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J W Frost

Publications and source records attributed to J W Frost.

At least 37 records · Page 2Linked to original sources
PubMed1996

Evidence for acetyl coenzyme A and cinnamoyl coenzyme A in the anaerobic toluene mineralization pathway in Azoarcus tolulyticus Tol-4.

A toluene-degrading denitrifier, Azoarcus tolulyticus Tol-4, was one of eight similar strains isolated from three petroleum-contaminated aquifer sediments. When the strain was grown anaerobically on toluene, 68% of the carbon from toluene was found as CO2 and 30% was found as biomass. Strain Tol-4 had a doubling time of 4.3 h, a Vmax of 50 micromol x min-1 x g of protein-1, and a cellular yield of 49.6 g x mol of toluene-1. Benzoate appeared to be an intermediate, since F-benzoates accumulated from F-toluenes and [14C]benzoate was produced from [14C]toluene in the presence of excess benzoate. Two metabolites, E-phenylitaconic acid (1 to 2%) and benzylsuccinic acid (<1%), accumulated from anaerobic toluene metabolism. These same products were also produced when cells were grown on hydrocinnamic acid and trans-cinnamic acid but were not produced from benzylalcohol, benzaldehyde, benzoate, p-cresol, or their hydroxylated analogs. The evidence supports an anaerobic toluene degradation pathway involving an initial acetyl coenzyme A (acetyl-CoA) attack in strain Tol-4, as proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for another toluene-degrading denitrifier, strain T1. Our findings support a modification of the proposed pathway in which cinnamoyl-CoA follows the oxidation of hydrocinnamoyl-CoA, analogous to the presumed oxidation of benzylsuccinic acid to form E-phenylitaconic acid. Cinnamic acid was detected in Tol-4 cultures growing in the presence of toluene and [14C]acetate. We further propose a second acetyl-CoA addition to cinnamoyl-CoA as the source of benzylsuccinic acid and E-phenylitaconic acid. This pathway is supported by the finding that monofluoroacetate added to toluene-growing cultures resulted in a significant increase in production of benzylsuccinic acid and E-phenylitaconic acid and by the finding that [14C]benzylsuccinic acid was detected after incubation of cells with toluene, [14C]acetate, and cinnamic acid. Evidence for anaerobic toluene metabolism by methyl group oxidation was not found, since benzylsuccinic acid and E-phenylitaconic acid were not detected after incubation with benzylalcohol and benzaldehyde, nor were benzylalcohol and benzaldehyde detected even in 14C trapping experiments.

Acetyl Coenzyme A↗
PubMed1996

Benzylfumaric, benzylmaleic, and Z- and E-phenylitaconic acids: synthesis, characterization, and correlation with a metabolite generated by Azoarcus tolulyticus Tol-4 during anaerobic toluene degradation.

E-Phenylitaconic acid has been isolated as a metabolite generated by Azoarcus tolulyticus Tol-4 along with benzylsuccinic acid during anaerobic degradation of toluene. Strain Tol-4 converted 1 to 2% of toluene carbon to E-phenylitaconate and benzylsuccinate (10:1). The identification of E-phenylitaconic acid was based on 1H nuclear magnetic resonance (NMR) characterization of degradation products derived from 13C-labeled toluene followed by comparison of spectroscopic and chromatographic data for the isolated, unlabeled metabolite with those for chemically synthesized benzylfumaric acid, benzylmaleic acid, E-phenylitaconic acid, and Z-phenylitaconic acid. Spectroscopic comparisons included 1H NMR, 13C NMR, and nuclear overhauser effect correlations. High-pressure liquid chromatography (HPLC) retention times and HPLC coinjections with synthetic dioic acids provided another reliable line of evidence for structure assignment. The formation of E-phenylitaconic acid differs from previous reports of benzylfumaric acid generation along with benzylsuccinic acid during anaerobic microbial degradation of toluene. This has important implications relevant to elaboration of the metabolic route for anaerobic toluene degradation by strain Tol-4 and related organisms. Similar amounts of E-phenylitaconic acid were also produced by seven other strains of A. tolulyticus.

Benzyl Compounds↗
PubMed1995

Biocatalytic syntheses of aromatics from D-glucose: renewable microbial sources of aromatic compounds.

Chemistry is moving into a new era in which renewable resources and starting materials such as D-glucose will likely be prominent features of industrial chemical manufacture. The keys to this progress are the design, development, and use of microbial biocatalysts. Aromatic biosynthesis serves as a paradigm for how biocatalysts can be manipulated to achieve the yield, rate, and purity criteria central to chemical manufacture. A disproportionate amount of the metabolic carbon flow of the biocatalyst must first be directed into the common pathway of aromatic amino acid biosynthesis. This review describes ways of achieving this goal through the traditional strategy of manipulating the catalytic activity of the first enzyme in the common pathway, as well as the amelioration of limitations in the in vivo availability of common-pathway enzyme substrates. The inability of individual enzymes to convert their substrate to product fast enough to avoid substrate accumulation further impedes carbon flow through the common pathway. This review also discusses identification and removal of these rate-limiting enzymes. Finally, we examine the creation of heterologous biocatalysts and how biocatalysis could be integrated with traditional chemical transformations to expand the number of organic chemicals that can be synthesized from glucose.

Amino Acids↗
PubMed1994

Biocatalytic desulfurization of arylsulfonates.

A microbial strain, Klebsiella oxytoca KS3D, has been isolated which is capable of exploiting arylsulfonates as a sole source of sulfur during growth. The desulfurization catalyzed by intact K. oxytoca KS3D results in the conversion of arylsulfonates into the corresponding phenols. Even arylsulfonates carrying substituents which significantly alter steric and electronic characteristics are substrates. Only a single regioisomer is produced from substituted arylsulfonates. Based on the products formed from the biocatalytic desulfurizations and incorporation of isotopic oxygen in phenolic product when the desulfurization is run under 18O-enriched oxygen, hydrolysis mechanisms can be eliminated from consideration. Two reaction types which might mimic the chemistry occurring during microbial desulfurization of arylsulfonates were examined. The first reaction involved conversion of appropriately substituted arylsulfonates into phenols by single electron reduction followed by reaction of the radical anions with molecular oxygen. A second reaction using intramolecular reaction of arylsulfonates and arylsulfones with alkoxy radicals failed to achieve desulfurization. In addition to mechanistic evaluation, desulfurization of arylsulfonates catalyzed by K. oxytoca KS3D is examined from the perspective of its relevance to desulfurization of the organosulfur components of coal and its possible use for industrial manufacture of phenols.

Arylsulfonates↗
PubMed1993

[Enrofloxacin (Baytril)--an alternative for psittacosis prevention and therapy in imported psittacines].

It was the aim of the presented work to test Enrofloxacin under field conditions of compulsory prophylaxis and treatment of psittacosis, as JUNG (1993) had already demonstrated the effectiveness of this antibiotic against Chlamydia psittaci. During official quarantine 22 groups comprising 2536 birds were treated with Baytril. With suitable feed like steamed maize and a dose of 500 mg/kg Enrofloxacin the mean blood values ranged from 0.9 to 4.1 micrograms/ml within the different species. Application via drinking water can only be advised as an alternative in sick birds with reduced food intake. Duration of excretion (limit was the minimal inhibitory dose of Enrofloxacin with Chlamydia psittaci, 0.125 micrograms/ml was depending on the uptake of Enrofloxacin and correlated with serum levels. With mean serum levels of 1.0 micrograms/ml the minimal inhibitory dose for Chlamydia psittaci was attained in all birds after 14 days. The tissue concentrations measured in Patagonian conures surpassed serum levels several-fold, with the exception of brain tissue. Out of 22 quarantine groups only two were naturally infected with Chlamydia. One group of 196 Senegal parrots could only be cured from chlamydial infection after substituting their normal mixed feed with medicated maize containing 1000 mg/kg Enrofloxacin. In 50 per cent of the Salmonella infected groups Salmonella could again be grown from faecal samples after the end of treatment. For the treatment of psittacosis mean blood levels of 0.5 micrograms/ml are imperative and duration of treatment should not be below 14 days. Satisfactory hygienic conditions provided Baytril is a valuable therapeutic drug against psittacosis.

Animals↗
PubMed1992

Isolation and characterization of 115 street rabies virus isolates from Ethiopia by using monoclonal antibodies: identification of 2 isolates as Mokola and Lagos bat viruses.

There were 115 isolates of rabies viruses recovered by tissue culture technique from 119 animal brains collected in Ethiopia. By using 17 selected antinucleocapsid monoclonal antibodies (MAbs), 113 isolates were classic street rabies viruses (serotype 1). An isolate of feline origin (Eth-16) was a Mokola virus (serotype 3) and another isolate (Eth-58, obtained from a rabid dog) was serotype 2 (Lagos bat virus). None of the 16 antiglycoprotein MAbs used neutralized the Eth-16 isolate, whereas Eth-58 was neutralized by 1 (TERA543). Antirabies vaccines prepared from Pitman-Moore and Pasteur virus strains protected mice against homologous challenge, but neither was protective against the 2 rabies-related virus isolates. The isolation of Mokola and Lagos bat viruses from domestic animals in eastern Africa is of public and veterinary concern mainly due to lack of effective vaccines against these agents and the difficulty of proper diagnosis.

Animals↗
PubMed1990

Evaluation of five different methods for routine diagnosis of rabies.

Brain tissue from 187 animals of different species was investigated by means of fluorescent antibody test, peroxidase anti-peroxidase technique, mouse inoculation test and cell culture technique for a diagnosis of rabies. With peroxidase anti-peroxidase technique the rabies specific reaction comprised inclusion bodies and a granular staining of the cytoplasm of affected cells. A specific positive reaction was found only in neurons, in which perikaryon as well as cell processes were affected. Fluorescent antibody test and peroxidase anti-peroxidase technique detected 98% each, mouse inoculation test 95% and cell culture technique 81% of the rabies positive animals. In conclusion, peroxidase anti-peroxidase technique allows a highly reliable diagnosis of rabies when only formalin-fixed and paraffin-embedded material is available. Histopathological alterations comprising Negri bodies, inflammatory and degenerative lesions were encountered in 53% of the rabies positive brains.

Animals↗
PubMed1989

Enzyme-linked immunosorbent assay (ELISA) using staphylococcal protein A for the measurement of rabies antibody in various species.

An ELISA was developed using staphylococcal protein A linked with horseradish peroxidase for detecting IgG antibody of rabies virus in human and carnivore sera (80 human, 270 fox, 40 cat, 35 marten, 5 badger and 4 polecat sera were tested in the present work). In comparison with the serum neutralization (SN) test in cell culture, close overall agreement was obtained particularly in human and cat sera (97.5%). Two post-vaccination human sera were found positive with ELISA values of 2.16 and 2.65 IU/0.2 ml, but with SN titers less than 1:10. All prevaccination human sera were found negative by both tests. Regression analysis on 30 post-vaccination human sera revealed better correlation between ELISA and SN test at a serum dilution of 1:100 than at lower serum dilutions of 1:80 or 1:20. The correlation coefficient (r) was 0.73 (p less than or equal to 0.0001).

Animals↗
PubMed1987

Genetic characterization of an Escherichia coli mutant deficient in organophosphonate biodegradation.

An E. coli mutant deficient in organophosphonate biodegradation has been complemented with a cosmid library prepared from a BamHI partial digest of wild-type E. coli W3110. Mutant E. coli SL724, when transformed with cosmid pSL163 and plasmid pSL263, regained the ability to exploit ethylphosphonate as a sole source of phosphorus during growth. In route to complementation, the Tn5 insert of SL724 was subcloned and restriction enzyme mapped. Complementing pSL163 and pSL263 were also characterized via restriction enzyme digests.

Cosmids↗
PubMed1987

Q fever vaccines for animals.

The results of different workgroups and those obtained in own investigations with Q fever vaccines for cattle and sheep are reviewed and discussed. In field trials Coxiella (C.) burnetii vaccines of inactivated whole cells (WC) in phase (ph) I as well as WC ph II vaccines prevented infections of cows exposed to naturally infected environments, provided they were vaccinated as noninfected calves. To monitor calves for this purpose the sensitive ELISA has superseded the complement fixation test (CF). WC ph I vaccines failed to prevent shedding by uninfected cattle and sheep which were vaccinated and subsequently challenged by parenteral inoculation with viable organisms, but reduced shedding, and prevented pathological lesions and clinical symptoms. The results obtained in field trials with a commercially available vaccine consisting of killed WC ph II of C. burnetii and inactivated WC of Chlamydia psittaci indicated that vaccination had significantly improved fertility in vaccinated herds. However, the mechanism of protection is unclear because the function of the chlamydial component in this vaccine is not yet defined. Cattle given this vaccine can be distinguished from naturally infected cattle because the former produce predominantly non-complement-binding IgG2, detectable by ELISA. This vaccine frequently causes undesirable local reactions. Antigens of C. burnetii (strain "Frankfurt", ph II) prepared by propagation in cell cultures and successive purification by guanidinium hydrochloride- and chloroform/methanol-extraction (CMR), as well as a major protein extracted from these CMR preparations represent potential vaccine candidates without such side effects.

Animals↗
PubMed1987

[The detection of antibodies against the feline infectious peritonitis (FIP) virus using the indirect immunofluorescence test].

The present study was carried out to detect FIP-virus antibodies in feline sera and peritoneal fluids of unknown history and origin. During a five years period a total of 1976 blood samples and exudates from cats were investigated by a recently developed immunofluorescence assay. 43 and 70 percent of sera and punctates were positive for FIP-virus-reacting antibodies. Special emphasis is given to the reliability of the test procedure. It was ascertained by comparing post mortem findings of pathognomonic diseased cats with fluorescence-serological results. The technique used could routinely be applied. It is assumed that the prevalence of FIP-virus infections in our cat population is spreading.

Animals↗
PubMed1986

A plant flavone, luteolin, induces expression of Rhizobium meliloti nodulation genes.

The symbiotic interaction of Rhizobium meliloti and alfalfa results in the formation of nitrogen-fixing root nodules. Rhizobium meliloti nodABC genes are required for the early host responses of cortical cell divisions and root hair curling. The induction of nodABC expression by alfalfa exudates demonstrates host-symbiont signaling at an early stage in nodule development. The inducer molecule for nodABC expression was isolated from plant exudate by constructing a nodABC-lacZ fusion to monitor the inducing activity. From ultraviolet-visible absorption spectra, proton nuclear magnetic resonance, and mass spectrometry, the inducer was determined to be 3',4', 5,7-tetrahydroxyflavone (luteolin). Luteolin is a normal secondary plant metabolite found throughout the plant kingdom that may serve to control nodABC expression during nodule development. This regulatory role for a flavone contrasts with the function of some flavonoids as defense compounds.

Flavonoids↗
PubMed1986

[Experiences with oral immunization of foxes against rabies in Hesse].

Since spring 1983 a field trial to immunize foxes by the oral route against rabies has been carried out in a heavily infected area of Hessen measuring about 1,500 km2. The vaccine strain SAD B 19, produced by the Federal Research Institute for Animal Virus Diseases at Tübingen and filled into 2 ml aliquots, was inserted into chicken heads and distributed by forest rangers in the trial area, 15 baits per km2. Up to the present day five baiting campaigns, two per year in spring and autumn, have been completed and in all 110,000 vaccine baits additionally marked with 150 mg tetracycline were layed out. Four baitings have been previously evaluated. With the exception of some rabies cases on the border to the unvaccinated region in the northwest, rabies was reduced impressively. A secondary outbreak in the region of Hausen/Kiedrich, caused by an extremely high density of the fox population, was eradicated by increasing the baits to 25 per km2. The number of rabies cases, which amounted to 238 within 15 months prior to the vaccination trial, went down from 38 in the first 6 months to 6 cases after the fourth baiting. As innocuity control 2,273 wild and domestic animals from the vaccination area were examined for the presence of the vaccine virus. The results were always negative indicating that rabies strain SAD B 19 had not established an infective cycle in wildlife. As could be shown by the marker studies, an average of 78 per cent of the foxes (variation of the baitings from 62 per cent to 82 per cent) had taken up one or more baits. Seroconversion had been achieved in 65 per cent of the foxes (variation from 43 per cent to 70 per cent) as demonstrated by the presence of neutralizing antibodies. From experiences in Switzerland it can be expected, that rabies will be extinguished, if 50 per cent of the population are successfully immunized. This value has been surpassed in the field trial in Hessen. These very promising results have led to the project of an enlargement of the vaccination area to 12,000 km2. The neighbouring states of Rheinland-Pfalz and Nordrhein-Westfalen have joined in this effort, which has started in autumn 1985 with a new artificial vaccine bait developed at the Tübingen research institute.

Administration, Oral↗
PubMed1984

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate: chemical synthesis and isolation from Escherichia coli auxotrophs.

A new chemical synthesis of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate is described and contrasted to isolation of the same molecule from the growth medium of several different auxotrophic strains of Escherichia coli. The higher yielding chemical synthesis begins with 2-deoxyglucose while the less time-intensive biological approach proceeds directly from glucose. Growth and accumulation aspects of whole cell biological synthesis are discussed along with various aspects of the biological purification protocol. Both approaches can be utilized to produce substantial quantities of methyl (methyl 3-deoxy-D-arabino-heptulopyranosid)onate, a key intermediate for semisynthetic 3-deoxy-D-arabino-heptulosonic acid 7-phosphate and a number of its derivatives.

Deoxyglucose↗
PubMed1984

Dehydroquinate synthase from Escherichia coli: purification, cloning, and construction of overproducers of the enzyme.

Dehydroquinate synthase has been purified 9000-fold from Escherichia coli K-12 (strain MM294). The synthase is encoded by the aroB gene, which is carried by plasmid pLC29-47 from the Carbon-Clarke library. Construction of an appropriate host bearing pLC29-47 results in a strain that produces 20 times more enzyme than strain MM294. Subcloning of the aroB gene behind a tac promoter results in E. coli transformants that produce 1000 times more enzyme than MM294: the synthase constitutes 5% of the soluble protein of the cell. A laborious isolation from 50 g of wild-type E. coli cells yields 80 micrograms of impure enzyme, whereas 50 g of cells containing the subcloned gene yields 150 mg of homogeneous enzyme in a two-column purification. Dehydroquinate synthase is a monomeric protein of Mr 40 000-44 000. The chromosomal enzyme from E. coli K-12, the cloned enzyme encoded by the plasmid pLC29-47, and the subcloned inducible enzyme encoded by pJB14 all comigrate on polyacrylamide gel electrophoresis under denaturing conditions.

Chromatography, Gel↗