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.

Synthesis of aminoshikimic acid.

5-amino-5-deoxyshikimic acid (aminoshikimic acid) was synthesized from glucose using recombinant Amycolatopsis mediterranei and also synthesized by a tandem, two-microbe route employing Bacillus pumilus and recombinant Escherichia coli.

Plasma concentrations of flecainide acetate, a new antiarrhythmic agent, in humans.

Flecainide, a new antiarrhythmic agent, was given to eight healthy men to ascertain plasma drug levels and to assess tolerance for the drug. Each subject received a single intravenous (IV) dose (0.5, 1, 1.5, or 2 mg/kg) of flecainide, and plasma levels of unchanged flecainide were measured by gas-liquid chromatography. After an initial rapid distribution phase, the drug's plasma half-life (terminal elimination phase) ranged from seven to 15 hours (mean, 11 hours); half-life was apparently independent of dose level. Immediately after IV administration, the relatively high plasma drug levels seen during the short distribution phase were associated with minor and transient side effects in some subjects receiving the two higher doses; overall, the drug was well tolerated. Plasma flecainide levels 60 minutes after administration (after distribution) ranged from 52 to 300 ng/ml and were reasonably proportional to dose level. The drug's relatively long plasma half-life in humans indicates that plasma levels will be maintained for prolonged periods; thus flecainide should provide sustained therapeutic activity in patients with cardiac arrhythmias.

Inhibitor Ionization as a Determinant of Binding to 3-Dehydroquinate Synthase.

Phosphinomethyl and carboxymethyl monoacids along with succinyl, malonyl ether, malonyl, and hydroxymalonyl diacids were substituted for phosphorylmethyl, phosphonoethyl, and phosphonomethyl groups in carbocyclic inhibitors of DHQ synthase. All but one of the carbocyclic inhibitors were synthesized via intermediacy of a 2,3-butane bisacetal-protected 3-dehydroquinic acid. Carbaphosphinate (K(i) = 20 x 10(-)(6) M) was a modest competitive inhibitor of DHQ synthase, while carbaacetate was a linear mixed-type inhibitor (K(i) = 3 x 10(-)(6) M, K(i)' = 20 x 10(-)(6) M). Carbasuccinate (K(i) = 5 x 10(-)(6) M), carbamalonate ether (K(i) = 7 x 10(-)(6) M), carbamalonate (K(i) = 0.7 x 10(-)(6) M), and carbahydroxymalonate (K(i) = 0.3 x 10(-)(6) M) were all competitive inhibitors. Carbaacetate was the only inhibitor that was not oxidized by DHQ synthase. On the basis of these data, carbocyclic inhibitors with malonyl and hydroxymalonyl groups are apparently bound by DHQ synthase as tightly as carbocyclic inhibitors possessing phosphorylmethyl and phosphonoethyl moieties.

Microbial synthesis of 3-dehydroshikimic acid: a comparative analysis of D-xylose, L-arabinose, and D-glucose carbon sources.

3-Dehydroshikimic acid is a hydroaromatic precursor to chemicals ranging from L-phenylalanine to adipic acid. The concentration and yield of 3-dehydroshikimic acid microbially synthesized from various carbon sources has been examined under fed-batch fermentor conditions. Examined carbon sources included D-xylose, L-arabinose, and D-glucose. A mixture consisting of a 3:3:2 molar ratio of glucose/xylose/arabinose was also evaluated as a carbon source to model the composition of pentose streams potentially resulting from the hydrolysis of corn fiber. Escherichia coli KL3/pKL4.79B, which overexpresses feedback-insensitive DAHP synthase, synthesizes higher concentrations and yields of 3-dehydroshikimic acid when either xylose, arabinose, or the glucose/xylose/arabinose mixture is used as a carbon source relative to when glucose alone is used as a carbon source. E. coli KL3/pKL4.124A, which overexpresses transketolase and feedback-insensitive DAHP synthase, synthesizes higher concentrations and yields of 3-dehydroshikimic acid when the glucose/xylose/arabinose mixture is used as the carbon source relative to when either xylose or glucose is used as a carbon source. Observed high-titer, high-yielding synthesis of 3-dehydroshikimic acid from the glucose/xylose/arabinose mixture carries significant ramifications relevant to the employment of corn fiber in the microbial synthesis of value-added chemicals.

Benzene-free synthesis of adipic acid.

Strains of Escherichia coli were constructed and evaluated that synthesized cis,cis-muconic acid from D-glucose under fed-batch fermentor conditions. Chemical hydrogenation of the cis,cis-muconic acid in the resulting fermentation broth has also been examined. Biocatalytic synthesis of adipic acid from glucose eliminates two environmental concerns characteristic of industrial adipic acid manufacture: use of carcinogenic benzene and benzene-derived chemicals as feedstocks and generation of nitrous oxide as a byproduct of a nitric acid catalyzed oxidation. While alternative catalytic syntheses that eliminate the use of nitric acid have been developed, most continue to rely on petroleum-derived benzene as the ultimate feedstock. In this study, E. coli WN1/pWN2.248 was developed that synthesized 36.8 g/L of cis,cis-muconic acid in 22% (mol/mol) yield from glucose after 48 h of culturing under fed-batch fermentor conditions. Optimization of microbial cis,cis-muconic acid synthesis required expression of three enzymes not typically found in E. coli. Two copies of the Klebsiella pneumoniae aroZ gene encoding DHS dehydratase were inserted into the E. coli chromosome, while the K. pneumoniae aroY gene encoding PCA decarboxylase and the Acinetobacter calcoaceticus catA gene encoding catechol 1,2-dioxygenase were expressed from an extrachromosomal plasmid. After fed-batch culturing of WN1/pWN2.248 was complete, the cells were removed from the broth, which was treated with activated charcoal and subsequently filtered to remove soluble protein. Hydrogenation of the resulting solution with 10% Pt on carbon (5% mol/mol) at 3400 kPa of H2 pressure for 2.5 h at ambient temperature afforded a 97% (mol/mol) conversion of cis,cis-muconic acid into adipic acid.

Modulation of phosphoenolpyruvate synthase expression increases shikimate pathway product yields in E. coli.

Product yields in microbial synthesis are ultimately limited by the mechanism utilized for glucose transport. Altered expression of phosphoenolpyruvate synthase was examined as a method for circumventing these limits. Escherichia coli KL3/pJY1.216A was cultured under fed-batch fermentor conditions where glucose was the only source of carbon for the formation of microbial biomass and the synthesis of product 3-dehydroshikimic acid. Shikimate pathway byproducts 3-deoxy-D-arabino-heptulosonic acid, 3-dehydroquinic acid, and gallic acid were also generated. An optimal expression level of phosphoenolpyruvate synthase was identified, which did not correspond to the highest expression levels of this enzyme, where the total yield of 3-dehydroshikimic acid and shikimate pathway byproducts synthesized from glucose was 51% (mol/mol). For comparison, the theoretical maximum yield is 43% (mol/mol) for synthesis of 3-dehydroshikimic acid and shikimate pathway byproducts from glucose in lieu of amplified expression of phosphoenolpyruvate synthase.

Altered glucose transport and shikimate pathway product yields in E. coli.

Different glucose transport systems are examined for their impact on phosphoenolpyruvate availability as reflected by the yields of 3-dehydroshikimic acid and byproducts 3-deoxy-d-arabino-heptulosonic acid, 3-dehydroquinic acid, and gallic acid synthesized by Escherichia coli from glucose. 3-Dehydroshikimic acid is an advanced shikimate pathway intermediate in the syntheses of a spectrum of commodity, pseudocommodity, and fine chemicals. All constructs carried plasmid aroF(FBR) and tktA inserts encoding, respectively, a feedback-insensitive isozyme of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase and transketolase. Reliance on the native E. coli phosphoenolpyruvate:carbohydrate phosphotransferase system for glucose transport led in 48 h to the synthesis of 3-dehydroshikimic acid (49 g/L) and shikimate pathway byproducts in a total yield of 33% (mol/mol). Use of heterologously expressed Zymomonas mobilis glf-encoded glucose facilitator and glk-encoded glucokinase resulted in the synthesis in 48 h of 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 41% (mol/mol). Recruitment of native E. coli galP-encoded galactose permease for glucose transport required 60 h to synthesize 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 43% (mol/mol). Direct comparison of the impact of altered glucose transport on the yields of shikimate pathway products synthesized by E. coli has been previously hampered by different experimental designs and culturing conditions. In this study, the same product and byproduct mixture synthesized by E. coli constructs derived from the same progenitor strain is used to compare strategies for increasing phosphoenolpyruvate availability. Constructs are cultured under the same set of fermentor-controlled conditions.

Phosphoenolpyruvate availability and the biosynthesis of shikimic acid.

The impact of increased availability of phosphoenolpyruvate during shikimic acid biosynthesis has been examined in Escherichia coli K-12 constructs carrying plasmid-localized aroF(FBR) and tktA inserts encoding, respectively, feedback-insensitive 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase and transketolase. Strategies for increasing the availability of phosphoenolpyruvate were based on amplified expression of E. coli ppsA-encoded phosphoenolpyruvate synthase or heterologous expression of the Zymomonas mobilis glf-encoded glucose facilitator. The highest titers and yields of shikimic acid biosynthesized from glucose in 1 L fermentor runs were achieved using E. coli SP1.lpts/pSC6.090B, which expressed both Z. mobilis glf-encoded glucose facilitator protein and Z. mobilis glk-encoded glucose kinase in a host deficient in the phosphoenolpyruvate:carbohydrate phosphotransferase system. At 10 L scale with yeast extract supplementation, E. coli SP1.lpts/pSC6.090B synthesized 87 g/L of shikimic acid in 36% (mol/mol) yield with a maximum productivity of 5.2 g/L/h for shikimic acid synthesized during the exponential phase of growth.

Safety tests with Flury HEP strain 675 in wild-living European mammals.

For the evaluation of residual pathogenicity of Flury HEP strain 675, the vaccinal virus was orally administered to 207 animals belonging to 15 wild-living and 1 domestic species. Rabies virus antigen could be demonstrated by FA method in 20 animals. Reisolation of the virus was possible from 23 animals within the first two weeks after application, the titres being very low. During the observation period of 100 days no symptoms of rabies could be observed in the animals tested.

Effectivity of oral vaccination of foxes against rabies with Flury HEP strain 675.

A group of 14 adult foxes was orally vaccinated with 2 ml Flury HEP strain 675, titre 10(8.2 TCID50. Each fox seroconverted neutralizing antibody titres ranging between 1:40 and 1:640. These foxes survived an intramuscular challenge with 8000 mouse LD50 pathogenic rabies virus, whereas six out of seven unprotected control foxes died within 28 days. This study was accompanied by virus isolation, fluorescence microscopic examination and neutralization tests.

[Enrofloxacin (Baytril)--an alternative for psittacosis prevention and therapy in imported psittacines]. / Enrofloxacin (Baytril)--eine Alternative in der Psittakoseprophylaxe und -therapie bei importierten Psittaciden.

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.

[Coxiella burnetii as zoonotic pathogen with special regard to food hygiene]. / Coxiella bumetii als Zoonoseerreger unter besonderer Berücksichtigung der Lebensmittelhygiene.

In Hesse, Germany, bulk milk of farms producing raw milk cheese is examined by PCR for Coxiella burnetii yearly. In 2003 the pathogen has been detected unusually frequent. By means of two examples the hygienic measures are shown, which were initiated by the veterinary administration. To detect Coxiella burnetii means always the preoccupation with unsolved questions. It is particularly uncertain, whether there is a risk of oral infection for the human being. From the point of view of food hygiene, surveys are needed urgently to work out a risk assessment. Based on this a uniform risk management and a reasonable risk communication can be fixed.