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J W Frost

Publications and source records attributed to J W Frost.

At least 19 recordsLinked to original sources
PubMed2000

Synthesis of gallic acid: Cu(2+)-mediated oxidation of 3-dehydroshikimic acid.

With the elaboration of high-yielding, high-titer syntheses of 3-dehydroshikimic acid from glucose using recombinant Escherichia coli, oxidation of this hydroaromatic becomes a potential route for synthesis of gallic acid. Conversion of 3-dehydroshikimic acid into gallic acid likely proceeds via initial enolization of an alpha-hydroxycarbonyl and oxidation of the resulting enediol. 3-Dehydroshikimate enolization in water was catalyzed by inorganic phosphate while Zn(2+) was used to catalyze enolization in acetic acid. Enediol oxidation employed Cu(2+) as either the stoichiometric oxidant or as a catalyst in the presence of a cooxidant. Gallic acid was produced in a yield of 36% when 3-dehydroshikimic acid in phosphate-buffered water reacted for 35 h with H2O2 and catalytic amounts of CuSO(4). 3-Dehydroshikimate-containing, phosphate-buffered culture supernatants reacted with stoichiometric amounts of CuCO(3)Cu(OH)(2) and Cu(x)(H(3-x)(PO4)(2) to give gallic acid in yields of 51% in 5 h and 43% in 12 h, respectively. Solutions of 3-dehydroshikimic acid in acetic acid reacted with stoichiometric amounts of Cu(OAc)(2) to afford a 74% yield of gallic acid in 36 h. Acetic acid solutions of 3-dehydroshikimic acid could also be oxidized by air using catalytic quantities of Cu(OAc)(2). ZnO accelerated these oxidations leading to a 67% yield of gallic acid in 4 h when an acetic acid solution of 3-dehydroshikimic acid was reacted with O(2) and a catalytic amount of Cu(OAc)(2).

Catalysis↗
PubMed1999

Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinant Escherichia coli.

3-Dehydroshikimic acid (DHS), in addition to being a potent antioxidant, is the key hydroaromatic intermediate in the biocatalytic conversion of glucose into aromatic bioproducts and a variety of industrial chemicals. Microbial synthesis of DHS, like other intermediates in the common pathway of aromatic amino acid biosynthesis, has previously been examined only under shake flask conditions. In this account, synthesis of DHS using recombinant Escherichia coli constructs is examined in a fed-batch fermentor where glucose availability, oxygenation levels, and solution pH are controlled. DHS yields and titers are also determined by the activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. This enzyme's expression levels, sensitivity to feedback inhibition, and the availability of its substrates, phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P), dictate its in vivo activity. By combining fed-batch fermentor control with amplified expression of a feedback-insensitive isozyme of DAHP synthase and amplified expression of transketolase, DHS titers of 69 g/L were synthesized in 30% yield (mol/mol) from D-glucose. Significant concentrations of 3-dehydroquinic acid (6.8 g/L) and gallic acid (6.6 g/L) were synthesized in addition to DHS. The pronounced impact of transketolase overexpression, which increases E4P availability, on DHS titers and yields indicates that PEP availability is not a limiting factor under the fed-batch fermentor conditions employed.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1998

Structure of dehydroquinate synthase reveals an active site capable of multistep catalysis.

Dehydroquinate synthase (DHQS) has long been regarded as a catalytic marvel because of its ability to perform several consecutive chemical reactions in one active site. There has been considerable debate as to whether DHQS is actively involved in all these steps, or whether several steps occur spontaneously, making DHQS a spectator in its own mechanism. DHQS performs the second step in the shikimate pathway, which is required for the synthesis of aromatic compounds in bacteria, microbial eukaryotes and plants. This enzyme is a potential target for new antifungal and antibacterial drugs as the shikimate pathway is absent from mammals and DHQS is required for pathogen virulence. Here we report the crystal structure of DHQS, which has several unexpected features, including a previously unobserved mode for NAD+-binding and an active-site organization that is surprisingly similar to that of alcohol dehydrogenase, in a new protein fold. The structure reveals interactions between the active site and a substrate-analogue inhibitor, which indicate how DHQS can perform multistep catalysis without the formation of unwanted by-products.

Alcohol Oxidoreductases↗
PubMed1997

Study on the suitability of sow colostrum for the serological diagnosis of porcine reproductive and respiratory syndrome (PRRS).

Serum and colostrum from 73 sows were collected. The serum samples were tested by Immuno. Peroxidase Monolayer Assay (IPMA) and the corresponding colostrum samples with the indirect Immuno fluorescent Antibody (IFA) technique. All serum positive sows were colostrum positive and all colostrum negative were serum negative. Eight sows only reacted positively in the colostral testing. Compared to the serum standard test the specificity was 82.6% and the sensitivity 100%. The observed agreement between both tests was 89.2%. In addition all serum samples were also tested with the IF test (IFT). Of the eight sows which were negative in the IPMA serum test and positive in the IFA colostrum test, three were found positive when the serum was tested with IFA. Consequently, the observed agreement was higher at 93.2%. After the suitability of colostrum for porcine reproductive and respiratory syndrome (PRRS) diagnosis was demonstrated, 1915 colostrum samples collected from 135 different farms were tested in a comparative study with the IPMA and IFA techniques. Of the 1915 colostrum samples 139 were positive with both IPMA and IFA. With IPMA only, 43 samples were positive compared with 192 samples found positive with the IFA technique. A total of 1541 samples were negative in both tests. The observed agreement between both tests was 87.5%. The quotient of the observed agreement minus chance agreement and the maximum possible agreement beyond chance level (Kappa Quotient) was 0.49. In 90% of the farms that tested IFA positive there was a seroconversion of more than 50% of all colostrum tested. By comparison only 29% of the IPMA positive farms were positive with more than 50%. Based on the epidemiological findings on PRRS it was concluded that the IFA technique indicates a higher sensitivity for the detection of PRRS virus antibodies in sow colostrum. Finally the possible advantages and disadvantages of sow colostrum testing and serum testing are discussed.

Animals↗
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↗