Detection of metabolic fluxes of o and h atoms into intracellular water in Mammalian cells

Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue.     

Synthesis and secretion of transferrin by cultured mouse hepatoma cells.

The mouse hepatoma cell (Hepa-1) in tissue culture has been shown to synthesize and secrete three electrophoretically distinct transferrins. Each of these forms of transferrin has a molecular weight of 77,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentration of each form is indicated by its staining intensity, which is highest in the form with the fastest mobility and lowest in the form with the slowest mobility. The relative rate of transferrin synthesis has been determined in log-phase and stationary-phase cells; the data indicate that the relative rate of synthesis increases twofold in stationary-phase cells. When the incorporation of [3H]leucine into transferrin reaches steady state, the rate of secretion is equal to the rate of synthesis; the rate of secretion also increases twofold in stationary-phase cells. Our studies also show that transferrin synthesis accounts for 0.98% of the total protein synthesis in log-phase cells and for 1.8% in stationary-phase cells. This is the level of synthesis that has been determined by in vivo studies. We conclude that after continuous culture for several years these hepatoma cells have maintained one of the characteristics of the differentiated liver cell, namely, the ability to synthesize and secrete transferrin.     

A low-pH-inducible, stationary-phase acid tolerance response in Salmonella typhimurium.

Acid is an important environmental condition encountered by Salmonella typhimurium during its pathogenesis. Our studies have shown that the organism can actively adapt to survive potentially lethal acid exposures by way of at least three possibly overlapping systems. The first is a two-stage system induced in response to low pH by logarithmic-phase cells called the log-phase acid tolerance response (ATR). It involves a major molecular realignment of the cell including the induction of over 40 proteins. The present data reveal that two additional systems of acid resistance occur in stationary-phase cells. One is a pH-dependent system distinct from log-phase ATR called stationary-phase ATR. It was shown to provide a higher level of acid resistance than log-phase ATR but involved the synthesis of fewer proteins. Maximum induction of stationary-phase ATR occurred at pH 4.3. A third system of acid resistance is not induced by low pH but appears to be part of a general stress resistance induced by stationary phase. This last system requires the alternative sigma factor, RpoS. Regulation of log-phase ATR and stationary-phase ATR remains RpoS independent. Although the three systems are for the most part distinct from each other, together they afford maximum acid resistance for S. typhimurium.     

Escherichia coli produces linoleic acid during late stationary phase.

Escherichia coli produces linoleic acid in the late stationary phase. This was the case whether the cultures were grown aerobically or anaerobically on a supplemented glucose-salts medium. The linoleic acid was detected by thin-layer chromatography and was measured as the methyl ester by gas chromatography. The linoleic acid methyl ester was identified by its mass spectrum. Lipids extracted from late-stationary-phase cells generated thiobarbituric acid-reactive carbonyl products when incubated with a free radical initiator. In contrast, extracts from log-phase or early-stationary-phase cells failed to do so, in accordance with the presence of polyunsaturated fatty acid only in the stationary-phase cells.     

OmpR regulates the stationary-phase acid tolerance response of Salmonella enterica serovar typhimurium

Tolerance to acidic environments is an important property of free-living and pathogenic enteric bacteria. Salmonella enterica serovar Typhimurium possesses two general forms of inducible acid tolerance. One is evident in exponentially growing cells exposed to a sudden acid shock. The other is induced when stationary-phase cells are subjected to a similar shock. These log-phase and stationary-phase acid tolerance responses (ATRs) are distinct in that genes identified as participating in log-phase ATR have little to no effect on the stationary-phase ATR (I. S. Lee, J. L. Slouczewski, and J. W. Foster, J. Bacteriol. 176:1422-1426, 1994). An insertion mutagenesis strategy designed to reveal genes associated with acid-inducible stationary-phase acid tolerance (stationary-phase ATR) yielded two insertions in the response regulator gene ompR. The ompR mutants were defective in stationary-phase ATR but not log-phase ATR. EnvZ, the known cognate sensor kinase, and the porin genes known to be controlled by OmpR, ompC and ompF, were not required for stationary-phase ATR. However, the alternate phosphodonor acetyl phosphate appears to play a crucial role in OmpR-mediated stationary-phase ATR and in the OmpR-dependent acid induction of ompC. This conclusion was based on finding that a mutant form of OmpR, which is active even though it cannot be phosphorylated, was able to suppress the acid-sensitive phenotype of an ack pta mutant lacking acetyl phosphate. The data also revealed that acid shock increases the level of ompR message and protein in stationary-phase cells. Thus, it appears that acid shock induces the production of OmpR, which in its phosphorylated state can trigger expression of genes needed for acid-induced stationary-phase acid tolerance.     

Synthesis and Secretion of Transferrin by Cultured Mouse Hepatoma Cells

The mouse hepatoma cell (Hepa-1) in tissue culture has been shown to synthesize and secrete three electrophoretically distinct transferrins. Each of these forms of transferrin has a molecular weight of 77,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentration of each form is indicated by its staining intensity, which is highest in the form with the fastest mobility and lowest in the form with the slowest mobility. The relative rate of transferrin synthesis has been determined in log-phase and stationary-phase cells; the data indicate that the relative rate of synthesis increases twofold in stationary-phase cells. When the incorporation of [³H]leucine into transferrin reaches steady state, the rate of secretion is equal to the rate of synthesis; the rate of secretion also increases twofold in stationary-phase cells. Our studies also show that transferrin synthesis accounts for 0.98% of the total protein synthesis in log-phase cells and for 1.8% in stationary-phase cells. This is the level of synthesis that has been determined by in vivo studies. We conclude that after continuous culture for several years these hepatoma cells have maintained one of the characteristics of the differentiated liver cell, namely, the ability to synthesize and secrete transferrin.     

Production and Repair of Radiochemical Damage in Escherichia coli Deoxyribonucleic Acid; Its Modification by Culture Conditions and Relation to Survival

Late log-phase Escherichia coli B/r cells are 1.6 times more sensitive to killing by X rays than are stationary-phase cells when grown in Brain Heart Infusion (BHI) + glucose. The number of single-chain breaks formed per krad is the same for log- and stationary-phase cells. Stationary-phase cells show a somewhat greater ability to repair single-chain breaks (especially after high doses of X rays) than do log-phase cells. The rapidity and extent of postirradiation deoxyribonucleic acid (DNA) degradation are greater in log-phase cells than in stationary-phase cells. The enhanced viability exhibited by stationary-phase cells thus appears to correlate both with enhanced single-chain break repair and the reduced degradation of DNA. Cells grown to stationary phase in peptone medium (PO cells) are 3.4 times more sensitive to killing by X rays than cells grown to stationary phase in peptone medium supplemented with glucose and phosphate buffer (PG cells). The yield of single-strand breaks is the same for both types of cells (but the absolute yield is about two times higher than in the cells grown in BHI + glucose). The kinetics for the repair of single-chain breaks are the same for both types of cells for about 30 min. After this time period, further repair ceases in the PO cells but continues in the PG cells, provided that glucose is present in the medium. Postirradiation DNA degradation is both more rapid and more extensive in PO cells than in PG cells whether or not glucose is present in the postirradiation incubation medium. The survival of stationary-phase E. coli B/r grown in PO or PG medium is likewise unaffected by the presence of glucose in the plating medium, and thus correlates better with the lower DNA degradation seen in the PG cells than with the increased strand rejoining, since this latter process requires the presence of glucose.     

Induction of acid tolerance at neutral pH in log-phase Escherichia coli by medium filtrates from organisms grown at acidic pH

Escherichia coli grown at pH 5·0 became acid-tolerant (acid-habituated) but, in addition, neutralized medium filtrates from cultures of E. coli grown to log-phase or stationary-phase at pH 5·0 (pH 5·0 filtrates) induced acid tolerance when added to log-phase E. coli growing at pH 7·0. In contrast, filtrates from pH 7·0-grown cultures were ineffective. The pH 5·0 filtrates were inactivated by heating in a boiling water-bath but there was less activity loss at 75 °C. Protease also inactivated such filtrates, which suggested that a heat-resistant protein (or proteins) in the filtrates was essential for the induction of acid tolerance. Filtrates from cells grown at pH 5·0 plus phosphate or adenosine 3':5'-cyclic monophosphate (cAMP) were much less effective in inducing acid tolerance, while the conversion of pH 7·0-grown log-phase cells to acid tolerance by pH 5·0 filtrates was inhibited by cAMP and bicarbonate. It seems likely that the acid tolerance response (acid habituation) involved the functioning of the extracellular protein(s) as protease reduces tolerance induction if added during acid habituation. Most inducible responses are believed to involve the functioning of only intracellular reactions and components ; the present results suggest that this is not the case for acid habituation, as an extracellular protein (or proteins) is needed for induction.     

Luciferase inactivation in the luminous marine bacterium Vibrio harveyi.

Luciferase was rapidly inactivated in stationary-phase cultures of the wild type of the luminous marine bacterium Vibrio harveyi, but was stable in stationary-phase cultures of mutants of V. harveyi that are nonluminous without exogenous aldehyde, termed the aldehyde-deficient mutants. The inactivation in the wild type was halted by cell lysis and was slowed or stopped by O2 deprivation or by addition of KCN and NaF or of chloramphenicol. If KCN and NaF or chloramphenicol were added to a culture before the onset of luciferase inactivation, then luciferase inactivation did not occur. However, if these inhibitors were added after the onset of luciferase inactivation, then luciferase inactivation continued for about 2 to 3 h before the inactivation process stopped. The onset of luciferase inactivation in early stationary-phase cultures of wild-type cell coincided with a slight drop in the intracellular adenosine 5'-triphosphate (ATP) level from a relatively constant log-phase value of 20 pmol of ATP per microgram of soluble cell protein. Addition of KCN and NaF to a culture shortly after this drop in ATP caused a rapid decrease in the ATP level to about 4 pmol of ATP per microgram whereas chloramphenicol added at this same time caused a transient increase in ATP level to about 25 pmol/microgram. The aldehyde-deficient mutant (M17) showed a relatively constant log-phase ATP level identical with that of the wild-type cells, but rather than decreasing in early stationary phase, the ATP level increased to a value twice that in log-phase cells. We suggest that the inactivation of luciferase is dependent on the synthesis of some factor which is produced during stationary phase and is itself unstable, and whose synthesis is blocked by chloramphenicol or cyanide plus fluoride.     

Variations in the natural abundance of oxygen-18 and deuterium in plant carbohydrates

Variations in the natural abundance of ¹⁸O and ²H in plant cellulose are influenced by the isotopic composition of the water directly involved in metabolism—the metabolic water fraction. The isotopic distinction between the metabolic source water and total tissue water must reflect the formation of isotopic gradients within the tissue that are influenced by the rate of water turnover, by properties of the water conducting system and by environmental conditions. It seems that the ¹⁸O abundance in the metabolic water is conserved in cellulose with a relatively constant isotope effect. The relationship of the ²H abundance between metabolic water and cellulose is more complex. Hydrogen incorporated into photosynthetic products during primary reduction steps is highly depleted in ²H. However, a large proportion of these hydrogens are subsequently replaced by exchange with water, leading to ²H enrichment during heterotrophic metabolism. Deciphering the oxygen isotope ratio of cellulose could help in providing insights into the carbon and oxygen fluxes exchanged between plants and the atmosphere. This is because the ¹⁸O abundance in cellulose records the ¹⁸O abundance in the metabolic water, which in turn, controls the oxygen isotopic signatures of the CO₂ and O₂ released by plants into the atmosphere. The hydrogen isotope effects associated with carbohydrate metabolism provide insights into the autotrophic state of a plant tissue. This is because the hydrogen isotope ratio of carbohydrates must reflect the net effects of the two opposing isotope effects associated with photosynthesis and heterotrophic metabolism.     

The influence of diet and water on the stable oxygen and hydrogen isotope composition of Chironomidae (Diptera) with paleoecological implications

Stable oxygen and hydrogen isotope analyses of fossil aquatic organisms, such as the chitinous head capsules of chironomid larvae (Chironomidae: Diptera), are promising proxies for inferring paleoecological conditions. In order for analyses of stable oxygen (δ¹⁸O) and hydrogen isotope ratios (δ²H) of fossil chironomid head capsules to be used effectively in paleoecological research, it is necessary to understand the factors controlling their stable oxygen and hydrogen composition. We cultured chironomid larvae in two isotopically distinct waters under controlled, replicated laboratory conditions. Chironomid larvae were fed on identical diets, to examine the degree to which water and diet influence the δ¹⁸O and δ²H of these organisms. We used a two-end member mixing model to determine the proportional contributions of oxygen and hydrogen from water to the oxygen and hydrogen of chironomid larvae. Our experiment demonstrated that 69.0 ± 0.4% of oxygen and 30.8 ± 2.6% of hydrogen in chironomid larvae are derived from habitat water. Our results show that oxygen isotopes from chironomid remains can better constrain past habitat water isotopic changes compared to hydrogen, due to 69% of the chironomid oxygen being influenced by habitat water. Our data add to a small but growing suite of comparative data on the sources of oxygen and hydrogen in animal tissues, and provide the first such analyses from aquatic insects.     

Headspace analyses of H labeling of acetone: Enabling studies of fatty acid oxidation in vivo

We demonstrate that one can measure low levels of ²H labeling (e.g., <0.025% excess ²H) by exchanging hydrogen (deuterium) in water with acetone and subjecting samples to gas chromatography–pyrolysis–isotope ratio mass spectrometry. This analytical method circumvents the need to use typical off-line reduction methods that convert water to hydrogen gas prior to isotope ratio mass spectrometry or the need to purchase extra peripheral devices that would permit the direct analysis of water labeling. This method enables routine measurements of fatty acid oxidation in rodents; that is, one administers a ²H-labeled fatty acid(s) and then quantifies the production of ²H-labeled water.     

Human Fatty Acid Transport Protein 2a/Very Long Chain Acyl-CoA Synthetase 1 (FATP2a/Acsvl1) Has a Preference in Mediating the Channeling of Exogenous n-3 Fatty Acids into Phosphatidylinositol

The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M_r 70,000) and FATP2b (M_r 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14–C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation.     

Isotope ratios of cellulose from plants having different photosynthetic pathways

Hydrogen and carbon isotope ratios of cellulose nitrate and oxygen isotope ratios of cellulose from C₃, C₄, and Crassulacean acid metabolism (CAM) plants were determined for plants growing within a small area in Val Verde County, Texas. Plants having CAM had distinctly higher deuterium/hydrogen (D/H) ratios than plants having C₃ and C₄ metabolism. When hydrogen isotope ratios are plotted against carbon isotope ratios, each photosynthetic mode separates into a distinct cluster of points. C₄ plants had many D/H ratios similar to those of C₃ plants, so that hydrogen isotope ratios cannot be used to distinguish between these two photosynthetic modes. Portulaca mundula, which may have a modified photosynthetic mode between C₄ and CAM, had a hydrogen isotope ratio between those of the C₄ and CAM plants. When oxygen isotope ratios are plotted against carbon isotope ratios, no distinct clustering of the C₄ and CAM plants occurs. Thus, oxygen isotope ratios are not useful in distinguishing between these metabolic modes. A plot of hydrogen isotope ratios versus oxygen isotope ratios for this sample set shows considerable overlap between oxygen isotope ratios of the different photosynthetic modes without a concomitant overlap in the hydrogen isotope ratios of CAM and the other two photosynthetic modes. This observation is consistent with the hypothesis that higher D/H ratios in CAM plants relative to C₃ and C₄ plants are due to isotopic fractionations occurring during biochemical reactions.     

Isotopic Fractionation of Hydrogen in Plants

The naturally-occurring stable isotopes deuterium and hydrogen are fractionated by a number of physical and biological processes. Deuterium has a tendency to precipitate out first from a moist air mass. Thus ground water will become isotopically lighter with an increase in latitude, altitude, or distance inland. Water taken up by the plant from the soil undergoes little change until evapotranspiration results in leaf water becoming isotopically heavier. Thus hydrogen isotopes in plants can reveal something of geography (groundwater) and climate. Hydrogen isotopes undergo little fractionation by passage through the food chain, although plant parasites tend to be enriched in D as compared to their hosts, possibly due to higher rates of transpiration in the parasitic plants. The splitting of water in photosynthesis results in the lighter isotope being incorporated into organic matter. An even larger isotopic fractionation results during lipid synthesis and other processes involving the pyruvate dehydrogenase complex. Differences in metabolic pathway between species can be detected by D/H ratios. Hydrogen isotopic differences can be detected between CAM, C₄, and C₃ species. Within C₄ plants, the NADP-ME plants are isotopically distinguishable from NAD-ME and PEP-CK plants.     

Regulation of transcription of the Escherichia coli phosphoenolpyruvate carboxykinase locus: studies with pck-lacZ operon fusions

Mutants of Escherichia coli containing genetic fusions of lacZ to the pck (phosphoenolpyruvate carboxykinase) locus were isolated by using Mu d(lacZ Ampr) bacteriophage. Synthesis of beta-galactosidase in these strains is regulated by cyclic AMP and glucose (catabolite repression). Synthesis of beta-galactosidase by pck-lacZ fusions was induced in log-phase cells growing on gluconeogenic media, was repressed by glucose, and was also induced up to 100-fold at the onset of stationary phase in LB medium. This stationary-phase induction required cyclic AMP and some other unknown regulatory signal.     

Flow-microfluorometric analysis of Escherichia coli, Rhizobium meliloti, and Rhizobium japonicum at different stages of the growth cycle.

The applicability of flow-microfluorometry (FMF) to the study of bacterial samples was investigated on cultures of Rhizobium meliloti, Rhizobium japonicum, and Escherichia coli using fluorescent and light-scattering signals. This technique which analyzes individual bacterial cells in a population was used to monitor the relative change in nucleic acid content and cell size during the growth cycle of the three microorganisms which were known to have different growth rates. Early log-phase E. coli cells contained at least eightfold more nucleic acid and were significantly larger than the stationary-phase cells. Cultures of early log-phase R. meliloti cells contained three to four-fold more nucleic acid and were slightly larger than cells in the stationary phase. Rhizobium japonicum had very little change in either parameter. In general, the amount of change in both cell size and nucleic acid content upon initiation of log-phase growth was related to the overall growt rate of the organisms, with E. coli experiencing the greatest change and R. japonicum the least. Results obtained by FMF analysis, therefore, were consistent with observations reported by earlier workers. Cultures of R. meliloti also were used to demonstrate that the intensity of the fluorescent signals was sensitive to digestion by DNase and RNase and to prolonged storage and fixation. The potential use of FMF in the study of microorganisms is discussed.     

Quantitative 2H NMR at natural abundance can distinguish the pathway used for glucose fermentation by lactic acid bacteria

For any given metabolic pathway, isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom can be defined. By using quantitative deuterium NMR, the redistribution of deuterium at natural abundance in lactic acid produced by the bacterial fermentation of glucose has been determined for each non-labile hydrogen atom of glucose or water and the hydrogen atoms of lactic acid. Distinct differences are observed in the lactic acid isolated from Lactococcus lactis and Leuconostoc mesenteroides that can be interpreted in terms of the different fermentative pathways used. Specifically, the affiliations observed between the H1, H3, and H4 positions of glucose with methyl and hydroxymethylene of lactic acid can give quantitative information on whether the glycolytic or the reductive pentose-phosphate pathway was involved in glucose catabolism.     

Glucose Transport in Stationary-Phase Cultures of an Asporogenous Strain of Bacillus licheniformis

The sporulation-deficient industrial organism Bacillus licheniformis HWL10 possesses two distinct glucose transport systems in log-phase cells, a glucose phosphotransferase system (PTS) and a non-PTS mechanism. The strain continues to take up glucose at a significant though reduced rate during prolonged stationary-phase incubation, but only the PTS is active.