerase, and phosphofructokinase, as well as a hexokinase genomic DNA, have been isolated and characterized. These sequences constitute important molecular tools for biological oceanographers.

Actins

Rapid enzyme assays investigating the variation in the glycolytic pathway in field-caught populations of Fundulus heteroclitus.

Variation in enzyme expression may be important in evolutionary adaptation, yet is seldom studied. Furthermore, no studies have examined the expression of all enzymes in a defined metabolic pathway. Enzyme concentration is a measure of enzyme expression and was ascertained by assaying maximal activity. Presented here is an analysis of variation of maximal enzyme activity for all the enzymes in a single metabolic pathway, glycolysis, from three clinically distributed populations of the fish, Fundulus heteroclitus. Techniques for rapidly analyzing maximal enzyme activity for all the enzymes of an entire metabolic pathway from many individuals are described. The high degree of repeatability (mean coefficient of variation for replicates, 4.4%) and sensitivity (less than 3 mg of tissue is required to measure all 10 enzymes) of these assays demonstrate the utility of such an approach for analyzing variation among populations for a large numbers of enzymes. Results from these studies indicate that (1) the average coefficient of variation for all enzyme determinations within a population is 45.3% and (2) between populations, the activity of 5 of the 10 glycolytic enzymes are significantly different. This considerable variation occurs even in populations where there is little allelic variation. These data demonstrating substantial variation in enzyme expression support the idea that changes in gene regulation may be as important as, or even more important than, changes in biochemical kinetic parameters in evolutionary processes.

Alleles

Inability of interferon-gamma and aminoguanidine to alter Cryptosporidium parvum infection in mice with severe combined immunodeficiency.

Severe combined immunodeficiency (scid) mice have been useful in identifying specific host defense systems responsible for containing and eradicating Cryptosporidium parvum infection. Adult scid mice were given C. parvum oocysts and treated weekly with monoclonal antimurine interferon-gamma (anti-IFN-gamma). Anti-IFN-gamma-treated mice had more cryptosporidia seen in the intestines and had more severe morphologic changes associated with disease than control mice. To assess the mechanism of this effect, infected adult BALB/c and scid mice were treated with the nitric oxide synthase inhibitor, aminoguanidine. Infection in aminoguanidine-treated mice was not significantly different from that in control mice. Next, the effects of pharmacologic doses of IFN-gamma (10,000 IU) on the course of cryptosporidiosis in newborn scid mice were evaluated. IFN-gamma did not reverse the initial susceptibility of neonatal scid mice to cryptosporidiosis and continued treatment with IFN-gamma (10,000 IU weekly) did not alter survival. We conclude that IFN-gamma does not exert its anticryptosporidial effect by stimulation of nitric oxide production. Deficient IFN-gamma production by neonatal lymphocytes does not appear to be responsible for the increased severity of infection observed in neonatal animals. Also, IFN-gamma may not be useful in treating immunocompromised patients with cryptosporidiosis.

Amino Acid Oxidoreductases

Cryptosporidiosis in adult and neonatal mice with severe combined immunodeficiency.

Cryptosporidium parvum causes protracted diarrhoea in immunodeficient hosts. To characterize the role that T and B lymphocytes play in the eradication of the parasite from the intestinal mucosa, the course of infection in mice with severe combined immunodeficiency (SCID) was studied. Twenty-nine SCID and 26 BALB/c adult mice received 10(6) oocysts intragastrically. The course of infection in the two strains was similar until 2 months after inoculation, when moderate numbers of organisms were identified in the villous and crypt mucosa of the ileum and proximal colon of SCID mice. Three months after inoculation, SCID mice developed wasting and progressive intestinal and biliary tract disease. At 5 months, mortality of 72 and 0 per cent, respectively, was observed in the SCID and BALB/c mice. Twenty-four SCID and 26 BALB/c neonatal mice were also inoculated with C. parvum. Cryptosporidiosis occurred in SCID and BALB/c mice within 2 weeks of inoculation. Subsequently, BALB/c, but not SCID mice, eradicated the parasite from their intestinal mucosa. SCID mice developed progressively severe cryptosporidiosis which killed all animals within 7 weeks. Responses mediated by B or T cells, or both, appeared to play a role in eradicating C. parvum from the intestinal mucosa, since SCID mice were more severely affected than BALB/c mice. The different course of infection in adult and neonatal SCID mice indicated that other age-related factors also played a role in containing C. parvum infection.

Animals

Evolutionary adaptation to different thermal environments via transcriptional regulation.

Populations of the teleost fish Fundulus heteroclitus are subjected to the clinical variation in environmental temperatures that occurs along the eastern seacoast of North America. In concordance with this change in temperature is the clinal variation in the enzyme concentration of the heart-type lactate dehydrogenase (LDH-B; E.C.1.1.1.27). Previously we have shown that the compensating change in the LDH-B enzyme concentration is due to a change in the amount of LDH-B mRNA, but we did not define whether this was due to differences in mRNA stability or to differences in rate of transcription. The results presented here help clarify the molecular mechanism responsible for the variation in Ldh-B gene expression: the rate of transcription from the Ldh-B locus is significantly different between populations, and this difference is responsible for the compensatory change in LDH-B enzyme concentration.

Adaptation, Biological

Mineralization of sulfonated azo dyes and sulfanilic acid by Phanerochaete chrysosporium and Streptomyces chromofuscus.

Five 14C-radiolabeled azo dyes and sulfanilic acid were synthesized and used to examine the relationship between dye substitution patterns and biodegradability (mineralization to CO2) by a white-rot fungus and an actinomycete. 4-Amino-[U-14C]benzenesulfonic acid and 4-(3-sulfo-4-aminophenylazo)-[U-14C]benzenesulfonic acid were used as representative compounds having sulfo groups or both sulfo and azo groups. Such compounds are not known to be present in the biosphere as natural products. The introduction of lignin-like fragments into the molecules of 4-amino-[U-14C]benzenesulfonic acid and 4-(3-sulfo-4-aminophenylazo)-[U-14C]benzenesulfonic acid by coupling reactions with guaiacol (2-methoxyphenol) resulted in the formation of the dyes 4-(3-methoxy-4-hydroxyphenylazo)-[U-14C]benzenesulfonic acid and 4-(2-sulfo-3'-methoxy-4'-hydroxy-azobenzene-4-azo)-[U-14C]benzenesulf oni c acid, respectively. The synthesis of acid azo dyes 4-(2-hydroxy-1-naphthylazo)-[U-14C]benzenesulfonic acid and 4-(4-hydroxy-1-naphthylazo)-[U-14C]benzenesulfonic acid also allowed the abilities of these microorganisms to mineralize these commercially important compounds to be evaluated. Phanerochaete chrysosporium mineralized all of the sulfonated azo dyes, and the substitution pattern did not significantly influence the susceptibility of the dyes to degradation. In contrast, Streptomyces chromofuscus was unable to mineralize aromatics with sulfo groups and both sulfo and azo groups. However, it mediated the mineralization of modified dyes containing lignin-like substitution patterns. This work showed that lignocellulolytic fungi and bacteria can be used for the biodegradation of anionic azo dyes, which thus far have been considered among the xenobiotic compounds most resistant to biodegradation.(ABSTRACT TRUNCATED AT 250 WORDS)

Azo Compounds

Influence of aromatic substitution patterns on azo dye degradability by Streptomyces spp. and Phanerochaete chrysosporium.

Twenty-two azo dyes were used to study the influence of substituents on azo dye biodegradability and to explore the possibility of enhancing the biodegradabilities of azo dyes without affecting their properties as dyes by changing their chemical structures. Streptomyces spp. and Phanerochaete chrysosporium were used in the study. None of the actinomycetes (Streptomyces rochei A10, Streptomyces chromofuscus A11, Streptomyces diastaticus A12, S. diastaticus A13, and S. rochei A14) degraded the commercially available Acid Yellow 9. Decolorization of monosulfonated mono azo dye derivatives of azobenzene by the Streptomyces spp. was observed with five azo dyes having the common structural pattern of a hydroxy group in the para position relative to the azo linkage and at least one methoxy and/or one alkyl group in an ortho position relative to the hydroxy group. The fungus P. chrysosporium attacked Acid Yellow 9 to some extent and extensively decolorized several azo dyes. A different pattern was seen for three mono azo dye derivatives of naphthol. Streptomyces spp. decolorized Orange I but not Acid Orange 12 or Orange II. P. chrysosporium, though able to transform these three azo dyes, decolorized Acid Orange 12 and Orange II more effectively than Orange I. A correlation was observed between the rate of decolorization of dyes by Streptomyces spp. and the rate of oxidative decolorization of dyes by a commercial preparation of horseradish peroxidase type II, extracellular peroxidase preparations of S. chromofuscus A11, or Mn(II) peroxidase from P. chrysosporium. Ligninase of P. chrysosporium showed a dye specificity different from that of the other oxidative enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Azo Compounds

Comparison of extracellular peroxidase- and esterase-deficient mutants of Streptomyces viridosporus T7A.

Peroxidase-deficient mutants of the lignin-degrading bacterium Streptomyces viridosporus T7A were screened for their production of acid-precipitable polymeric lignin, extracellular peroxidases and esterases, and immunoreactivities against a polyclonal antibody produced against electrophoretically purified peroxidase isoform P3 of wild-type S. viridosporus. The mutants showed diminished abilities to solubilize lignin and produce acid-precipitable polymeric lignin. Their peroxidase activities were decreased, and their esterase production patterns were altered. Western immunoblots demonstrated that the mutants produced proteins immunologically reactive with the antibody, but with different mobilities from those of wild-type proteins. These findings confirm a direct role for peroxidases in lignin solubilization. They also indicate a possible role for esterases.

Esterases

Bioremediation of soils contaminated with the herbicide 2-sec-butyl-4,6-dinitrophenol (dinoseb).

A novel soil treatment method for achieving the removal of dinoseb (2-sec-butyl-4,6-dinitrophenol) from contaminated soils was investigated. One soil contained dinoseb as the major contaminant, although several other hazardous compounds were also present. A second soil was highly contaminated with dinoseb. Dinoseb was not degraded in these soils under the aerobic conditions at each site. Pretreatment of the soils by the addition of a starchy potato-processing by-product and flooding with phosphate buffer stimulated the consumption of oxygen and nitrate from the soils, thereby lowering the redox potential and creating anaerobic conditions. Anaerobiosis (Eh less than -200 mV) promoted the establishment of an anaerobic microbial consortium that degraded dinoseb completely, without the formation of the polymerization products seen under aerobic or microaerophilic conditions. When dinoseb was present at low concentrations in a chronically contaminated soil, the natural microflora was capable of establishing anaerobic conditions and degrading dinoseb as a result of starch degradation. Inoculation of this soil with an aerobic starch-degrading microorganism and then an acclimated, anaerobic, dinoseb-degrading consortium did not improve dinoseb degradation. In a second acutely contaminated soil, these inoculations improved dinoseb degradation rates over those of uninoculated controls.

2,4-Dinitrophenol

Influence of 2,4,6-trinitrotoluene (TNT) concentration on the degradation of TNT in explosive-contaminated soils by the white rot fungus Phanerochaete chrysosporium.

The ability of Phanerochaete chrysosporium to bioremediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5- triazine; 3,000 ppm) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives extracted from the soil were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Mycelium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt extract broth, spore-inoculated cultures mineralized 10% of added [14C]TNT (5 ppm) in 27 days at 37 degrees C. No mineralization occurred during [14C]TNT biotransformation by mycelium-inoculated cultures, although the TNT was transformed.

Basidiomycota

Selection and isolation of bacteria capable of degrading dinoseb (2-sec-butyl-4,6-dinitrophenol).

Dinoseb (2-sec-butyl-4,6-dinitrophenol) has been a widely used herbicide that persists in some contaminated soils, and has been found in groundwaters, causing health and environmental hazards. Persistence in some soils may stem from a lack of dinoseb-degrading organisms. We established a chemostat environment that was strongly selective for aerobic (liquid phase) and anaerobic (sediment phase) bacteria able to degrade dinoseb. The chemostat yielded five taxonomically diverse aerobic isolates that could transform dinoseb to reduced products under microaerophilic or denitrifying conditions, but these organisms were unable to degrade the entire dinoseb molecule, and the transformed products formed multimeric material. The chemostat also yielded an anaerobic consortium of bacteria that could completely degrade dinoseb to acetate and CO2 when the Eh of the medium was less than -200 mV. The consortium contained at least three morphologically different bacterial species. HPLC analysis indicated that dinoseb was degraded sequentially via several as yet unidentified products. Degradation of these intermediates was inhibited by addition of bromoethane sulfonic acid. GC-MS analysis of metabolites in culture medium suggested that regiospecific attacks occurred non-sequentially on both the nitro groups and the side-chains of dinoseb. The consortium was also able to degrade 4,6-dinitro-o-cresol, 3,5-dinitrobenzoic acid, 2,4-dinitrotoluene, and 2,6-dinitrotoluene via a similar series of intermediate products. The consortium was not able to degrade 2,4-dinitrophenol. To our knowledge, this is the first report of strictly anaerobic biodegradation of an aromatic compound containing a multicarbon, saturated hydrocarbon side chain.

2,4-Dinitrophenol

Synthesis and properties of lignin peroxidase from Streptomyces viridosporus T7A.

The production of lignin peroxidase by Streptomyces viridosporus T7A was studied in shake flasks and under aerobic conditions in a 7.5-L batch fermentor. Lignin peroxidase synthesis was found to be strongly affected by catabolite repression. Lignin peroxidase was a non-growth-associated, secondary metabolite. The maximum lignin peroxidase activity was 0.064 U/mL at 36 h. In order to maximize lignin peroxidase activity, optimal conditions were determined. The optimal incubation temperature, pH, and substrate (2,4-dichlorophenol) concentration for the enzyme assays were 45 degrees C, 6, and 3 mM, respectively. Stability of lignin peroxidase was determined at 37, 45, and 60 degrees C, and over the pH range 4-9.

Enzyme Repression

Immunologic relatedness of extracellular ligninases from the actinomycetes Streptomyces viridosporus T7A and Streptomyces badius 252.

Four isoforms of the extracellular lignin peroxidase of the ligninolytic actinomycete Streptomyces viridosporus T7A (ALip-P1, P2, P3, and P4) were individually purified by ultrafiltration and ammonium sulfate precipitation, followed by electro-elution using polyacrylamide gel electrophoresis. Three of the purified peroxidases were compared for their immunologic relatedness by Western blot analysis using a polyclonal antibody preparation produced in rabbits against pure isoform P3. The anti-P3 antibody was also tested for its reactivity towards a lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium and another ligninolytic actinomycete Streptomyces badius 252. Results showed that peroxidases ALip-P1 through ALip-P3 are immunologically related to one another. The peroxidases of S. badius, but not the peroxidase of P. chrysosporium, also reacted with the antibody, thus indicating that the lignin peroxidases of S. viridosporus and S. badius are immunologically related. Based upon its specific affinity, lignin peroxidase isoform ALip-P3 of S. viridosporus was readily purified using an anti-P3 antibody affinity column.

Blotting, Western

Cloning and expression of a lignin peroxidase gene from Streptomyces viridosporus in Streptomyces lividans.

A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. BglII-digested genomic DNA (4-10 kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. Transformants expressing pIJ702 with insert DNA were selected based upon the appearance of thiostrepton resistant (tsrr)/mel-colonies on regeneration medium. Lignin peroxidase-expressing clones were isolated from this population by screening of transformants on a tsr-poly B-411 dye agar medium. In the presence of H2O2 excreted by S. lividans, colonies of lignin peroxidase-expressing clones decolorized the dye. Among 1000 transformants screened, 2 dye-decolorizing clones were found. One, pIJ702/TK64.1 (TK64.1), was further characterized. TK64.1 expressed significant extracellular 2,4-dichlorophenol (2.4-DCP) peroxidase activity (= assay for S. viridosporus lignin peroxidase). Under the cultural conditions employed, plasmidless S. lividans TK64 had a low background level of 2.4-DCP oxidizing activity. TK64.1 excreted an extracellular peroxidase not observed in S. lividans TK64, but similar to S. viridosporus lignin peroxidase ALip-P3, as shown by activity stain assays on nondenaturing polyacrylamide gels. The gene was located on a 4 kb fragment of S. viridosporus genomic DNA. When peroxidase-encoding plasmid, pIJ702.LP, was purified and used to transform three different S. lividans strains (TK64, TK23, TK24), all transformants tested decolorized poly B-411. When grown on lignocellulose in solid state processes, genetically engineered S. lividans TK64.1 degraded the lignocellulose slightly better than did S. lividans TK64. This is the first report of the cloning of a bacterial gene coding for a lignin-degrading enzyme.

Cloning, Molecular