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PubMed1996

Concentrating breath samples using liquid nitrogen: a reliable method for the simultaneous determination of ethane and pentane.

The measurement of ethane and pentane in breath offers a sensitive and noninvasive means to assess in vivo lipid peroxidation in animals and humans. However, numerous technical obstacles inherent in collecting and concentrating air-breath samples have limited the wider application of these measurements for the assessment of in vivo lipid peroxidation. We have developed a relatively simple, inexpensive, rapid, and reliable method to collect, concentrate, and measure breath ethane and total-body pentane from rats. This method, which concentrates alkanes from 4 liters of collected air-breath on adsorbant cooled to -174 degrees C, was found to be superior to similar cryofocusing techniques at -130 degrees C, which fail to effectively trap highly volatile ethane from large volumes of air. We found ethane evolves predominantly through breath, whereas a significant amount of pentane evolves from sources other than breath. Mean evolution rate for ethane was 1.08 pmol/100 g body wt/min. Pentane evolution rates displayed more inter-rat and day-to-day variability with a mean of 0.52 pmol/100 g body wt/min. We also found that excreted rat feces exude large amounts of ethane and pentane.

Animal Population Groups↗
PubMed1995

Detection of hydroxyl radical in intact cells of Chlorella vulgaris.

Using ESR with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin-trapping reagent, we measured the levels of free radical species generated from living cells of Chlorella vulgaris var. vulgails (IAM C-534). To investigate the production of free radicals in the living Chlorella vulgaris cells, the influence of DMPO toward the intact cells of the Chlorella vulgaris using the O2 evolution rate was first studied as a guide. Since the O2 evolution rate was not changed by DMPO, it was judged that DMPO has no toxicity toward the intact cells of Chlorella vulgaris. Only hydroxyl radicals (.OH) were detected as the DMPO-OH adduct in the suspension of intact cells of Chlorella vulgaris irradiated with visible light. Moreover, since production of .OH was inhibited by some hydroxyl radical scavengers such as KI and ethanol, production of .OH was proved to be due to hydroxyl radicals. It was also clear that the intensity of .OH increased with increasing irradiation intensity of visible light. Therefore, it was suggested that .OH might be one of the photoinhibition factors of the intact Chlorella vulgaris cells in severe light conditions.

Chlorella↗
PubMed1992

Higher rate of evolution of X chromosome alpha-repeat DNA in human than in the great apes.

The rate of introduction of neutral mutations is lower in man than in other primates, including the chimpanzee. This species is generally regarded as our closest relative among the great apes. We present here an analysis of sequences of X chromosomal alphoid repetitive DNA from man and the great apes, which supports the closer relationship between man and chimpanzee and indicates a considerably increased rate of recombination in the human repeat DNA. These results indicate that the 'molecular clock' is running more quickly in man.

Animals↗
PubMed2005

Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state.

The Escherichia coli K-12 strain TG1 was grown at 28 degrees C in aerobic glucose-limited continuous cultures at dilution rates ranging from 0.044 to 0.415 h(-1). The rates of biomass formation, the specific rates of glucose, ammonium and oxygen uptake and the specific carbon dioxide evolution rate increased linearly with the dilution rate up to 0.3 h(-1). At dilution rates between 0.3 h(-1) and 0.4 h(-1), a strong deviation from the linear increase to lower specific oxygen uptake and carbon dioxide evolution rates occurred. The biomass formation rate and the specific glucose and ammonium uptake rates did not deviate that strongly from the linear increase up to dilution rates of 0.4 h(-1). An increasing percentage of glucose carbon flow towards biomass determined by a reactor mass balance and a decreasing specific ATP production rate concomitant with a decreasing adenylate energy charge indicated higher energetic efficiency of carbon substrate utilization at higher dilution rates. Estimation of metabolic fluxes by a stoichiometric model revealed an increasing activity of the pentose phosphate pathway and a decreasing tricarboxylic acid cycle activity with increasing dilution rates, indicative of the increased NADPH and precursor demand for anabolic purposes at the expense of ATP formation through catabolic activities. Thus, increasing growth rates first result in a more energy-efficient use of the carbon substrate for biomass production, i.e. a lower portion of the carbon substrate is channelled into the respiratory, energy-generating pathway. At dilution rates above 0.4 h(-1), close to the wash-out point, respiration rates dropped sharply and accumulation of glucose and acetic acid was observed. Energy generation through acetate formation yields less ATP compared with complete oxidation of the sugar carbon substrate, but is the result of maximized energy generation under conditions of restrictions in the tricarboxylic acid cycle or in respiratory NADH turnover. Thus, the data strongly support the conclusion that, in aerobic glucose-limited continuous cultures of E. coli TG1, two different carbon limitations occur: at low dilution rates, cell growth is limited by cell-carbon supply and, at high dilution rates, by energy-carbon supply.

Aerobiosis↗
PubMed2001

Nitrous oxide and dinitrogen emissions from soil under different water regimes and straw amendment.

In a laboratory study, soil amended with and without wheat straw (2.8 g kg(-1) soil) was incubated under 70% water holding capacity (WHC), continuously flooded and flooded/drained cycle conditions at 30 degrees C for 51 days. Dinitrogen and N2O evolution and ammonia volatilisation were measured during the incubation. Extractable NH4+-N and NO3--N were determined at the end of the incubation. Entrapped N2, N2O, and dissolved NH4+-N and NO3--N in drainage water were measured in the flooded/drained cycle treatment when the floodwater was drained. The results indicated that N loss through ammonia volatilisation was undetected in all treatments due to the low soil pH value (pHH2O= 5.87) and no air movement. The recovery of urea-15N as N2 was lowest in the continuously flooded treatments (0.75% and 0.96% with and without straw amendment, respectively), highest in the 70% WHC treatments (5.65% and 4.41%, respectively), and intermediate in the flooded/drained cycle treatments (1.79% and 2.65%, respectively). The recovery of urea-15N as N2O was in the same order as that of N2, negligible in the continuously flooded treatments, 0.01% and 0.07% in the flooded/drained cycle treatments, and 1.29% and 2.23% in the 70% WHC treatments, respectively. Peak N2O evolution rates were observed after the floodwater was drained but no substantial evolution was found after the soil was reflooded following drained periods. However, peak N2 evolution rates were observed after the onset of both drainage and re-flooding. Considerable quantities of N2 but no detectable N2O were entrapped in the flooded soil.

Agriculture↗
PubMed2004

Monitoring the progress of infection and recombinant protein production in insect cell cultures using intracellular ATP measurement.

Several monitoring methods used to predict viable cell density have been the subject of extensive studies, including oxygen uptake rate, carbon dioxide evolution rate, optical density, NADH-dependent fluorescence and relative permittivity measurement. We propose intracellular ATP determination by bioluminescence assay to monitor the progress of baculovirus infection and recombinant protein production in insect cell cultures. We found that the ATP content in viable cells increased after virus addition. The increase in the ATP level was observed until the maximum recombinant protein accumulation was reached. At maximum product yield, the specific ATP content significantly decreased. Results obtained in both batch and fed-batch cultures demonstrated that the specific ATP level could be considered as a good indicator of recombinant protein productivity. Monitoring the cellular ATP content after viral infection makes it possible to define the optimum time for product harvest. The main advantage of applying the ATP assay as an index of the progress of infection and recombinant protein synthesis is its short time and sensitivity.

Adenosine Triphosphate↗
PubMed2006

Phenotypes and fed-batch fermentation of ubiquinone-overproducing fission yeast using ppt1 gene.

Ubiquinone (UQ), a component of the electron transfer system in many organisms, has been widely used for pharmaceuticals and cosmetics. In this study, we cloned and overexpressed the full-length ppt1 (MTppt1) gene, which encodes p-hydroxybenzoate:polyprenyltransferase and ERppt1 gene, which was modified to be localized on endoplasmic reticulum in fission yeast. The yeast MTppt1 and ERppt1 transgenic lines showed about 3.7 and 5.1 times increment in UQ content and the recombinant yeasts with a higher UQ level are more resistant to H(2)O(2), Cu(2+) and NaCl, and interestingly their growth was also faster than the wild type at lower temperature. For large-scale cultivation, the direct feedback control of glucose using an on-line ethanol concentration monitor for ubiquinone production of yeast ERppt1 by high-cell-density fermentation was investigated and the fermentation parameters (e.g., dissolved oxygen, pH, ethanol concentration, oxygen uptake rate, carbon dioxide evolution rate and respiration quotient) were also discussed. After 90 h cultures, the yeast dry cell weight reached 57 gl(-1) and the ubiquinone yield reached 23 mgl(-1). In addition, plasmid stability was maintained at high level throughout the fermentation.

Biomass↗
PubMed1994

Mutation of chlorophyll ligands in the chlorophyll-binding CP47 protein as studied in a Synechocystis sp. PCC 6803 photosystem I-less background.

Site-directed mutations have been introduced to replace conserved histidine residues in the chlorophyll-binding protein CP47 of photosystem II (PS II) in a PS I-less/apcE-background strain of the cyanobacterium Synechocystis sp. PCC 6803. In thylakoids isolated from such a system, the degree of loss of the 695-nm fluorescence emission maximum at 77 K compared to that at 685 nm generally was consistent with the decrease in oxygen evolution rates measured at saturating light intensity. Taking into account that in the absence of CP47 and PS I some chlorophyll remains detectable in cells, the relative 695-nm fluorescence emission and the rate of oxygen evolution also correlate with the relative amount of chlorophyll per cell and with the number of PS II reaction centers on a chlorophyll basis. Interestingly, the 77 K fluorescence excitation spectra monitoring 695-nm emission of thylakoids from the CP47 His-to-Tyr mutants in a photosystem I-less/apcE-background showed increases in the 413- and 531-nm absorption regions, compared to spectra of thylakoids from the background strain. These wavelengths coincide with absorption maxima of pheophytin. No increase in the 531-nm excitation band was observed in thylakoids from mutants lacking PS II or with a His-to-Asn mutation. These results are interpreted to indicate that replacement of conserved histidine residues by tyrosine in CP47 leads to the loss of Mg2+ from chlorophyll, resulting in the formation of pheophytin, or to the binding of pheophytin (rather than chlorophyll) at a particular pigment-binding site of CP47 during biogenesis and assembly of the protein. It was observed that the light-harvesting efficiency of CP47 His mutants was lower judging from the light intensity dependence of electron transport and analysis of fluorescence decay kinetics. This suggests that the presence of pheophytin in the antenna decreases antenna efficiency.

Chlorophyll↗
PubMed1996

Autonomous metabolic oscillation in continuous culture of Saccharomyces cerevisiae grown on ethanol.

When the yeast Saccharomyces cerevisiae was grown under aerobic continuous culture conditions with a medium containing ethanol as carbon source, an autonomous sustained metabolic oscillation appeared. This oscillation was observed in rates and concentrations of various parameters such as, ethanol, oxygen uptake rate, carbon dioxide evolution rate, NaOH addition rate for pH control, acetate, and intracellular pH. No changes were observed in concentrations of stock carbohydrates. Intracellular pH changes were out of phase with oxygen uptake rate, which was reverse of the results with glucose-based oscillation. These results suggested that changes in glycolytic flux and intracellular pH were not regulating the oscillation. Analysis suggested that one of the oscillatory regulation points was located in the ethanol assimilation pathway.

Acetates↗
PubMed2005

Dynamics of mammalian chromosome evolution inferred from multispecies comparative maps.

The genome organizations of eight phylogenetically distinct species from five mammalian orders were compared in order to address fundamental questions relating to mammalian chromosomal evolution. Rates of chromosome evolution within mammalian orders were found to increase since the Cretaceous-Tertiary boundary. Nearly 20% of chromosome breakpoint regions were reused during mammalian evolution; these reuse sites are also enriched for centromeres. Analysis of gene content in and around evolutionary breakpoint regions revealed increased gene density relative to the genome-wide average. We found that segmental duplications populate the majority of primate-specific breakpoints and often flank inverted chromosome segments, implicating their role in chromosomal rearrangement.

Animals↗
PubMed1977

Two-carbon assimilative capacity and the induction of isocitrate lyase in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was grown on 10% glucose medium and subsequently transferred to fresh medium containing 2- and 3-carbon substrates. Under these conditions, the yeast rapidly acquired an oxidative capacity, as evidenced by oxygen uptake rates and 14CO2 evolution rates during respiration on ethanol or (14C)acetate. The assimilative capacity for 2-carbon substrates developed more slowly and followed the induction of isocitrate lyase. Washed yeast transferred to the basic medium containing no added carbon substrate possessed only low levels of isocitrate lyase after a 6-h adaptation. After 6 h, isocitrate lyase was present at high levels in cells transferred to a range of ethanol concentrations but was present in only low amounts in cells transferred to acetate. The role of ethanol as an inducer of isocitrate lyase is discussed.

Acetates↗
PubMed2006

High-cell-density fermentation for ergosterol production by Saccharomyces cerevisiae.

The direct feedback control of glucose using an on-line ethanol concentration monitor for ergosterol production by high-cell-density fermentation was investigated and the fermentation parameters (e.g., pH, dissolved oxygen, ethanol concentration, oxygen uptake rate, carbon dioxide evolution rate and respiratory quotient) were analyzed. Controlling glucose feeding rate in accordance with ethanol concentration and adjusting pH with ammonia during the fermentation process were effective fed-batch methods for ergosterol production. The fermentation parameters well described the variation of the whole fermentation process. Cultivation in a 5 l fermentor was carried out under the following conditions: culture temperature, 30 degrees C; pH, 5.5; agitation speed, 600 rpm; fermentation time, 60 h; controlling ethanol concentration below 1% and keeping respiratory quotient (RQ) at approximately 1.0. Under these conditions, the yeast dry weight reached 120 g/l and the ergosterol yield reached 1500 mg/l.

Bioreactors↗
PubMedDate not supplied

Real-time determination of microbial activity of pasteurized fluid milk using a novel microrespirometer method.

The effectiveness of the rapid CO2 evolution rate (CER) method was evaluated by using a novel noninstrumental microrespirometer to determine the microbial activity of pasteurized milk and comparing it with traditional culturing methods in homogeneous milk samples. Three different kinds of milk (skim, 1% fat, and whole) stored at 2 temperatures (4 degrees and 7 degrees C) were measured daily for CER, aerobic plate count (APC), and psychrotrophic bacterial count. The mean initial rates of CO2 evolution for all 3 samples stored at the 2 temperatures ranged from 3.42 to 3.71 microL/h/mL and increased to 29 microL/h/mL and above on the final day of the experiment. Regression analysis showed a high correlation (R = 0.98-0.99) between the APC and CER results in combined milk samples. A cut-off value of CER (25 microL/h/mL) for milk spoilage at refrigeration temperatures was identified. The real-time CER method shows promise as a potential alternative to the traditional culture method.

Animals↗
PubMed1987

Species variation in the coronary collateral circulation during regional myocardial ischaemia: a critical determinant of the rate of evolution and extent of myocardial infarction.

To determine residual flow to ischaemic tissue, which is the primary determinant of the rate of development and ultimate size of the myocardial infarct resulting from coronary artery occlusion, the coronary collateral circulation was quantified during acute myocardial ischaemia in eight species in vivo using the radiolabelled microsphere technique. In each case, a prominent branch of the left coronary artery was ligated, and within 5 min microspheres (141Ce labelled, 15 micron diameter) were injected intra-atrially. Hearts were then excised, frozen, and sliced perpendicular to the septum. Using autoradiograms as a guide, tissue samples were obtained from non-ischaemic and ischaemic tissue and the radioactivity of the ischaemic samples measured and expressed as a percentage of the activity in the non-ischaemic myocardium. In the guinea pig heart, despite ligation of a major artery, no zone of significant underperfusion was detected. In the hearts from other species, coronary collateral flow (as a percentage (mean(SEM)) of non-ischaemic flow) was: dog 15.9(1.8) (n = 6); cat 11.8(1.1) (n = 16); rat 6.1(0.7) (n = 6); ferret 2.4(0.6) (n = 6); baboon 2.1(0.3) (n = 6); rabbit 2.0(0.5) (n = 9); pig 0.6(0.2) (n = 6). The dog and cat hearts both possessed transmural gradients of collateral flow with greatest delivery in the epicardium. The patterns of flow distribution in the guinea pig heart were further examined in a Langendorff perfused preparation. Blue dye was injected into the coronary circulation and its distribution over 5 s recorded on cine film. After ligation of the left anterior descending or circumflex arteries, or both, the perfusion field of these arteries was seen to fill retrogradely within seconds. It is concluded that a wide spectrum of collateral flow exists between various mammalian species, a fact that should be taken into account in the study of the pathophysiology and control of regional ischaemia and myocardial infarction.

Animals↗
PubMed2005

Mathematical model for carbon dioxide evolution from the thermophilic composting of synthetic food wastes made of dog food.

The impacts of the aeration and the agitation on the composting process of synthetic food wastes made of dog food were studied in a laboratory-scale reactor. Two major peaks of CO(2) evolution rate were observed. Each peak represented an independent stage of composting associated with the activities of thermophilic bacteria. CO(2) evolutions known to correlate well with microbial activities and reactor temperatures were fitted successfully to a modified Gompertz equation, which incorporated three biokinetic parameters, namely, CO(2) evolution potential, specific CO(2) evolution rate, and lag phase time. No parameters that describe the impact of operating variables are involved. The model is only valid for the specified experimental conditions and may look different with others. The effects of operating parameters such as aeration and agitation were studied statistically with multivariate regression technique. Contour plots were constructed using regression equations for the examination of the dependence of CO(2) evolution potentials on aeration and agitation. In the first stage, a maximum CO(2) evolution potential was found when the aeration rate and the agitation parameter were set at 1.75 l/kg solids-min and 0.35, respectively. In the second stage, a maximum existed when the aeration rate and the agitation parameter were set at 1.8 l/kg solids-min and 0.5, respectively. The methods presented here can also be applied for the optimization of large-scale composting facilities that are operated differently and take longer time.

Animal Feed↗
PubMed1976

Evolution of Hawaiian drosophilidae. II. Patterns and rates of chromosome evolution in an antopocerus phylogeny.

The phylogenetic relationships of seven species of the genus Antopocerus (Family Drosophilidae) have been determined by means of a study of the metaphase configurations and polytene chromosomes. Based on biogeographical, behavioral and cytogenetic information A. longiseta from Molokai is tentatively identified as the primitive species of the genus. The metaphase karyotypes of all Antopocerus species are either five pairs of rod chromosomes and a pair of dots (5R1D), or six rods (6R). Heterochromatin additions converted the dots to rods. Chromosome breakpoints for inversions also are clustered at heterochromatic loci. The chromosome segments between heterochromatic loci may represent sets of functionally related loci, evolving as a unit. The rate of chromosomal inversion substitution is estimated in the origin of the taxon (probably a subgenus of Drosophila rather than a separate genus). It averages no greater than one substitution per 1,000 years, or one per 5,000 generations. The average genetic death rate per generation of one individual per hundred is required to achieve this substitution rate. The rate of inversion substitution during radiation of this taxon may be only 4.4 X 10(-3) times as fast as that present in forming the taxon. Alternatively, radiation may have required only 250,000 years if rates of substitution are the same as in the origination of the taxon. Average rates of substitution reflect genetic accidents, selection pressures and rates of adaptation to new niches, as well as the rate of encountering new niches. Rate of adaptation probably is much greater in this instance than rate of encountering new niches. Rate of adaptation probably is much greater in this instance than rate of encountering new niches. Therefore, the average rate of evolution reflects more nearly biogeographic and ecological factors than genetic factors.

Animals↗
PubMed2005

Protein interactions limit the rate of evolution of photosynthetic genes in cyanobacteria.

Using a bioinformatic approach, we analyzed the correspondence in genetic distance matrices between all possible pairwise combinations of 82 photosynthetic genes in 10 species of cyanobacteria. Our analysis reveals significant correlations between proteins linked in a conserved gene order and between structurally identified interacting protein scaffolds that coordinate the binding of cofactors involved in photosynthetic electron transport. Analyses of amino acid substitution rates suggest that the tempo of evolution of genes encoding core metabolic processes in the photosynthetic apparatus is highly constrained by protein-protein, protein-lipid, and protein-cofactor interactions (collectively called "protein interactions"). These interactions are critical for energy transduction, primary charge separation, and electron transport and effectively act as an internal selection pressure governing the conservation of clusters of photosynthetic genes in oxygenic prokaryotic photoautotrophs. Consequently, although several proteins within the photosynthetic apparatus are biophysically and physiologically inefficient, selection has not significantly altered the genes encoding these essential proteins over billions of years of evolution. In effect, these core proteins have become "frozen metabolic accidents."

Computational Biology↗
PubMed2003

Mutation rate and evolution of fluoroquinolone resistance in Escherichia coli isolates from patients with urinary tract infections.

Escherichia coli strains from patients with uncomplicated urinary tract infections were examined by DNA sequencing for fluoroquinolone resistance-associated mutations in six genes: gyrA, gyrB, parC, parE, marOR, and acrR. The 54 strains analyzed had a susceptibility range distributed across 15 dilutions of the fluoroquinolone MICs. There was a correlation between the fluoroquinolone MIC and the number of resistance mutations that a strain carried, with resistant strains having mutations in two to five of these genes. Most resistant strains carried two mutations in gyrA and one mutation in parC. In addition, many resistant strains had mutations in parE, marOR, and/or acrR. No (resistance) mutation was found in gyrB. Thus, the evolution of fluoroquinolone resistance involves the accumulation of multiple mutations in several genes. The spontaneous mutation rate in these clinical strains varied by 2 orders of magnitude. A high mutation rate correlated strongly with a clinical resistance phenotype. This correlation suggests that an increased general mutation rate may play a significant role in the development of high-level resistance to fluoroquinolones by increasing the rate of accumulation of rare new mutations.

Anti-Bacterial Agents↗