Phylogenetic studies on Corynebacterium bovis isolated from bovine mammary glands.

Coryneform bacteria are frequently isolated from bovine mastitis with the lipophilic species, and Corynebacterium bovis is the most frequently isolated organism of this group. However, previous studies on the phylogeny of corynebacteria have incorporated only a single reference strain. We examined the phylogeny of C. bovis using 47 strains isolated from bovine mammary glands. Phylogenetic studies were performed by direct sequencing of the 16S ribosomal RNA and comparison to sequences of reference strains. All strains identified as C. bovis demonstrated similarity of 98% or higher to the ribosomal RNA gene sequences of the type strain of C. bovis. Phylogenetic analyses indicated that all strains tested clustered with members of the Corynebacterium urealyticum group confirming that C. bovis is a legitmate member of the genus Corynebacterium. Further investigation into the diversity within the species using repetitive element palindrome PCR indicated only minor differences between the strains tested. Corynebacterium bovis ATCC 13722 demonstrated the highest similarity (95%) with Brevibacterium helvolum, indicating that this organism does not belong in the genus Corynebacterium.

Cloning and functional analysis of a beta-tubulin gene from a benomyl resistant mutant of Aspergillus parasiticus.

A genomic DNA library prepared from a benomyl resistant strain of Aspergillus parasiticus was screened with a Neurospora crassa beta-tubulin gene probe. A unique A. parasiticus genomic DNA fragment, thought to carry a mutant beta-tubulin gene (benr), was isolated. Two plasmids, pYT1 and pYTPYRG, carrying the putative benr gene or benr plus a second selectable marker (pyrG), respectively, were used to transform a benomyl sensitive strain of A. parasiticus (CS10) to determine if benr conferred benomyl resistance (BenR). BenR colonies were obtained with pYTPYRG, pYT1 or pYT1 cotransformed with pPG3J which carries a functional pyrG gene. No BenR colonies were obtained without added DNA or with pPG3J only (controls). Southern hybridization analysis of BenR and BenS transformants suggested that plasmid integration occurred most frequently at the chromosomal bens locus, however evidence for gene conversion and heterologous recombination was also observed. The predicted amino acid sequence of benr displayed a high degree of identity (> 93%) with other fungal beta-tubulin genes which confer benomyl resistance. Sequence analysis together with the genetic data suggested that benr encodes a functional mutant beta-tubulin.

[Implementation of stereotactic focal radiotherapy using 10 MV x-ray].

Stereotactic focal irradiation is also called stereotactic radiosurgery by some neurosurgeons. This irradiation is used for the treatment of brain AVM (arterio-venous malformation) and small tumor. Application of stereotactic focal irradiation was developed with CRW (Cosman-Roberts-Wells) stereotactic device and two dimensional (2D) computer treatment planning system using 10 MV x-ray from a linear accelerator. This process of irradiation includes: (a) Identification and localization of a target volume in CRW stereotactic frame by CT scan or angiography. (b) To verify the alignment, a linear accelerator was used as a simulator to take portal films in anterior and lateral views. This was done to ascertain the angles between arc therapy, to encompass the target volume, and to exclude the critical organs such as lens at 0 degrees, 45 degrees, 90 degrees and 315 degrees couch angles. (c) A 2D computer treatment planning system was used to generate an isodose curve distribution for each couch angle. Then this was used to calculate the monitor unit per degree for rotation treatment. (d) 10 MV x-ray was used to implement the stereotactic focal radiotherapy.

Phenothiazines as lipid peroxidation inhibitors and cytoprotective agents.

A series of phenothiazines was synthesized and evaluated as in vitro inhibitors of iron-dependent lipid peroxidation. The MIC (minimum tested concentration that gave greater than or equal to 50% inhibition) for 2-(10H-phenothiazin-2-yloxy)-N,N-dimethylethanolamine methanesulfonate (6) was 0.26 microM. Whereas methyl substitution at N-10 diminished activity nearly 100-fold, other structural modifications such as varying the amine group, the distance separating the amine substituent from the phenothiazine nucleus, and the linking group had little effect. Compound 6 was more effective than probucol, a known antioxidant, in blocking Cu2+ catalyzed oxidation of low-density lipoprotein (LDL) as measured by competitive scavenger receptor mediated degradation of 125I-labeled acetyl-LDL by mouse peritoneal macrophage cells in vitro. At a concentration of 5 microM, compound 6 also protected primary cultures of rat hippocampal neurons exposed to hydrogen peroxide (50 microM) when assessed 18 h later by fluorescein diacetate and propidium iodide uptake.

Replication and temperature-sensitive maintenance functions of lactose plasmid pSK11L from Lactococcus lactis subsp. cremoris.

The replication region of pSK11L, the lactose plasmid of Lactococcus lactis subsp. cremoris (L. cremoris) SK11, was isolated on a 14.8-kbp PvuII fragment by shotgun cloning into an Escherichia coli vector encoding erythromycin resistance and selection for erythromycin-resistant transformants of L. lactis subsp. lactis (L. lactis) LM0230. Deletion analysis and Tn5 mutagenesis of the resulting plasmid (pKMP1) further localized the replication region to a 2.3-kbp ScaI-SpeI fragment. DNA sequence analysis of this 2.3-kbp fragment revealed a 1,155-bp open reading frame encoding the putative replication protein, Rep. The replication origin was located upstream of rep and consisted of an 11-bp imperfect direct repeat and a 22-bp sequence tandemly repeated three and one-half times. The overall organization of the pSK11L replicon was remarkably similar to that of pCI305, suggesting that pSK11L does not replicate by the rolling-circle mechanism. Like pSK11L, pKMP1 was unstable in L. lactis LM0230. Deletion analysis allowed identification of several regions which appeared to contribute to the maintenance of pKMP1 in L. lactis LM0230. pKMP1 was significantly more stable in L. cremoris EB5 than in L. lactis LM0230 at all of the temperatures compared. This stability was lost by deletion of a 3.1-kbp PvuII-XbaI fragment which had no effect on stability in L. lactis LM0230. Other regions affecting stability in L. cremoris EB5 but not in L. lactis LM0230 were also identified. Stability assays conducted at various temperatures showed that pKMP1 maintenance was temperature sensitive in both L. lactis LM0230 and L. cremoris EB5, although the plasmid was more unstable in L. lactis LM0230. The region responsible for the temperature sensitivity phenotype in L. lactis LM0230 was tentatively localized to a 1.2-kbp ClaI-HindIII fragment which was distinct from the replication region of pSK11L. Our results suggest that the closely related L. lactis and L. cremoris subspecies behave differently regarding maintenance of plasmids.

Transformation of Aspergillus parasiticus with a homologous gene (pyrG) involved in pyrimidine biosynthesis.

The lack of efficient transformation methods for aflatoxigenic Aspergillus parasiticus has been a major constraint for the study of aflatoxin biosynthesis at the genetic level. A transformation system with efficiencies of 30 to 50 stable transformants per microgram of DNA was developed for A. parasiticus by using the homologous pyrG gene. The pyrG gene from A. parasiticus was isolated by in situ plaque hybridization of a lambda genomic DNA library. Uridine auxotrophs of A. parasiticus ATCC 36537, a mutant blocked in aflatoxin biosynthesis, were isolated by selection on 5-fluoroorotic acid following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene resulting in elimination of orotidine monophosphate (OMP) decarboxylase activity were detected by assaying cell extracts for their ability to convert [14C]OMP to [14C]UMP. Transformation of A. parasiticus pyrG protoplasts with the homologous pyrG gene restored the fungal cells to prototrophy. Enzymatic analysis of cell extracts of transformant clones demonstrated that these extracts had the ability to convert [14C]OMP to [14C]UMP. Southern analysis of DNA purified from transformant clones indicated that both pUC19 vector sequences and pyrG sequences were integrated into the genome. The development of this pyrG transformation system should allow cloning of the aflatoxin-biosynthetic genes, which will be useful in studying the regulation of aflatoxin biosynthesis and may ultimately provide a means for controlling aflatoxin production in the field.

Development of a homologous transformation system for Aspergillus parasiticus with the gene encoding nitrate reductase.

The nitrate reductase structural gene (niaD) and an niaD mutant strain were isolated from Aspergillus parasiticus and used to develop a homologous transformation system. A transformation frequency of 110 to 120 transformants per microgram linear DNA was obtained with the 10.9 kb plasmid pSL82, which contained the niaD gene of A. parasiticus. Plasmid pSL82 was also capable of complementing Aspergillus nidulans FGSC A691, a niaD mutant, though at lower frequencies. Southern hybridization analyses of A. parasiticus niaD transformants showed that the niaD gene of pSL82 had integrated into the fungal genome. In addition, vector (bacterial plasmid) sequences were also present in one of the niaD transformants.

Atrial natriuretic peptide receptor modulators: effects of disubstituted quinazolines on receptor binding and in vitro biological activity.

Prazosin (25 microM) was found to increase 125I-labeled rat atrial natriuretic peptide ([125I]rANP) receptor binding by 50% (SC50) in bovine adrenal zona glomerulosa membranes. A series of 2,4-disubstituted quinazolines was prepared in order to identify more potent analogues for additional in vitro testing. Compound 7 (N-[3-[[2-(diethyl-amino)-4-quinazolinyl]amino]propyl] guanidine dinitrate) from this series (3 microM) significantly decreased the EC50 for rANP-mediated inhibition of ACTH-stimulated aldosterone synthesis in rat adrenal glomerulosa cells. At a higher concentration (20 microM), compound 7 had no effect on particulate guanylate cyclase from rabbit glomeruli in either the presence or absence of rANP.

Cloning and characterization of the trpC gene from an aflatoxigenic strain of Aspergillus parasiticus.

The trpC gene in the tryptophan biosynthetic pathway was isolated from an aflatoxigenic Aspergillus parasiticus by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene complemented an E. coli trpC mutant deficient in indoleglycerolphosphate synthase (IGPS) activity as well as an Aspergillus nidulans mutant strain that was defective in all three enzymatic activities of the trpC gene (glutamine amidotransferase, IGPS, and PRAI), thus indicating the presence of a complete and functional trpC gene. The location and organization of the A. parasiticus trpC gene on the cloned DNA fragment were determined by deletion mapping and by hybridization to heterologous DNA probes that were prepared from cloned trpC genes of A. nidulans and Aspergillus niger. These experiments suggested that the A. parasiticus trpC gene encoded a trifunctional polypeptide with a functional domain structure organized identically to those of analogous genes from other filamentous fungi. The A. parasiticus trpC gene was expressed constitutively regardless of the nutritional status of the culture medium. This gene should be useful as a selectable marker in developing a DNA-mediated transformation system to analyze the aflatoxin biosynthetic pathway of A. parasiticus.

Ergolines as selective 5-HT1 agonists.

The synthesis and serotonin receptor subtype affinity of a series of ergolines are described. High selectivity for the 5-HT1 subtype was found with a number of 8-substituted (3 beta, 5 beta)-9,10-didehydro-6-methylergolines. The more potent and selective of these compounds increased the concentration of serotonin and decreased the concentration of 5-HIAA in rat brain and increased corticosterone concentration in rat serum. Oral administration of 13, (3 beta)-2,3-dihydrolysergine, produced long-lasting decreases in serotonin turnover. Compound 13 lacked substantial dopaminergic activity as measured by its effects on dopamine turnover in whole brain or striatum and its affinity for alpha-adrenergic binding sites was significantly less than for 5-HT1 binding sites. The increases in serum corticosterone concentrations produced by 13 were not blocked by the serotonin uptake inhibitor fluoxetine or by the serotonin synthesis inhibitor p-chlorophenylalanine, suggesting that 13 exerts its effects through direct stimulation of serotonin receptors.

Alpha-2-adrenoceptor-mediated effects of pergolide.

The alpha-adrenoceptor-mediated effects of pergolide were investigated in a number of biochemical and pharmacological test systems. In radioligand binding studies, pergolide more effectively competed for alpha 2-adrenoceptors than for alpha 1-adrenoceptors in membranes prepared from rat cerebral cortex. Consistent with this finding was the observation that pergolide was several orders of magnitude more effective in activating presynaptic alpha 2-adrenoceptors in rat vas deferens to inhibit stimulation-evoked [3H]-norepinephrine release than in activating postsynaptic alpha 1-adrenoceptors in the same tissue to produce a contractile response. Pergolide elicited a potent vasopressor response in pithed rats that was highly sensitive to antagonism by the selective alpha 2-adrenoceptor antagonist, yohimbine, and resistant to blockade by the selective alpha 1-adrenoceptor antagonist, prazosin, suggesting that pergolide selectively activates postsynaptic vascular alpha 2-adrenoceptors. The results of the present study indicate that the alpha-adrenoceptor-mediated effects commonly associated with pergolide result from selective stimulation of alpha 2-adrenoceptors.

N-substituted imidazolines and ethylenediamines and their action on alpha- and beta-adrenergic receptors.

A series of N-substituted imidazolines and ethylenediamines were synthesized and examined for their activity in alpha- and beta-adrenergic systems. The length of the intermediate side chain between the catechol and imidazoline ring or the amine of the ethylenediamine segment was shown to affect the adrenergic activity. N-[2-(3,4-Dihydroxyphenyl)ethyl]imidazoline hydrochloride (2) and N-[2-(3,4-dihydroxyphenyl)ethyl]ethylenediamine dihydrochloride (4), both with two methylene groups between the catechol and amine segment, were found to be somewhat selective for alpha 2-adrenergic receptors while 1-(3,4-dihydroxybenzyl)imidazoline hydrochloride (1) and N-2-(3,4-dihydroxybenzyl)ethylenediamine dihydrochloride (3), both with one methylene group between the catechol and amine segment, were more selective for alpha1-adrenergic receptors in a pithed rat model. Of the four compounds examined, only compound 2 showed significant direct activity on beta1- and beta2-adrenergic receptors.

Interactions of the enantiomers of 3-O-methyldobutamine with alpha- and beta-adrenoceptors in vitro.

The enantiomers of 3-O-methyldobutamine, a metabolite of dobutamine, were evaluated for their alpha- and beta-adrenoceptor mediated effects in vitro in a variety of isolated organs and in radioligand binding studies. Neither enantiomer of 3-O-methyldobutamine possessed alpha 1-adrenoceptor agonist activity in isolated guinea pig aorta. However, both enantiomers of 3-O-methyldobutamine were competitive alpha 1-adrenoceptor antagonists, with the (+)-enantiomer being approximately 10-fold more potent than the (-)-enantiomer as assessed either in guinea pig aorta or by displacement of 3H-prazosin binding from alpha 1-adrenoceptors in rat cerebral cortex. The alpha 1-adrenoceptor blocking activity of (+)-3-O-methyldobutamine was relatively potent and corresponded to a pA2 of 7.33 in guinea pig aorta and a -log Ki of 7.72 in radioligand binding studies. Neither enantiomer of 3-O-methyldobutamine possessed alpha 2-adrenoceptor agonist activity in field-stimulated guinea pig ileum. Although (+)-3-O-methyldobutamine weakly inhibited the twitch response in field-stimulated guinea pig ileum, the response was not blocked by the selective alpha 2-adrenoceptor antagonist, yohimbine, and was found to result from weak anticholinergic activity (pA2 = 5.06). Neither enantiomer of 3-O-methyldobutamine possessed beta 1-adrenoceptor agonist activity in guinea pig atria, however the (+)-enantiomer was a weak noncompetitive antagonist at beta 1-adrenoceptors. In contrast, both enantiomers of 3-O-methyldobutamine were weak beta 2-adrenoceptor agonists in rat uterus, however these weak effects were not highly stereoselective, which was also confirmed in radioligand binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of dimethoxy-substituted tolazoline derivatives with alpha 1- and alpha 2-adrenoreceptors in vitro.

The activities of a series of dimethoxy-substituted tolazoline derivatives were investigated at alpha 1-adrenoreceptors in guinea-pig aorta and alpha 2-adrenoreceptors in field-stimulated guinea-pig ileum. Radioligand binding studies to alpha 1- and alpha 2-adrenoreceptors in rat cerebral cortex were also performed to support the pharmacological findings. The 2,5- and 3,5-dimethoxy-substituted tolazoline derivatives were potent full agonists at alpha 1-adrenoreceptors in guinea-pig aorta, whereas the 2,3- and 3,4-dimethoxy-substituted tolazoline derivatives were inactive as alpha 1-adrenoreceptor agonists. 2,3-Dimethoxytolazoline was a partial agonist at alpha 2-adrenoreceptors in field-stimulated guinea-pig ileum. The intrinsic activity of 2,3-dimethoxytolazoline was similar to that of clonidine, but less than that of UK-14,304. The -log ED50 of 2,3-dimethoxytolazoline (7.66) was only 3- to 5-fold lower than that of clonidine or UK-14,304, indicating that this compound has relatively high potency as an alpha 2-adrenoreceptor agonist. The 2,5-, 3,5- and 3,4-dimethoxy-substituted tolazoline derivatives were inactive as alpha 2-adrenoreceptor agonists. 3,4-Dimethoxytolazoline was a moderately potent and selective alpha 2-adrenoreceptor antagonist in field-stimulated guinea-pig ileum. The results indicate that the dimethoxy-substituted tolazoline derivatives possess different pharmacological activities and selectivities at alpha 1- and alpha 2-adrenoreceptors, depending upon the positions of the dimethoxy substitutions. The 2,5- and 3,5-dimethoxytolazoline derivatives are potent and selective alpha 1-adrenoreceptor agonists, whereas 2,3-dimethoxytolazoline is a potent and selective alpha 2-adrenoreceptor agonist. 3,4-dimethoxytolazoline is a moderately potent and selective alpha 2-adrenoreceptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

The 3,4-catechol derivative of propranolol, a minor dihydroxylated metabolite.

The O,O-dibenzyl ether of the 3,4-catechol derivative of propranolol (11) was prepared to determine whether the catechol is a product of metabolic hydroxylation. 4-(Allyloxy)-1,2-naphthoquinone (5) was reduced with sodium dithionite and alkylated with benzyl chloride to produce ether 7. Osmium tetroxide oxidation of 7 afforded glycol 8. Subsequent monotosylation, oxirane formation with KOH, and opening with isopropylamine afforded benzyl ether 11. Although hydrogenolysis was successful, catechol 3 was rapidly oxidized to the corresponding o-quinone (12). Reduction of 12 with sodium bisulfite afforded 3, which was derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) to serve as a standard for the metabolic experiments. Gas chromatography-mass spectrometry of the Me3Si ethers of the products of metabolism of pseudoracemic propranolol (made up of equal molar (2R)-propranolol-d0/(2S)-propranolol-3',3'-d2) in the presence of the rat liver 9000g supernatant fraction showed four dihydroxylated metabolites, of which catechol 3 was in smallest amount, approximately 9% of the sum of dihydroxylated metabolites. Each of the four dihydroxylated propranolols arises stereoselectively from the 2R enantiomer of propranolol (by 1.15- to 2-fold), as determined by parent ion intensities at m/z 507 vs. 509. Quinone 12 was a nonselective competitive beta-adrenoceptor antagonist, being about 16-fold less potent than propranolol in both beta 1 and beta 2 assays.

Interactions of three inotropic agents, ASL-7022, dobutamine and dopamine, with alpha- and beta-adrenoceptors in vitro.

Three inotropic agents, ASL-7022, dobutamine and dopamine, were evaluated for their alpha- and beta-adrenoceptor mediated effects in vitro in a variety of isolated organs and in radioligand binding studies. All compounds were alpha 1-adrenoceptor agonists in rat and guinea pig aortae, but the rank orders of potency were exactly opposite in these two tissues. Only the rank potency order of dobutamine greater than ASL-7022 greater than dopamine obtained in rat aorta was consistent with the results obtained in radioligand binding studies to alpha 1-adrenoceptors in rat cerebral cortex and to previous results obtained in vivo in the pithed rat. The results obtained in guinea pig aorta did not parallel the radioligand binding studies in rat brain or our previous results in pithed rat, and suggests that species differences exist between postsynaptic vascular alpha 1-adrenoceptors in rat and guinea pig aorta, consistent with previous conclusions. ASL-7022 was found to be a potent alpha 2-adrenoceptor agonist in field-stimulated guinea pig ileum, and was approximately 10-fold more potent than dobutamine in this respect, which was also confirmed by radioligand binding studies to alpha 2-adrenoceptors in rat cerebral cortex. The beta 1-adrenoceptor mediated effects of these compounds were evaluated in guinea pig atria, where the rank order of potency was dobutamine greater than ASL-7022 greater than dopamine. An identical rank order of affinity was established for these compounds by displacement of 3H-dihydroalprenolol from beta 1-adrenoceptors in rat cerebral cortex. The beta 1-adrenoceptor mediated effects of dobutamine and ASL-7022 in guinea pig atria were completely direct in nature and not secondary to the release of endogenous catecholamines. In contrast, a major component of the beta 1-adrenoceptor mediated tachycardia produced by dopamine in guinea pig atria was indirect in nature as evidenced by the marked attenuation in potency that occurred following catecholamine depletion with reserpine. All three compounds elicited beta 2-adrenoceptor mediated inhibition of tone in rat uterus, with the rank order of potency being ASL-7022 greater than dobutamine greater than dopamine. Again, this rank order of beta 2-adrenoceptor potency was also reflected in beta 2-adrenoceptor affinity as assessed by displacement of 3H-dihydroalprenolol from beta 2-adrenoceptors in rat cerebellum. Based on these results, it may be concluded that for alpha-adrenoceptors, dobutamine is a selective alpha 1-adrenoceptor agonist, ASL-7022 is a selective alpha 2-adrenoceptor agonist, and dopamine is a nonselective alpha-adrenoceptor agonist.(ABSTRACT TRUNCATED AT 400 WORDS)