Cloning and expression in Escherichia coli of the obtusifoliol 14α-demethylase of Sorghum bicolor (L.) Moench, a cytochrome P450 orthologous to the sterol 14α-demethylases (CYP51) from fungi and mammals

Obtusifoliol 14β-demethylase from Sorghum bicolor (L.) Moench has been cloned using a gene-specific probe generated using PCR primers designed from an internal 14 amino acid sequence. The sequence identifies sorghum obtusifoliol 14α-demethylase as a cytochrome P450 and it is assigned to the CYP51 family together with the sterol 14α-demethylases from fungi and mammals. The presence of highly conserved regions in the amino acid sequences, analogous substrates and the same metabolic role demonstrate that the sterol 14α-demethylases are orthologous enzymes. The sterol 14α-demethylases catalyse an essential step in sterol biosynthesis as evidenced by the absence of a 14α-methyl group in all known functional sterols. A functional sorghum obtusifoliol 14α-demethylase was expressed at high levels in Escherichia coli and purified using an efficient method based on temperature-induced Triton X-114 phase partitioning. The recombinant purified enzyme produced a type I spectrum with obtusifoliol as substrate. Reconstitution of purified recombinant enzyme with sorghum NADPH—cytochrome P450 reductase in dilaurylphosphatidylcholine micelles confirms that obtusifoliol 14α-demethylase catalyses the 14α-demethylation of obtusifoliol to 4α-methyl-5α-ergosta-8,14,24(28)-trien-3β-ol as evidenced by GC—MS. The isolation of a cDNA clone encoding the plant sterol 14α-demethylase, combined with the previously isolated cDNA clones for fungal and mammalian sterol 14α-demethylases, provides an important tool in the rational design of specific inhibitors towards the individual sterol 14α-demethylases.     

PHYLOGENYAND HISTORICAL ECOLOGY OF THE DESMARESTIACEAE (PHAEOPHYCEAE) SUPPORT A SOUTHERN HEMISPHERE ORIGIN1

Phylogenetic relationships in the Desmarestiales (Phaeophyceae) were inferred among the monotypic Arthrocladia (Arthrocladiaceae) and 27 isolates from Desmarestiaceae, representing 17 taxa of Desmarestia and the monotypic Antarctic genera Himantothallus and Phaeurus. Phaeurus and Arthrocladia were used as outgroups. Parsimony analyses of nuclear ribosomal DNA internal transcribed spacer (ITS1 and ITS2) sequences, in which gaps were both included and excluded, yielded well-resolved trees with a consistent general branching pattern. A parallel analysis of nine morphological and life-history characters and three ecological characters yielded a similar tree but provided little resolution in the terminal clades. The position of the monotypic Arthrocladia villosa within the Desmarestiales is consistent with monophyly for the order, but its position as the most primitive desmarestialean is not resolvable from the molecular data set. The basal position of Phaeurus, the Antarctic Desmarestia species, and Himantothallus is consistent with the hypothesis of a Southern Hemisphere origin for the family Desmarestiaceae. The more recent Northern Hemisphere “aculeata” clade evolved from an Antarctic ancestor. A “D. aculeata-like” species was ancestral to a lineage characterized by annual sporophytes with high sulfuric acid content, which radiated into many species, widely distributed in both hemispheres. Mapping of morphological and ecological characters onto the molecular tree confirm the informativeness of sulfuric acid-containing vacuoles and unilocular sporangial types. There is good congruence between phylogenetic tree topology and temperature impints in relation to biogeographic distribution, supporting the theory that temperature tolerance is a conservative trait.     

Nucleotide sequence analysis of genes encoding a toluene/benzene-2-monooxygenase from Pseudomonas sp. strain JS150.

It was previously shown by others that Pseudomonas sp. strain JS150 metabolizes benzene and alkyl- and chloro-substituted benzenes by using dioxygenase-initiated pathways coupled with multiple downstream metabolic pathways to accommodate catechol metabolism. By cloning genes encoding benzene-degradative enzymes, we found that strain JS150 also carries genes for a toluene/benzene-2-monooxygenase. The gene cluster encoding a 2-monooxygenase and its cognate regulator was cloned from a plasmid carried by strain JS150. Oxygen (18O2) incorporation experiments using Pseudomonas aeruginosa strains that carried the cloned genes confirmed that toluene hydroxylation was catalyzed through an authentic monooxygenase reaction to yield ortho-cresol. Regions encoding the toluene-2-monooxygenase and regulatory gene product were localized in two regions of the cloned fragment. The nucleotide sequence of the toluene/benzene-2-monooxygenase locus was determined. Analysis of this sequence revealed six open reading frames that were then designated tbmA, tbmB, tbmC, tbmD, tbmE, and tbmF. The deduced amino acid sequences for these genes showed the presence of motifs similar to well-conserved functional domains of multicomponent oxygenases. This analysis allowed the tentative identification of two terminal oxygenase subunits (TbmB and TbmD) and an electron transport protein (TbmF) for the monooxygenase enzyme. In addition to these gene products, all the tbm polypeptides shared significant homology with protein components from other bacterial multicomponent monooxygenases. Overall, the tbm gene products shared greater similarity with polypeptides from the phenol hydroxylases of Pseudomonas putida CF600, P35X, and BH than with those from the toluene monooxygenases of Pseudomonas mendocina KR1 and Burkholderia (Pseudomonas) pickettii PKO1. The relationship found between the phenol hydroxylases and a toluene-2-monooxygenase, characterized in this study for the first time at the nucleotide sequence level, suggested that DNA probes used for surveys of environmental populations should be carefully selected to reflect DNA sequences corresponding to the metabolic pathway of interest.     

Modeling the leech heartbeat elemental oscillator I. Interactions of intrinsic and synaptic currents

We have developed a biophysical model of a pair of reciprocally inhibitory interneurons comprising an elemental heartbeat oscillator of the leech. We incorporate various intrinsic and synaptic ionic currents based on voltage-clamp data. Synaptic transmission between the interneurons consists of both a graded and a spike-mediated component. By using maximal conductances as parameters, we have constructed a canonical model whose activity appears close to the real neurons. Oscillations in the model arise from interactions between synaptic and intrinsic currents. The inhibitory synaptic currents hyperpolarize the cell, resulting in activation of a hyperpolarization-activated inward currentI _h and the removal of inactivation from regenerative inward currents. These inward currents depolarize the cell to produce spiking and inhibit the opposite cell. Spike-mediated IPSPs in the inhibited neuron cause inactivation of low-threshold Ca⁺⁺ currents that are responsible for generating the graded synaptic inhibition in the opposite cell. Thus, although the model cells can potentially generate large graded IPSPs, synaptic inhibition during canonical oscillations is dominated by the spike-mediated component.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

A circular waveguide irradiation system for nonhuman primates: design and dosimetry

A 275-MHz exposure system, consisting of a circular waveguide irradiator and a transparent plastic animal cage, has been developed to accommodate rhesus monkeys weighing up to 15 kg. The vertically oriented waveguide is composed primarily of stainless steel and is fitted with an inner cage fabricated from a tubular section of acrylic plastic. Circularly polarized electromagnetic energy at 275 MHz, either pulsed or continuous wave (CW), can be propagated from the removable top section of the waveguide. The cage is designed to function as the monkey's permanent home. It is fitted with a lever-actuated behavioral performance device on which the monkey responds according to a predetermined schedule to obtain a daily food ration. The system can be adapted to provide for the collection of metabolic and physiologic data as well. Dosimetric measurements were conducted with six rhesus monkeys weighing 3.0-7.2 kg and with a 4-kg model. The dosimetric results show that about one-third of the net incident energy is absorbed by a subject in this system at a normalized specific absorption rate (SAR) of 0.33 (W/kg)/(mW/cm2).     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria.

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Studies on the subunits of human myeloperoxidase.

The subunit composition of human myeloperoxidase was studied with the use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration. The subunit pattern observed depended on the manner in which the enzyme was treated before analysis. Reduction before heat treatment in detergent led to two main protein species (Mr 57 000 and 10 500), whereas reduction during or after heat treatment yielded an additional species of Mr 39 000. Heating without any reductive pretreatment yielded the 39 000-Mr form as the major electrophoretic species. Carbohydrate staining showed large amounts of sugar on the 57 000-Mr species and little on the 10 500-Mr form. Significant amounts of haem were associated with this latter subunit. Haem also seemed to be associated with the 57 000-Mr form but not with the 39 000-Mr one. These three subunit forms were isolated and their amino acid composition analysed. The 57 000-Mr and 39 000-Mr forms had very similar amino acid composition and yielded an apparently identical collection of fragments on incubation with CNBr. Once separated, the subunits could not be interconverted. Generally, minor amounts of other molecular-mass forms were observed. The nature of the various molecular-mass forms originating from myeloperoxidase is discussed.     

Perfusion rate dependent prostaglandin release from isolated rabbit kidneys

Isolated rabbit kidneys were perfused with 37°C Krebs-Henseleit solution aerated with 95% O₂ + 5% CO₂. Perfusion rate was varied from 1 to 10 ml/min. This was accompanied by parallel changes of perfusion pressure, prostaglandin excretion and release of radioactivity from kidneys with ¹⁴C-arachidonic acid incorporated into the tissue lipid pool. It is suggested that enhancement of perfusion rate raises the intrarenal pressure which increases renal prostaglandin release due to increased substrate availability.     

Separation of P-labeled inorganic phosphate from rat liver homogenates

A method has been developed for the separation of radioactive inorganic phosphate from rat liver homogenates by a combination of ion-exchange and precipitation chromatography. The method has been applied to normal rat liver.