A method for long-term measurement of respiration in intertidal fishes during simulated intertidal conditions

Aquatic and aerial respiration of the amphibious fishes Lipophrys pholis and Periophthalmus barbarus were examined using a newly designed flow-through respirometer system. The system allowed long-term measurements of oxygen consumption and carbon dioxide release during periods of aquatic and aerial respiration. The M o ₂ of L. pholis , measured at 15° C, was 2·1 μmol O₂ g^–1 h^–1 during aquatic and 1·99 μmol O₂ g^–1 h^–1 during aerial exposure. The corresponding values of the M co₂ were 1.67 and 1.59 μmol O₂ g^–1 h^–1 respectively, giving an aquatic respiratory exchange ratio (RER) of 0·80 and an aerial RER of 0·79. The M o₂ of P. barbarus , measured at 28°C, was 4·05 μmol O₂ g^–1 h^–1 during aquatic and 3·44 μmol O₂ g^–1 h^–1 during aerial exposure. The corresponding values of the Mco₂ were 3·29 μmol CO₂ g^–1 h^–1 and 2·63 μmol CO₂ g^–1 h^–1 respectively, giving an aquatic RER of 0·81 and an aerial RER of 0·77. While exposed to air for at least 10 h, both species showed no decrease in metabolic rate or carbon dioxide release. The RER of these fishes equalled their respiratory quotient. After re-immersion an increased oxygen consumption, due to the payment of an oxygen debt, could not be detected.     

Resting Energy Expenditure in Obese African American and Caucasian Women

The prevalence of obesity among African American women approaches 50% and greatly exceeds rates for Caucasian women. In addition, black women lose less weight than white during obesity treatment and gain more weight when untreated. This study assessed resting energy expenditure (REE) and body composition in obese white (n=122) and black (n=44) women to explore the relationship between biological variables and these observed differences. REE and body composition were assessed by indirect calorimetry and densitometry, respectively, before weight loss. REE was significantly lower in black subjects (1637.6 ± 236.9 kcal/d) than in white (1731.4 ± 262.0) (p=0.04). REE remained significantly lower in blacks than whites after adjusting for body weight (p=0.02). REE, adjusted for fat-free mass, was also significantly lower in blacks than whites (p<0.0001), although the overestimation of fat-free mass by densitometry in blacks may have contributed to this finding. There were no differences between the groups in respiratory quotient. These results suggest that a decreased REE may exist in obese black women, and it may be related to the observed differences between black and white women in the prevalence of obesity and in the response to weight loss treatment. These crosssectional findings await confirmation in longitudinal studies.     

Separation of intact and damaged hepatocytes in sucrose following non-enzymatic liver perfusion

This study deals with isolation of rat hepatocytes by a non-enzymatic method and the separation of intact and damaged cells in sucrose medium. Low speed centrifugation in isotonic sucrose medium of a hepatocyte suspension obtained by mechanical desaggregation of liver pre-perfused with EDTA solution results in the formation of a cell pellet which contains two different layers. A darker layer contains hepatocytes with intact plasma membranes. Their respiratory activity and xenobiotic metabolism are close to those of the cells isolated by collagenase perfusion. The study of distribution of lipophilic cation tetraphenylphosphonium (TPP⁺) indicates a predominantly mitochondrial localization of TPP⁺ in the intact cells following non-enzymatic and collagenase isolation. Hepatocytes in the upper layer have damaged plasma membranes. As a result they lose the potential to accumulate TPP⁺, and have low rates of endogenous respiration and biotransformation activity. Addition of exogenous NADPH restores the capability to metabolize xenobiotics. Washing and incubation of these hepaticytes in an intracellular type medium results in restoration of uncoupler-stimulated oxygen consumption and generation of membrane potential in the presence of a succinate substrate. These properties are close to those of hepatocytes permeabilized by digitonin treatment. Thus, the procedure allows the simultaneous isolation of both intact and permeabilized hepatocytes with functionally active intracellular structures without the use of relatively expensive chemicals such as collagenase and Percoll.Abbreviations 4-OHBP 4-hydroxybiphenyl - BP biphenyl - BSA bovine serum albumin - DNP 2,4-dinitrophenol - EDTA ethylendiamintetraacetate - NADPH nicotinamide adenine dinucleotide phosphate reduced - p-NA p-nitroanisole - p-NPh p-nitrophenol - TPP⁺ tetraphenylphosphonium     

苯并噻唑醌类化合物对呼吸链酶系的抑制作用

合成了2-氯-5-正十二硫烷基-6-甲基-4,7-苯并噻唑醌(2-Cl-DMMDBT)和2-氯-5-正丁烷氨基-6-甲基-4,7-苯并噻唑醌(2-Cl-BAMDBT)两种化合物,研究了它们对线粒体呼吸链酶系的抑制作用.结果表明:2-Cl-DMMDBT和2-C1-BAMDBT对琥珀酸氧化酶及泛醌氧化酶的电子传递活性均表现一定的抑制作用,而对细胞色素氧化酶无作用,说明二者的抑制作用发生在泛醌反应区.二者对NADH氧化酶的抑制行为略有不同,2-Cl-DMMDBT是一个逐渐加强的过程,最终可致酶活性完全抑制,而2-Cl-BAMDBT则表现为瞬间抑制.比较了2-Cl-DMMDBT和2-Cl-BAMDBT对琥珀酸氧化酶的抑制能力,长侧链的2-Cl-DMMDBT比短侧链的2-Cl-BAMDBT抑制能力强很多.     

棉铃虫的呼吸代谢

棉铃虫的耗氧量随着幼虫的生长发育而增加。当发育状态基本相同时,在同一龄期内,幼虫耗氧量与虫体鲜重呈直线相关;在不同龄期之间,耗氧量则与虫体鲜重的0.97次方成正比。幼虫的代谢速率随着龄期的增加而稳定地降低。在20—35℃范围内,温度每升高10℃,幼虫的代谢速率约增加一倍。蛹期的代谢速率呈典型的“U”形曲线变化。从卵期及幼虫期到蛹期的呼吸商变动在0.75—0.96之间。     

The role of Mg2+ in the regulation of the structural and functional steady-states in rat liver mitochondria

A possible relationship between mitochondrial Mg²⁺ levels, structural configurations, and functional steady states has been studied in rat liver mitochondria. The results show that the concentration of mitochondrial Mg²⁺ in respiratory state 4 is definitely higher than in respiratory state 3. The metabolic transition from state 3 to state 4 and vice-versa is associated with reversible influx-efflux of about 10 nmol of Mg²⁺ per mg protein. The net uptake of this aliquot of Mg²⁺ is a necessary condition in order for the metabolic transition to state 4, both structurally and functionally, to occur. This process requires a threshold concentration of external Mg²⁺ greater than 5 mM. The phosphorylative mechanism does not appear to depend on the presence or absence of external Mg²⁺. The role of Mg²⁺ on the attainment and maintenance of the structural and functional steady state 4 seems to be correlated with its regulatory effect on the concentration of the mitochondrial P_i.     

Luminol-dependent chemiluminescence in bovine eosinophils and neutrophils: Differential increase of intracellular and extracellular chemiluminescence induced by soluble stimulants

Luminol chemiluminescence was used to detect activation of the respiratory burst oxidase in bovine eosinophils and neutrophils. Extracellular and intracellular chemiluminescence were measured by supplementing the medium with horseradish peroxidase and catalase, respectively. Pure bovine eosinophils (> 90%), maximally stimulated with 1 nmol/l phorbol 12-myristate-13-acetate (PMA) showed ten times more extracellular luminol-dependent chemiluminescence (CL) than maximally stimulated pure bovine neutrophils (> 96%). Extracellular CL from eosinophils was preferably induced over intracellular CL by both PMA (27-fold difference) and platelet-activating factor (PAF) at 2 μmol/l (9-fold difference), but not by calcium ionophore A23187 (15 μmol/l). Time course information was used in the following experiments to distinguish between the mode of action of various stimulants. A progressively longer lag period was observed in eosinophil suspensions treated with decreasing doses of PMA, whereas platelet-activating factor induced a dose-dependent increase in the maximum response with no change in time to peak CL. The time course of extracellular CL was almost identical to intracellular CL for all stimulants tested, providing no evidence to suggest that extracellular CL stems from a different enzyme system than intracellular CL. Eosinophils generated most extracellular CL when stimulated with PMA, whereas neutrophils were most efficiently stimulated with A23187, which induced intracellular CL in eosinophils as well as in neutrophils. This accords with the greater tendency of neutrophils to ingest and kill microorganisms, whereas eosinophils are armed to destroy large extracellular targets.     

A unified stoichiometric model for oxidative and oxidoreductive growth of yeasts

Yeasts degrade glucose through different metabolic pathways, where the choice of the pathway is dependent on the nature of the limitation in the various substrates. When oxygen is limiting in addition to glucose, yeasts often grow according to a mixture of oxidative and reductive metabolism. Oxygen may be limiting either by supply or by inherent biological restrictions such as the respiratory bottleneck in Saccharomyces cerevisiae or by both. A unified model incorporating both supply and biological limitations is proposed for the quantitative prediction of growth rates, consumption and production rates, as well as key metabolite concentrations during mixed oxidoreductive metabolism occuring as a result of such oxygen limitations. This simple unstructured model can be applied to different yeast strains while at the same time requiring a minimum number of measured parameters. "Estimators" are utilized in order to predict the presence of supply-side or biological limitations. The values of these estimators also characterize the relative importance of oxidative to total metabolism. Results from the aerobic and oxygen-limited chemostat cultures were used to corroborate the model predictions. During these experiments, the heat released by the yeast cultures was also monitored on-line. The model correctly predicted the overall stoichiometry, steady-state concentrations, and rates including heat dissipation rates measured in the various situations of oxygen limitations. Direct continuous measurements such as heat can be used in conjunction with the unified model for on-line proces control. (c) 1992 John Wiley & Sons, Inc.     

Vertical profiles of CH4 concentrations,dissolved substrates and processes involved in CH4 production in a flooded Italian rice field

Vertical profiles were measured in soil cores taken from flooded rice fields in the Po valley during July and August 1990. Methane concentrations generally increased with depth and reached maximum values of 150–500 μM in 5–13 cm depth. However, the shape of the profiles was very different when studying different soil cores. The CH₄ content of gas bubbles showed a similar variability which apparently was due to spatial rather than temporal inhomogeneities. Similar inhomogeneities were observed in the vertical profiles of acetate, propionate, lactate, and formate which showed maximum values of 1500, 66, 135, and 153, μM, respectively. However, maxima and minima of the vertical profiles of the different substates usually coincided in one particular soil core. Large inhomogeneities in the vertical profiles were also observed for the rates of total CH₄ production, however, the percentage contribution of H₂/CO₂ to CH₄ production was relatively homogeneous at 24 ± 7% (SD). Similarly, the H₂ content of gas bubbles was relatively constant at 93.3 ± 9.6 ppmv when randomly sampled in the rice field at different times of the day. A small contribution (6%) of H₂/CO₂ to acetate production was also observed. Vertical profiles of the respiratory index (RI) for [2-¹⁴C] acetate showed that acetate was predominantly degraded by methanogenesis in 5–11 cm depth, but by respiration in the surface soil (3 cm depth) and in soil layers below 13–16 cm depth which coincided with a transition of the colour (grey to reddish) and the physical characteristics (porosity, density) of the soil. The observations indicate that the microbial community which degrades organic matter to CH₄ is in itself relatively homogenous, but operates at highly variable rates within the soil structure. Author for correspondence     

Selective isolation and culture of a proliferating epithelial cell population from the hamster trachea

Summary A reliable cell isolation technique was developed to allow the cultivation of cells from the hamster respiratory tract. Repeated thermolysin treatments and gradient centrifugation yielded a cell culture completely free from contamination by fibroblasts. Viable cells could be isolated from as little tissue as a single hamster trachea, but in vitro proliferation occurred only if the hamster was less than 4 months of age. The cultured cells could be repeatedly passaged and subcultured for weeks by employing normal tissue culture techniques. Morphologically, the monolayers appeared to be a homogeneous population of epithelial cells, and successful cloning of freshly isolated single cells resulted in apparently identical cultures. The epithelial origin of these cells was also suggested by continued growth in minimum essential medium withd-valine substituted forl-valine. The relative ease with which this cell type can be isolated, cultured, and manipulated in vitro should encourage its application as a model of the respiratory epithelium. This research was supported by Public Health Service Grant P50-HL 19171 and Research Career Development Award 1-K04-AI 00178 to J. B. B.