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PubMed2006

Molecular characterization of MG isolates using RAPD and PFGE isolated from chickens in Brazil.

In the present study, 27 primers were screened under different cycles by random amplified polymorphic DNA (RAPD) method. Mathematical models were used for analysis of the genetic relationships among strains, including vaccinal, reference strains and nine field isolates previously characterized as Mycoplasma gallisepticum (MG)F by RAPD and pulsed field gel electrophoresis (PFGE). The PFGE was considered as laborious, expensive and time-consuming than RAPD method. These methods improved the typeability for epidemiological studies of MG with regard to differentiation from vaccinal and field strains.

Animals↗

PubMed1992

Prejunctional actions of tacrine on autonomic neuroeffector transmission in rabbit isolated pulmonary artery and rat isolated atria.

1. This study investigated the effects of tacrine (1,2,3,4-tetrahydro-9-aminoacridine) on the resting and stimulation-induced (SI) release of radioactive substances from isolated preparations of rat atria and rabbit pulmonary artery in which the noradrenergic transmitter stores had been labelled with [3H]-noradrenaline, and from rat atrial preparations in which cholinergic transmitter stores had been labelled with [3H]-acetylcholine. In addition, the effect of tacrine on the uptake of [3H]-noradrenaline by noradrenergic nerves in rat atria was determined. 2. Tacrine produced concentration-dependent increases in the resting efflux of radioactivity from both the [3H]-noradrenaline-loaded artery and atrial preparations. Blockade of neuronal amine transport with desipramine reduced the release of radioactivity evoked by tacrine from atria but not that evoked from artery preparations. Inhibition of monoamine oxidase by pargyline pretreatment markedly reduced the tacrine-evoked release of radioactivity in both atrial and artery preparations. 3. The radioactivity released from [3H]-noradrenaline-labelled rat atrial preparations by 30 mumol/L tacrine consisted entirely of the deaminated metabolite [3H]-DOPEG. The evoked release of [3H]-DOPEG from atria was reduced by approximately 50% by desipramine (1 mumol/L). When atrial monoamine oxidase had been inhibited by pargyline treatment in vivo and in vitro, 30 mumol/L tacrine evoked the release of [3H]-noradrenaline instead of [3H]-DOPEG. However, the amounts of [3H]-noradrenaline released by tacrine when monoamine oxidase was inhibited were only about 25% of the amounts of [3H]-DOPEG released in untreated atria. 4. Tacrine, in concentrations of 1 and 10 mumol/L, enhanced the release of radioactivity evoked by field stimulation of [3H]-noradrenaline-loaded rabbit pulmonary artery preparations. This effect was unaltered by desipramine or pretreatment with pargyline. However, in artery preparations pretreated with pargyline, a high concentration of tacrine (100 mumol/L) markedly reduced SI efflux. In contrast to the findings with artery preparations, tacrine (1-30 mumol/L) did not alter SI efflux in rat atrial preparations. 5. It is concluded that tacrine displaces noradrenaline from intraneuronal transmitter stores of sympathetically-innervated tissues, and that the displaced amine is totally metabolized by monoamine oxidase before leaving the nerve terminals. When deamination of neuronal cytoplasmic noradrenaline is prevented, only a portion of the noradrenaline displaced from storage vesicles passes to the extracellular space. It is likely that the transfer of cytoplasmic noradrenaline out of the terminals is limited by the activity of the amine transport mechanism.

Acetylcholine↗

PubMed1996

Isolation and characterization of Vibrio alginolyticus isolated from diseased kuruma prawn, Penaeus japonicus.

Outbreaks of serious mortality among cultured kuruma prawns (Penaeus japonicus) with white spotted syndrome in the carapace occurred in the summer of 1993 in I-Lan, Taiwan. A swarming bacterium, strain Swy, was isolated from the hepatopancreas of the moribund prawns using tryptic soy agar supplemented with 1% NaCl and/or thiosulphate citrate bile salt sucrose agar. This strain was characterized and identified as Vibrio alginolyticus on the basis of a number of biochemical tests. The Swy strain was virulent to both kuruma prawns (P. japonicus) and tiger prawns (P. monodon) with LD50 values of 4.43 x 10(4) and 1.57 x 10(5) cfu g body weight-1, respectively.

Animals↗

PubMed1988

Effects of noradrenaline on the isolated human bronchus. Comparison with the isolated guinea pig trachea.

The pharmacodynamic activity of noradrenaline was evaluated comparatively in vitro on isolated human bronchi and on guinea pig tracheal spirals. Noradrenaline exerted a contractile effect on both preparations under resting tone and in the presence of propranolol 10(-6) M; maximal noradrenaline-induced contraction was 15-20% of maximal acetylcholine (ACh)-induced contraction. Without propranolol, the contractile effect of noradrenaline was negligible when the preparations were under resting tone and absent when they were precontracted with ACh. In contrast, noradrenaline exerted a strongly relaxant effect on both human bronchi (-log ED50 5.24 +/- 0.17; N = 5) and guinea pig tracheae (-log ED50 6.15 +/- 0.29; N = 8). With maximal contraction induced by ACh 3.10(-3) M the -log ED50 of both preparations were shifted to the right by functional antagonism and became 4.72 +/- 0.17 and 5.31 +/- 0.11, respectively. The pKD values of noradrenaline, calculated according to Furchgott and Bursztyn (1967), were 4.79 +/- 0.04 in human bronchi (N = 5) and 4.77 +/- 0.16 in guinea-pig tracheae (N = 8). In the presence of cocaine plus phenoxybenzamine these values were not significantly modified in human bronchi and only slightly modified in guinea pig tracheae. It is concluded that noradrenaline induces a strong beta-adrenergic response and a negligible alpha-adrenergic response from both human bronchi and guinea pig tracheae in vitro.

Animals↗

PubMed2004

Tissue collection, transport and isolation procedures required to optimize human hepatocyte isolation from waste liver surgical resections. A multilaboratory study.

BACKGROUND: The European Center for Validation of Alternative Methods (ECVAM) has funded a prevalidation study in three laboratories (France, USA and UK) on the use of human hepatocyte cultures to predict cytochrome P-450 induction. AIMS AND METHODS: As first stage of this prevalidation study, the purpose of the present work was to set criteria for optimization and harmonization of hepatocyte isolation from human tissue among laboratories to establish a routine procedure. This was achieved by combining and/or comparing the data generated by the two independent European laboratories (France and UK). RESULTS: The results confirmed that surgical waste material is a valuable source for obtaining high quality hepatocytes under certain pre-, intra- and post-operative conditions: cell yield of viable hepatocytes was not significantly affected by age and sex of patients, nor indications for resection, steatosis or cholestasis. Cold ischeamia up to 5 hours did not influence viable cell yield allowing transport of material. CONCLUSION: The use of biopsy sizes between 50-100 g, cannulation with 2-4 cannulae, digestion with collagenase-containing digestion medium at a flow rate of 25 ml/cannula for 20 minutes, with cut surface being glued in order to reform Glisson's capsule, should optimize the total yield of viable human hepatocytes obtained per preparation of waste liver surgical resections.

Adult↗

PubMed1982

Isolation and properties of isolated nuclei from the Florida red tide dinoflagellate Gymnodinium breve (Davis).

A simple and rapid method is described for the isolation of nuclei from the Florida red tide dinoflagellate Gymnodinium breve. The nuclei are free of cytoplasmic contamination and are active in endogenous RNA synthesis. The ratio of DNA : RNA : acid-soluble protein : acid-insoluble protein is 1:0.39:0.13:0.63, respectively, and each nucleus contains ca. 113 picograms of DNA. Electrophoretic analysis of the acid-soluble proteins reveals the presence of two histone-like proteins with molecular weights of 12,000 and 13,000.

Animals↗

PubMed1978

Differences in postsynaptic alpha-adrenoceptor populations between isolated cat urethra and various other isolated tissues.

This study was undertaken with the aim of determining whether the postsynaptic alpha-adrenergic receptor population of the cat urethra differed from that of other isolated tissues (rabbit aorta and rat vas deferens) and if so the possibility of selectively affect these receptors. For this purpose the substances 2-methylammonio-1-(spiro[cyclopentane-1,1'-indene]-3'-yl)ethanol (KABI 2023), noradrenaline (NA) and dopamine (DA) were used. KABI 2023 and NA acted as full agonists on all three tissues investigated. DA was a full agonist on the vas deferens but was almost inactive on urethra. The contractile response of urethra to KABI 2023 was of an alpha-adrenergic nature, as it could be blocked with phentolamine. Compared with NA, KABI 2023 showed a 10 times higher selectivity for the receptors of urethra than for those of aorta. Affinity constants (log KB) for phentolamine and haloperidol with use of the various agonists were estimated. The affinity of phentolamine was found to be significantly different when using NA and KABI 2023 as agonists on the urethra but not on the aorta. Corresponding findings were obtained with haloperidol. On the vas deferens a greater difference in log KB values than that on the urethra was found. On the basis of the results, it is suggested that the population of postsynaptic alpha-adrenoceptors in the urethra (cat) differs from that in the aorta (rabbit). On the vas deferens a heterogenicity of postsynaptic receptors seem to exist which makes the interpretation of the results more difficult on this organ. In the presence of phentolamine and haloperidol the maximum responses to NA were potentiated on the aorta and vas deferens, but not on the urethra. Investigations to evaluate this difference are now in progress.

Animals↗

PubMed1971

Release of endogenous noradrenaline from an isolated muscular artery. Release of endogenous noradrenaline from an isolated muscular artery.

1. The release of noradrenaline by field stimulation of vasoconstrictor nerves has been studied in isolated preparations of the main uterine artery of the guinea-pig.2. In preparations from virgin animals stimulation with trains of 3000 square pulses at 5 and 25 pulses/sec resulted in mean overflows of 0.56 ng/g.pulse and 1.53 ng/g.pulse respectively.3. Inhibition of monoamine oxidase and catechol-O-methyltransferase had no consistent effect on overflow at either stimulation frequency.4. Desmethylimipramine (10(-5)M) caused, on the average, a 2.4-fold increase in overflow following stimulation at 5 pulses/sec while phenoxybenzamine (10(-5)M) caused a 3.8-fold increase. Neither of these drugs caused a significant alteration of the overflow during stimulation at 25 pulses/sec.5. Treatment of the tissues with desmethylimipramine plus normetanephrine (4.5 x 10(-4)M) caused no more increase in overflow than treatment with desmethylimipramine alone.6. It is concluded that enzymatic metabolism of noradrenaline at the synapse is of little functional importance in this tissue, and that the most important mechanism of transmitter inactivation is by nervous re-uptake. Although phenoxybenzamine was more effective than desmethylimipramine in increasing transmitter overflow, no evidence was obtained to support the view that this effectiveness was due partly to blockade of ;Uptake 2'.7. There was sometimes very low overflow of noradrenaline from arteries taken from animals in the last week of pregnancy. In these instances overflow following stimulation at 5 pulses/sec was not increased by phenoxybenzamine treatment of the tissue.8. Methylene blue and fluorescence microscopic techniques indicated that the terminal adrenergic axons in each artery possess approximately 8.74 x 10(5) varicosities. The mean tissue content of noradrenaline was found to be 9.6 mug/g or 29 ng/artery. These results have been correlated with known morphological and electrophysiological data to derive a peak post-junctional concentration of noradrenaline during transmission of about 4 x 10(-4)M.9. The fraction of total noradrenaline content of the artery released per pulse (under the influence of phenoxybenzamine) had a mean value of 2.2 x 10(-4).

Animals↗

PubMed2004

[Evaluation of isolation media for the detection of Bartonella henselae--isolation of Bartonella henselae from domestic cats].

Bartonella henselae is a causative agent of cat scratch disease. We preliminarily tested four media for the bacterial growth, including agar plates with sheep, horse or rabbit blood, and chocolate agar. Of these media, rabbit blood and chocolate agar plate were found to be more excellent for the growth than the medium with sheep or horse blood. Blood samples from 60 domestic cats in Yamaguchi Prefecture were then cultured using 7% rabbit blood agar plates and BACTEC9050 (BD), automated blood culture microbial detection system. B. henselae was isolated from six of the 60 (10%) blood samples. Tiny colonies of B. henselae were visible on the agar medium after one week of culture at 35 degrees C in the 5% CO2 atmosphere. BACTEC 9050 detected B. henselae in one of the 10 blood samples and it took two weeks to detect the bacteria automatically, though gram stain failed to show organisms in the blood culture bottle. In conclusion, rabbit blood or chocolate agar and incubation of agar media more than one week and of BACTEC more than two weeks are recommended for the detection of B. henselae.

Agar↗

PubMed2006

Mechanism of drug resistance in clonally related clinical isolates of Vibrio fluvialis isolated in Kolkata, India.

The molecular mechanisms of drug resistance in 19 strains of Vibrio fluvialis isolated from 1998 to 2002 in Kolkata, India, were investigated. Class 1 integrons were detected in eight strains, and four strains were found to carry SXT integrases. In the presence of carbonyl cyanide m-chlorophenylhydrazone or reserpine, all nalidixic acid- and ciprofloxacin-resistant strains became sensitive, suggesting that drug efflux plays a major role in quinolone resistance in V. fluvialis. It was further seen that strains which had MICs of > 25 microg/ml for nalidixic acid had a sense mutation (Ser to Ile) at position 83 of the quinolone resistance-determining region of gyrA. All except one of the integron- and SXT integrase-bearing strains belonged to the same ribotype.

Anti-Bacterial Agents↗

PubMed1983

Isolation and Characterization of Methanomicrobium paynteri sp. nov., a Mesophilic Methanogen Isolated from Marine Sediments.

A new mesophilic methanogenic bacterial species isolated from marine sediments collected in the Cayman Islands is described. Cells are small rods occuring singly without filaments, are not motile, and do not possess flagella. Colonies are semitransparent and off-white in color. After 2 weeks of incubation at 37 degrees C colonies are 1 to 2 mm in size, circular, and have entire edges. Only hydrogen-carbon dioxide is a substrate for growth and methane formation. Cells can tolerate a variety of organic secondary buffers (bicarbonate-CO(2) being the primary buffer). Cells do not require yeast extract or Trypticase, but do require acetate, for growth. The optimum growth temperature is 40 degrees C. The optimum sodium concentration is 0.15 M. The optimum pH for growth is 7.0. The minimum generation time is 4.8 h. The DNA base composition is 44.9 mol% guanine plus cytosine. The name Methanomicrobium paynteri is proposed in honor of M. J. B. Paynter. The type strain is G-2000 (=ATCC 33997, =DSM 2545).

Journal Article↗

PubMed2003

Similarity of tetracycline resistance genes isolated from fish farm bacteria to those from clinical isolates.

Tetracycline-resistant (Tet(r)) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tet(r) gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tet(r) genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tet(r) strains transferred Tet(r) genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tet(r) strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.

Animals↗

PubMed1980

Isolation, characterization, and biological properties of a tuberculin-active peptidoglycan isolated from the culture filtrate of Mycobacterium tuberculosis.

A water-soluble tuberculin-active peptidoglycan (TAPG) with a molecular weight of ca. 28,000 to 30,000 was isolated from the culture filtrate of Mycobacterium tuberculosis. TAPG was approximately four to five times more potent than tuberculin purified protein derivative S in guinea pigs sensitized with M. tuberculosis or M. bovis (freeze-dried BCG). It showed little or no cross-reactivity at a dose of 0.1 to 0.4 microgram in guinea pigs sensitized with M. kansasii, M. scrofulaceum, M. intracellulare, or M. avium. TAPG did not show any adjuvant activity when injected in guinea pigs in a water-in-oil emulsion containing ovalbumin. TAPG, in Freund incomplete adjuvant, proved to be an effective immunogen for inducing delayed hypersensitivity in guinea pigs. Chemical analysis of TAPG showed that it contains proline, glutamic acid, alanine, diaminopimelic acid, tyrosine, threonine, glucosamine, and the reducing sugars, arabinose and galactose. In immunoelectrophoretic studies with reference M. tuberculosis H37Rv antiserum, TAPG did not show any precipitin bands.

Adjuvants, Immunologic↗

PubMed1981

Adherence of Streptococcus sanguis clinical isolates to smooth surfaces and interactions of the isolates with Streptococcus mutans glucosyltransferase.

Streptococcus sanguis isolated from human dental plaque were grown in Todd-Hewitt broth. Cells were collected by centrifugation and lyophilized after extensive washing with water. The cell-associated glucosyltransferase (GTase) activities of S. sanguis strains were assayed with [14C]sucrose. Strain differences in GTase activity were significant within the same serotype or biotype or both. The ability of S. sanguis cells to adhere to smooth glass surfaces was generally weak, irrespective of significant cell-associated GTase activity synthesizing water-insoluble, gel-like glucans. Resting cells of most S. sanguis strains bound extracellular GTase from Streptococcus mutans strain B13 (serotype d), resulting in the strong adherence of the S. sanguis cells to smooth glass surfaces in the presence of sucrose. Conversely, S. mutans B13 cells also could bind extracellular GTase from some strains of S. sanguis examined. The sucrose-dependent adherence of S. mutans cells was not altered, although S. sanguis strains from which the extracellular GTases were obtained did not produce significant adherence in the presence of sucrose. In view of these findings, it was suggested that S. mutans GTase could affect the adherence of S. sanguis to smooth tooth surfaces in the oral cavity.

Binding Sites↗

PubMed1999

Isolation and chemical characterization of a capsular polysaccharide antigen shared by clinical isolates of Enterococcus faecalis and vancomycin-resistant Enterococcus faecium.

Enterococci are a common cause of serious infections, especially in newborns, severely immunocompromised patients, and patients requiring intensive care. To characterize enterococcal surface antigens that are targets of opsonic antibodies, rabbits were immunized with various gentamicin-killed Enterococcus faecalis strains, and immune sera were tested in an opsonophagocytic assay against a selection of clinical isolates. Serum raised against one strain killed the homologous strain (12030) at a dilution of 1:5,120 and mediated opsonic killing of 33% of all strains tested. In addition, this serum killed two (28%) of seven vancomycin-resistant Enterococcus faecium strains. Adsorption of sera with the homologous strain eliminated killing activity. The adsorbing antigens were resistant to treatment with proteinase K and to boiling for 1 h, but were susceptible to treatment with sodium periodate, indicating that the antigen inducing opsonic activity is a polysaccharide. Antibodies in immune rabbit sera reacted with a capsule-like structure visualized by electron microscopy both on the homologous E. faecalis strain and on a vancomycin-resistant E. faecium strain. The capsular polysaccharides from E. faecalis 12030 and E. faecium 838970 were purified, and chemical and structural analyses indicated they were identical glycerol teichoic acid-like molecules with a carbohydrate backbone structure of 6-alpha-D-glucose-1-2 glycerol-3-PO4 with substitution on carbon 2 of the glucose with an alpha-2-1-D-glucose residue. The purified antigen adsorbed opsonic killing activity from immune rabbit sera and elicited high titers of antibodies (when used to immunize rabbits) that both mediated opsonic killing of bacteria and bound to a capsule-like structure visualized by electron microscopy. These results indicate that approximately one-third of a sample of 15 E. faecalis strains and 7 vancomycin-resistant E. faecium strains possess shared capsular polysaccharides that are targets of opsonophagocytic antibodies and therefore are potential vaccine candidates.

Animals↗

PubMed1972

New centrifugation technique for isolating enzymes from large cell structures: isolation and characterization of two Bacillus subtilis autolysins.

Bacillus subtilis cell walls can be centrifuged through a linear gradient of 0 to 2 m LiCl and 10 to 25% sucrose so that different autolysins are removed by different salt concentrations and banded in separate positions as the walls pass through the gradient. Using this technique we have found that B. subtilis cell walls are isolated with two autolytic enzymes attached. One autolysin, a glycosidase, can be eluted from walls with 0.5 m LiCl, has a pH optimum between 5 and 8, is relatively heat-sensitive, and has a molecular weight of 60,000. The other autolysin, an alanine amidase, can be eluted from walls with 1.5 m LiCl, has a pH optimum around 8, is relatively heat-stable, has a molecular weight of 35,000, and is present in quantities ten times greater than the glycosidase.

Alanine↗

PubMed1973

Isolation and partial characterization of temperature-sensitive mutants in ten loci that affect cell membrane synthesis in Escherichia coli: isolation and genetic sorting.

Four hundred temperature-sensitive (ts) mutants were isolated by the (3)H-glycerol-3-phosphate membrane suicide procedure of Cronan, Ray, and Vagelos and were sorted into 13 groups by the rapid mapping procedures of Low. Recombination and complementation studies on representative members of each group suggested that the ts mutations of all 13 groups are present in genetically different complementation groups. Biochemical studies suggested that 10 of these ts mutations affected cell membrane synthesis. In this paper, the genetic data are presented in detail so that the limits of the Low rapid mapping procedures can be assessed, and in an accompanying paper the partial biochemical characterization of the ts mutations is described.

Cell Membrane↗

PubMed1978

Microbial oxidation of methane and methanol: isolation of methane-utilizing bacteria and characterization of a facultative methane-utilizing isolate.

A methane-utilizing organism capable of growth both on methane and on more complex organic substrates as a sole source of carbon and energy, has been isolated and studied in detail. Suspensions of methane-grown cells of this organism oxidized C-1 compounds (methane, methanol, formaldehyde, formate); hydrocarbons (ethane, propane); primary alcohols (ethanol, propanol); primary aldehydes (acetaldehyde, propionaldehyde); alkenes (ethylene, propylene); dimethylether; and organic acids (acetate, malate, succinate, isocitrate). Suspensions of methanol-or succinate-grown cells did not oxidize methane, ethane, propane, ethylene, propylene, or dimethylether, suggesting that the enzymatic systems required for oxidation of these substrates are induced only during growth on methane. Extracts of methane-grown cells contained a particulate reduced nicotinamide adenine dinucleotide-dependent methane monooxygenase activity. Oxidation of methanol, formaldehyde, and primary alcohols was catalyzed by a phenazine methosulfate-linked, ammonium ion-requiring methanol dehydrogenase. Oxidation of primary aldehydes was catalyzed by a phenazine methosulfate-linked, ammonium ion-independent aldehyde dehydrogenase. Formate was oxidized by a nicotinamide adenine dinucleotide-specific formate dehydrogenase. Extracts of methane-grown, but not succinate-grown, cells contained the key enzymes of the serine pathway, hydroxypyruvate reductase and malate lyase, indicating that the enzymes of C-1 assimilation are induced only during growth on C-1 compounds. Glucose-6-phosphate dehydrogenase was induced during growth on glucose. Extracts of methane-grown cells contained low levels of enzymes of the tricarboxylic acid cycle, including alpha-keto glutarate dehydrogenase, relative to the levels found during growth on succinate.

Alcohol Oxidoreductases↗