In vivo 19F-NMR study of halothane distribution in brain

Halothane distribution and elimination from rabbit brain was studied in vivo using 19F-NMR spectroscopy. Two exponential decay functions for the anesthetic were observed in the clearance curve. They are assigned to halothane in brain held in two distinct chemical environments characterized by different chemical shifts, and half-lives (25 and 320 min). A nonvolatile halothane metabolite with a half-life of several days was found to be present in rabbit brains. The in vivo results were corroborated by ex vivo experiments on excised brain tissue. Halothane was distributed in all of the major cell subfractions, whereas the metabolite was present predominantly in the cytoplasm.     

环境因子对厌氧微生物脱卤的影响研究进展

有机卤代物在工农业生产中的广泛应用及不合理处置与排放导致其成为地下环境中普遍存在的一类污染物,严重威胁地下生态系统功能、饮用水安全和人类健康。有机卤呼吸细菌在有机卤污染场地的原位生物修复中起到了至关重要的作用。本文简要概述了影响和制约有机卤呼吸微生物生长代谢及脱卤活性的一些关键环境因子,如pH、温度、盐度、电子供受体和氧气等,并且对有机卤呼吸细菌未来的研究方向提出展望,以期为污染场地原位生物修复工程的有效实施提供理论与技术参考。     

Characterization of the acclimation period before anaerobic dehalogenation of halobenzoates

The acclimation periods prior to detectable dehalogenation of halogenated benzoates in anaerobic lake sediments ranged from 3 weeks to 6 months. These acclimation periods were reproducible over time and among sampling sites and were characteristic of the chemical tested. The lengthy acclimation period appears to represent an induction phase in which little or no aryl dehalogenation is observed, followed by an exponential increase in activity typical of an enrichment response. Continuous growth from the time of the first exposure to the chemical is inconsistent with the extremely low per-cell activities estimated for the early days of the acclimation period and the fact that the dehalogenation yields no carbon to support microbial growth. The finding of a characteristic acclimation time for each chemical argues against nutritional deficiency, inhibition, or predation as an explanation for this phase of metabolism, while the reproducibility of the findings with time and space and among replicates argues against genetic changes as the explanation. The acclimation times did correlate with the eventual dehalogenation rates. This may reflect the general energy limitations in the anaerobic communities and suggests that those chemicals with faster dehalogenation rates provide more energy for the induction and growth phases of the active population.     

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B₁ and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.     

Characterization of the acclimation period before anaerobic dehalogenation of halobenzoates.

The acclimation periods prior to detectable dehalogenation of halogenated benzoates in anaerobic lake sediments ranged from 3 weeks to 6 months. These acclimation periods were reproducible over time and among sampling sites and were characteristic of the chemical tested. The lengthy acclimation period appears to represent an induction phase in which little or no aryl dehalogenation is observed, followed by an exponential increase in activity typical of an enrichment response. Continuous growth from the time of the first exposure to the chemical is inconsistent with the extremely low per-cell activities estimated for the early days of the acclimation period and the fact that the dehalogenation yields no carbon to support microbial growth. The finding of a characteristic acclimation time for each chemical argues against nutritional deficiency, inhibition, or predation as an explanation for this phase of metabolism, while the reproducibility of the findings with time and space and among replicates argues against genetic changes as the explanation. The acclimation times did correlate with the eventual dehalogenation rates. This may reflect the general energy limitations in the anaerobic communities and suggests that those chemicals with faster dehalogenation rates provide more energy for the induction and growth phases of the active population.     

In vivo and in vitro anaerobic mating in Candida albicans

Candida albicans cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at 37 degrees C, block production of farnesol, and permit in vitro mating at 37 degrees C at efficiencies of up to 84%. Aerobically, farnesol prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized gastrointestinal (GI) tract, could serve as niches for C. albicans mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells, confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear, and prototrophic, and they contained the heterologous hisG marker from one of the parental strains.     

Rapid dehalogenation of 2,4,6-trichlorophenol at alkaline pH by an anaerobic enrichment culture

An enrichment culture, derived from the anaerobic stage of a two-step sequential anaerobic-aerobic reactor system which mineralized 2,4,6-trichlorophenol, stoichiometrically converted 2,4,6-trichlorophenol to 4-chlorophenol. Dehalogenation occurred only in alkaline media (pH 8–9) at concentrations of substrate up to 1 mmol 1¹. Formate plus acetate or trypticase could serve as electron donors. Neither vitamins nor trace elements were required in a chloride-free defined medium. The dehalogenating organism was oxygen-resistant, but was not active in media which were oxidized with respect to resazurin indicator dye. Most probable number counts of the dehalogenating cultures showed that the dehalogenating organisms were present in very small numbers, yet catalysed dehalogenation at rates considerably faster than other dehalogenating organisms described in the literature.     

Reductive dehalogenation of dichloroanilines by anaerobic microorganisms in fresh and dichlorophenol-acclimated pond sediment

We investigated the transformation of 2,4-dichloroaniline (2,4-DiCA) and 3,4-DiCA to monochloroanilines (CA) in anaerobic pond sediment. Dechlorination of 3,4-DiCA to 3-CA started after a lag period of 3 weeks and was complete after an additional 5 weeks. Although 2,4-DiCA disappeared over 8 weeks, the appearance of a CA product could not be detected. In contrast, anaerobic bacteria in pond sediment acclimated to dehalogenate 2,4-dichlorophenol (2,4-DiCP) or 3,4-DiCP rapidly dechlorinated 2,4-DiCA and 3,4-DiCA without any lag time. By comparison, anaerobic sediment bacteria acclimated to 3,4-DiCA rapidly degraded 3,4-DiCP without a lag. In all cases, the CA products were stable for the duration of the experiments. It is concluded that cross-acclimation occurred.     

Reductive dehalogenation of dichloroanilines by anaerobic microorganisms in fresh and dichlorophenol-acclimated pond sediment.

We investigated the transformation of 2,4-dichloroaniline (2,4-DiCA) and 3,4-DiCA to monochloroanilines (CA) in anaerobic pond sediment. Dechlorination of 3,4-DiCA to 3-CA started after a lag period of 3 weeks and was complete after an additional 5 weeks. Although 2,4-DiCA disappeared over 8 weeks, the appearance of a CA product could not be detected. In contrast, anaerobic bacteria in pond sediment acclimated to dehalogenate 2,4-dichlorophenol (2,4-DiCP) or 3,4-DiCP rapidly dechlorinated 2,4-DiCA and 3,4-DiCA without any lag time. By comparison, anaerobic sediment bacteria acclimated to 3,4-DiCA rapidly degraded 3,4-DiCP without a lag. In all cases, the CA products were stable for the duration of the experiments. It is concluded that cross-acclimation occurred.     

In vivo study on medullary H(+)-sensitive neurons

Using the micro pressure ejection technique, we examined responses of medullary neurons with nonphasic discharges (164 units) to direct application of acidified mock cerebrospinal fluid (CSF, pH 6.85-7.05) in decerebrated spontaneously breathing cats. We found 16 H(+)-sensitive cells; they were excited promptly on application of approximately 500 pl of acidified mock CSF in the vicinity of the neuron under investigation, whereas they were unaffected by microejection of the control mock CSF (pH 7.25-7.60). Of the 16 H(+)-sensitive cells, 10 units were further found to be excited by transcapillary stimulation of the central chemoreceptors by using a method of intravertebral arterial injection of CO2-saturated saline. The discharges increased in a similar time course to that of ventilatory augmentation. Distributions of these 10 specific H(+)-sensitive cells were found in the vicinity of nucleus tractus solitarii as well as deep in the ventrolateral medulla. The present results suggest a possibility that pH-dependent central chemoreceptors, if any, would be located in two distinct medullary regions described in this study.     

In vitro study of the proteolytic activity of rumen anaerobic fungi

Abstract To better define the antigenic structure of the outer cell membranes of Legionellae, a panel of 6 monoclonal antibodies was raised against partially purified outer membranes of Legionella pneumophila serogroup 1, Corby strain. This study describes the purification and characterization of one of these monoclonal antibodies reacting with a 135-kDa protein, which was shown to be common to all 14 serogroups of Legionella pneumophila . It shows no cross-reactivity with 20 other Legionella species, or 9 other Gram-negative species tested by SDS-PAGE and Western blotting procedures. The epitope would appear to be predominantly surface exposed and, from preliminary detergent extraction studies, not peptidoglycan-associated.     

Photolysis of nitrosamines and nitrosamides at neutral pH: a spin-trap study

A model system has been used to study the types of radicals formed on denitrosation of N-nitroso compounds. Free radicals were formed at room temperature (22 degrees-23 degrees C) and neutral pH by photolytic cleavage of N-nitroso bonds and were partially characterized following their addition to the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenyl-nitrone (PBN). Carbon-centered radical adducts were obtained during nitrosamine photolysis and nitrogen-centered radical adducts during nitrosamide photolysis. Since both the nitrosamines and nitrosamides initially form nitrogen-centered radicals on photolysis, a secondary reaction or rearrangement must occur after initial N-nitroso bond cleavage in the nitrosamines. Mechanisms are proposed to account for these results.     

抗生素在体内外对厌氧菌和艰难梭菌细胞毒素生成的作用

氯林霉素、灭滴灵和甲砜霉素对大多数肠道厌氧菌的生长具抑制作用。氯林霉素还会破坏肠道菌群平衡,使原来受抑制的艰难梭菌得以定植,并在艰难梭菌浓度达10~8/g盲肠内含物时,检测到艰难梭菌细胞毒素。培养基中亚抑菌浓度的氯林霉素和灭滴灵会推迟艰难梭菌细胞霉素的生成。灭滴灵还可保护无菌小鼠及受氯林霉素处理的悉生小鼠免遭艰难梭菌细胞毒素的致死作用,从而证实了灭滴灵在伪膜性结肠炎临床治疗中的可用性。     

In vivo study of chloroplast volume regulation.

This paper describes a new technique that can be used to study chloroplast volume regulation in vivo. Nuclear magnetic resonance spectroscopy was used to measure relative amounts of chloroplast water in Acer platanoides leaves as they dried in air, and also in leaf disks exposed to aqueous polyethylene glycol, sucrose, or glycerol. The chloroplasts retained a constant quantity of water as leaf water potentials varied between -0.05 and -1.90 MPa, indicating that volume regulation was effective throughout this range. The chloroplasts lost water when the water potential fell below -1.90 MPa, except when leaf disks were exposed to glycerol, suggesting that the lower limit of effective volume regulation is determined by physiological levels of osmotic solutes and that glycerol can be used for chloroplast osmoregulation.     

Oxygen radicals produced by plant plasma membranes: an EPR spin-trap study

Plant plasma membranes are known to produce superoxide radicals, while the production of the hydroxyl radical, previously detected in complex plant tissues, is thought to occur in the cell wall. The mechanism of production of superoxide radicals by plant plasma membranes is, however, under dispute. It is shown, using electron paramagnetic resonance spectroscopy with a 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin-trap capable of differentiating between radical species, that isolated purified plasma membranes from maize roots produce hydroxyl radicals besides superoxide radicals. The results argue in favour of superoxide production through an oxygen and diphenylene iodonium-sensitive, NADH-dependent superoxide synthase mechanism, as well as through other unidentified mechanism(s). The hydroxyl radical is produced by an oxygen-insensitive, NADH-stimulated mechanism, which is enhanced in membranes in which the superoxide synthase is incapacitated by substrate removal or inhibition.     

In vivo study on alkylation site in DNA by the bifunctional dianhydrogalactitol

In vivo alkylation of Yoshida sarcoma cell DNA by ³H-labelled 1,2:5,6-dianhydrogalactitol (DAG) yielded N-7 monogalactitylguanines and 1,6-di-(guanin-7-yl)-galactitol, similar to the alkylated products obtained by in vitro reaction of DNA with dianhydrogalactitol in neutral solution. The ratio between monoalkylguanines and diguaninyl product was 2–2.5, slightly increasing with doses. Persistence of alkylated products in DNA was followed in function of time. There was no significant loss of either monoalkylated bases or diguaninyl derivative during the observation period i.e. 7–24 h after treatment. In contrast, the physical measurements of the amount of renaturable DNA showed a rapid opening of cross-links in the same period. Taking the presence of diguaninyl moiety as an indicator of cross-links in DNA, these two latter findings show an apparent contradiction which could be reconciled however by the mechanism proposed by Reid and Walker (Biochim. Biophys. Acta, 179 (1969) 179) for the removal of cross-linkage induced by HN₂. Accordingly, one arm of the cross-links is removed, probably enzymically, leaving the DNA non-renaturable, while the other arm of cross-link is still covalently attached to the DNA molecule rendering possible the detection of diguaninyl moiety in DNA at some later time. This concept for the removal of cross-links from DNA seems to be supported by our results too.     

In vivo 19F-NMR study of isoflurane elimination from brain

The time course of isoflurane elimination from rabbit brain was studied in vivo with 19F-NMR spectroscopy. Two exponential decay functions with different time constants were observed and assigned to two distinct brain compartments. Isoflurane has a 26 min time constant for one compartment (similar to a value of 25 min with halothane) but 174 min in the second one, compared with 320 min for halothane. The shorter half-life for isoflurane may be due to lower solubility of this agent in brain tissue. Comparison of isoflurane 19F chemical shifts in solvents in isolated brain lipids and in whole brain tissue indicates that the anesthetic present in the brain exists in a single environment (on the NMR time scale), which is a weighted average of both hydrophilic and hydrophobic environments.