Biomedical subjects

A G Miller

Publications and source records attributed to A G Miller.

At least 37 records · Page 2Linked to original sources

PubMed1988

Chlorophyll a Fluorescence Yield as a Monitor of Both Active CO(2) and HCO(3) Transport by the Cyanobacterium Synechococcus UTEX 625.

Simultaneous measurements have been made of inorganic carbon accumulation (by mass spectrometry) and chlorophyll a fluorescence yield of the cyanobacterium Synechococcus UTEX 625. The accumulation of inorganic carbon by the cells was accompanied by a substantial quenching of chlorophyll a fluorescence. The quenching occurred even when CO(2) fixation was inhibited by iodoacetamide and whether the accumulation of inorganic carbon resulted from either active CO(2) or HCO(3) (-) transport. Measurement of chlorophyll a fluorescence yield of cyanobacteria may prove to be a rapid and convenient means of screening for mutants of inorganic carbon accumulation.

Journal Article↗

PubMed1988

Active Transport of CO(2) by the Cyanobacterium Synechococcus UTEX 625 : Measurement by Mass Spectrometry.

Mass spectrometry has been used to confirm the presence of an active transport system for CO(2) in Synechococcus UTEX 625. Cells were incubated at pH 8.0 in 100 micromolar KHCO(3) in the absence of Na(+) (to prevent HCO(3) (-) transport). Upon illumination the cells rapidly removed almost all the free CO(2) from the medium. Addition of carbonic anhydrase revealed that the CO(2) depletion resulted from a selective uptake of CO(2), rather than a total uptake of all inorganic carbon species. CO(2) transport stopped rapidly (<3 seconds) when the light was turned off. Iodoacetamide (3.3 millimolar) completely inhibited CO(2) fixation but had little effect on CO(2) transport. In iodoacetamide poisoned cells, transport of CO(2) occurred against a concentration gradient of about 18,000 to 1. Transport of CO(2) was completely inhibited by 10 micromolar diethylstilbestrol, a membrane-bound ATPase inhibitor. Studies with DCMU and PSI light indicated that CO(2) transport was driven by ATP produced by cyclic or pseudocyclic photophosphorylation. Low concentrations of Na(+) (<100 microequivalents per liter), but not of K(+), stimulated CO(2) transport as much as 2.4-fold. Unlike Na(+)-dependent HCO(3) (-) transport, the transport of CO(2) was not inhibited by high concentrations (30 milliequivalents per liter) of Li(+). During illumination, the CO(2) concentration in the medium remained far below its equilibrium value for periods up to 15 minutes. This could only happen if CO(2) transport was continuously occurring at a rapid rate, since the continuing dehydration of HCO(3) (-) to CO(2) would rapidly raise the CO(2) concentration to its equilibrium value if transport ceased. Measurement of the rate of dissolved inorganic carbon accumulation under these conditions indicated that at least part of the continuing CO(2) transport was balanced by HCO(3) (-) efflux.

Journal Article↗

PubMed1988

Simultaneous Transport of CO(2) and HCO(3) by the Cyanobacterium Synechococcus UTEX 625.

A mass spectrometer was used to simultaneously follow the time course of photosynthetic O(2) evolution and CO(2) depletion of the medium by cells of the cyanobacterium Synechococcus leopoliensis UTEX 625. Analysis of the data indicated that both CO(2) and HCO(3) (-) were simultaneously and continuously transported by the cells as a source of substrate for photosynthesis. Initiation of HCO(3) (-) transport by Na(+) addition had no effect on ongoing CO(2) transport. This result is interpreted to indicate that the CO(2) and HCO(3) (-) transport systems are separate and distinctly different transport systems. Measurement of CO(2)-dependent photosynthesis indicated that CO(2) uptake involved active transport and that diffusion played only a minor role in CO(2) acquisition in cyanobacteria.

Journal Article↗

PubMed1988

Active Transport of Inorganic Carbon Increases the Rate of O(2) Photoreduction by the Cyanobacterium Synechococcus UTEX 625.

Chlorophyll a fluorescence of Synechococcus UTEX 625 was quenched during the transport of inorganic carbon, even when CO(2) fixation was inhibited by iodoacetamide. Measurements with a pulse modulation fluorometer showed that at least 75% of the quenching was due to oxidation of Q(a), the primary acceptor of photosystem II. Mass spectrometry revealed that transport of inorganic carbon increased the rate of O(2) photoreduction. Hence, O(2) could serve as an electron acceptor to allow oxidation of Q(a) even in the absence of CO(2) fixation.

Journal Article↗

PubMed1988

Characterization of the na-requirement in cyanobacterial photosynthesis.

The Na(+) requirement for photosynthesis and its relationship to dissolved inorganic carbon (DIC) concentration and Li(+) concentration was examined in air-grown cells of the cyanobacterium Synechococcus leopoliensis UTEX 625 at pH 8. Analysis of the rate of photosynthesis (O(2) evolution) as a function of Na(+) concentration, at fixed DIC concentration, revealed two distinct regions to the response curve, for which half-saturation values for Na(+) (K((1/2))[Na(+)]) were calculated. The value of both the low and the high K((1/2))(Na(+)) was dependent upon extracellular DIC concentration. The low K((1/2))(Na(+)) decreased from 1000 micromolar at 5 micromolar DIC to 200 micromolar at 140 micromolar DIC whereas over the same DIC concentration range the high K((1/2))(Na(+)) decreased from 10 millimolar to 1 millimolar. The most significant increases in photosynthesis occurred in the 1 to 20 millimolar range. A fraction of total photosynthesis, however, was independent of added Na(+) and this fraction increased with increased DIC concentration. A number of factors were identified as contributing to the complexity of interaction between Na(+) and DIC concentration in the photosynthesis of Synechococcus. First, as revealed by transport studies and mass spectrometry, both CO(2) and HCO(3) (-) transport contributed to the intracellular supply of DIC and hence to photosynthesis. Second, both the CO(2) and HCO(3) (-) transport systems required Na(+), directly or indirectly, for full activity. However, micromolar levels of Na(+) were required for CO(2) transport while millimolar levels were required for HCO(3) (-) transport. These levels corresponded to those found for the low and high K((1/2))(Na(+)) for photosynthesis. Third, the contribution of each transport system to intracellular DIC was dependent on extracellular DIC concentration, where the contribution from CO(2) transport increased with increased DIC concentration relative to HCO(3) (-) transport. This change was reflected in a decrease in the Na(+) concentration required for maximum photosynthesis, in accord with the lower Na(+)-requirement for CO(2) transport. Lithium competitively inhibited Na(+)-stimulated photosynthesis by blocking the cells' ability to form an intracellular DIC pool through Na(+)-dependent HCO(3) (-) transport. Lithium had little effect on CO(2) transport and only a small effect on the size of the pool it generated. Thus, CO(2) transport did not require a functional HCO(3) (-) transport system for full activity. Based on these observations and the differential requirement for Na(+) in the CO(2) and HCO(3) (-) transport system, it was proposed that CO(2) and HCO(3) (-) were transported across the membrane by different transport systems.

Journal Article↗

PubMed1987

Na-Stimulation of Photosynthesis in the Cyanobacterium Synechococcus UTEX 625 Grown on High Levels of Inorganic Carbon.

Photosynthesis of washed cells of Synechococcus UTEX 625 grown on 5% CO(2) was markedly stimulated (647 +/- 50%) at pH 8.0 by the addition of low concentrations of NaCl (concentration required for half-maximal response, K((1/2),) = 18 micromolar). Studies with KCl and Na(2)SO(4) showed that the stimulation was due to Na(+). Photosynthesis at pH 6.1 was only slightly stimulated by Na(+). The response of photosynthesis at pH 8.0 to [Na(+)] was strongly sigmoidal for dissolved inorganic carbon ([DIC] </= 500 micromolar). Cells grown with high total [DIC], but air-levels of CO(2), at pH 9.6 showed the same response to low [Na(+)]. The absence of Na(+) could be partially, but not completely overcome, by higher [DIC]. Various methods for examining CO(2) or HCO(3) (-) use (K((1/2)) (CO(2) ) determination; isotopic disequilibrium; and consideration of HCO(3) (-) dehydration rate) were consistent with CO(2) use by the cells, but HCO(3) (-) use could not be ruled out. Isotopic disequilibrium studies showed that CO(2) use was stimulated by Na(+). Cells grown on 5% CO(2) accumulated DIC against a concentration gradient by a process (or processes) dependent on Na(+). No evidence for uptake of Na(+) concomitant with DIC uptake could be found. The lack of O(2) evolution during the initial and most rapid period of DIC accumulation suggested that the required energy was obtained from cyclic photophosphorylation.

Journal Article↗

PubMed1986

Effects of dietary alteration of bicarbonate and magnesium on rat bone.

To study the effects of bicarbonate and magnesium on bone, mild acidosis and/or hypermagnesemia were produced in growing rats by feeding ammonium chloride and/or magnesium sulfate. Bone composition, quantitative histomorphometry, and mineral x-ray diffraction (XRD) characteristics were measured after 6 wk of treatment. The results demonstrated that both acidosis (decreased HCO3) and hypermagnesemia inhibited periosteal bone formation, and, when combined, results were summative; and the previously observed in vitro role of HCO3- and Mg2+ as inhibitors of crystal growth were confirmed in vivo. XRD measurements demonstrated that decreased plasma HCO3 resulted in larger crystals and increased Mg resulted in smaller crystals. However, the combined XRD effects of acidosis and hypermagnesemia resembled acidosis alone. It is postulated that the final composition and crystal structure of bone are strongly influenced by HCO3- and Mg2+, and the effects are mediated by the combined influence on both osteoblastic bone formation and the growth of hydroxyapatite.

Animals↗

PubMed1984

Biochemical and genetic analysis of variant mouse hepatoma cells which overtranscribe the cytochrome P1-450 gene in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Using the fluorescence-activated cell sorter, we have isolated a population of variant mouse hepatoma cells which have a markedly increased ability to metabolize benzo(a)pyrene. Compared with wild-type (Hepa 1c1c7) cells, the variant cells exhibit increased aryl hydrocarbon hydroxylase activity and increased responsiveness of the aryl hydrocarbon hydroxylase induction mechanism to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cell fusion experiments indicate that the variant phenotype is co-dominant with respect to wild-type. Filter hybridization analyses indicate that increased accumulation of cytochrome P1-450-specific mRNA accounts for the overproduction of aryl hydrocarbon hydroxylase activity. Measurements of RNA synthesis in isolated nuclei reveal that the variants exhibit an increased rate of transcription of the cytochrome P1-450 gene in response to TCDD. The variant cells contain no detectable alteration in their TCDD receptors, nor is the cytochrome P1-450 gene amplified in the variants. Filter hybridization analyses of restriction endonuclease-digested DNA indicate that the variant cytochrome P1-450 gene is relatively undermethylated, compared with the wild-type gene. We conclude that the variant cells contain an altered cis-acting genomic element(s) which regulates the expression of the cytochrome P1-450 gene.

Animals↗

PubMed1984

Growth and Photosynthesis of the Cyanobacterium Synechococcus leopoliensis in HCO(3)-Limited Chemostats.

Synechococcus leopoliensis was grown in HCO(3) (-)-limited chemostats. Growth at 50% the maximum rate occurred when the inorganic carbon concentration was 10 to 15 micromolar (or 5.6 to 8.4 nanomolar CO(2)). The O(2) to CO(2) ratios during growth were as high as 192,000 to 1. At growth rates below 80% the maximum rate, essentially all the supplied inorganic carbon was converted to organic carbon, and the cells were carbon limited. Carbon-limited cells used HCO(3) (-) rather than CO(2) for growth. They also exhibited a very high photosynthetic affinity for inorganic carbon in short-term experiments. Cells growing at greater than 80% maximum growth rate, in the presence of high dissolved inorganic carbon, were termed carbon sufficient. These cells had photosynthetic affinities that were about 1000-fold lower than HCO(3) (-)-limited cells and also had a reduced capacity for HCO(3) (-) transport. HCO(3) (-)-limited cells are reminiscent of the air-grown cells of batch culture studies while the carbon sufficient cells are reminiscent of high-CO(2) grown cells. However, the low affinity cells of the present study were growing at CO(2) concentrations less than air saturation. This suggests that supranormal levels of CO(2) not required to induce the physiological changes usually ascribed to high CO(2) cells.

Journal Article↗

PubMed1984

Na+ requirement for growth, photosynthesis, and pH regulation in the alkalotolerant cyanobacterium Synechococcus leopoliensis.

We have found that Na+ is required for the alkalotolerance of the cyanobacterium Synechococcus leopoliensis. Cell division did not occur at any pH in the absence of Na+, but cells inoculated into Na+-free growth medium at pH 6.8 did continue metabolic activity, and over a period of 48 h, the cells became twice their normal size. Many of these cells remained viable for at least 59 h and formed colonies on Na+ -containing medium. Cells grown in the presence of Na+ and inoculated into Na+ -free growth medium at pH 9.6 rapidly lost viability. An Na+ concentration of ca. 0.5 milliequivalents X liter-1 was required for sustained growth above pH 9.0. The Na+ requirement could be only partially met by Li+ and not at all by K+ or Rb+. Cells incubated in darkness in growth medium at pH 6.8 had an intracellular pH near neutrality in the presence or absence of Na+. When the external pH was shifted to 9.6, only cells in the presence of Na+ were able to maintain an intracellular pH near 7.0. The membrane potential, however, remained high (-120 mV) in the absence or presence of Na+ unless collapsed by the addition of gramicidin. Thus, the inability to maintain a neutral intracellular pH at pH 9.6 in the absence of Na+ was not due to a generalized disruption of membrane integrity. Even cells containing Na+ still required added Na+ to restore photosynthetic rates to normal after the cells had been washed in Na+ -free buffer at pH 9.6. This requirement was only partially met by Li+ and was not met at all by K+, Rb+, Cs+ Mg2+, or Ca2+. The restoration of photosynthesis by added Na+ occurred within 30 s and suggests a role for extracellular Na+. Part of our results can be explained in terms of the operation of an Na+/H+ antiporter activity in the plasma membrane, but some results would seem to require other mechanisms for Na+ action.

Chlorophyll↗

PubMed1983

Biochemical and genetic analysis of variant mouse hepatoma cells defective in the induction of benzo(a)pyrene-metabolizing enzyme activity.

We have analyzed wild type mouse hepatoma (Hepa 1c1c7) cells and variant cells which are defective in the induction of benzo(a)pyrene-metabolizing enzyme activity. One type of variant has no detectable basal or inducible aryl hydrocarbon hydroxylase activity. This class contains apparently normal cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin, but is unable to translocate the inducer-receptor complex to the nucleus. The second type of variant has low levels of basal and inducible aryl hydrocarbon hydroxylase activity. This class contains cytosolic receptors which are decreased either in their number or in their ability to bind 2,3,7,8-tetrachlorodibenzo-p-dioxin; translocation of the inducer-receptor complex to the nucleus is apparently normal. Cell fusions indicate that both variant phenotypes are recessive with respect to wild type. Complementation analyses indicate that the defects are located on different genes.

Animals↗

PubMed1983

Ethylated fluoresceins: assay of cytochrome P-450 activity and application to measurements in single cells by flow cytometry.

Four ethylated derivatives of fluorescein were synthesized and their metabolism by mouse liver homogenates and cultured mouse hepatoma cells was investigated. Two of the ethylfluoresceins are specifically metabolized by the polycyclic aromatic hydrocarbon inducible cytochrome P-450 to yield fluorescein, which is about 15 times more fluorescent than the ethylated compounds. One of these fluorogenic derivatives, ethoxyfluorescein ethyl ester, is useful for flow cytometric analysis and sorting of intact, viable cells on the basis of cytochrome P-450 activity. The structure-activity relationship of the ethylfluoresceins is discussed.

Animals↗

PubMed1982

Heterogeneity in the rate of benzo[a]pyrene metabolism in single cells: quantitation using flow cytometry.

We describe a method for quantitating heterogeneity in the rate of benzo[a]pyrene metabolism in single cells by using flow cytometry. We have used the technique to study the response of Hepa-1c1c7 mouse hepatoma cells to the microsomal enzyme inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cells responded in a relatively homogeneous fashion at different times of induction with a maximally inducing concentration of the inducer. However, the induction response could be heterogeneous at a submaximal inducer concentration. We found even higher heterogeneity of enzyme activity among low-activity variants derived from the Hepa-1c1c7 cell line. When cells of either high or low activity were isolated from such a clonal population, propagated, and reanalyzed, they displayed average enzyme activity and heterogeneity identical to the parental cells; therefore, the heterogeneity represents transient, nonheritable differences between cells within the population.

Animals↗

PubMed1982

Efficient metabolism of benzo(a)pyrene at nanomolar concentrations by intact murine hepatoma cells.

We have studied the metabolism of benzo(a)pyrene (BP) by intact mouse hepatoma cells, at nM concentrations of the carcinogen, using an assay in which we directly measure the rate of BP fluorescence disappearance. The rate of BP metabolism is half-maximal, at limiting cell dilution, when the concentration of BP is about 4 nM. This apparent Km for BP metabolism is much lower than those reported previously for several reasons. (a) Partitioning of BP into cells markedly influences kinetic measurements, and we account for these effects. (b) Enzyme inducers can competitively inhibit BP metabolism and thus may introduce artifacts into kinetic measurements. (c) Under the conditions of this assay, phenolic BP metabolites are produced but do not accumulate, due to their further metabolism; therefore, assays of BP metabolism which measure the production of phenols, such as the commonly used aryl hydrocarbon hydroxylase assay, may markedly underestimate the rate of BP metabolism when intact cells and low substrate concentrations are used. Our results show that cells can efficiently metabolize BP when exposed to BP concentrations similar to those present in the environment.

Animals↗

PubMed1982

Syncope.

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Dental Care↗

PubMed1981

Novel variants in benzo(a)pyrene metabolism. Isolation by fluorescence-activated cell sorting.

We have used the fluorescence-activated cell sorter to measure the metabolism of the carcinogenic polycyclic aromatic hydrocarbon, benzo(a)pyrene (BP), in individual, viable mouse hepatoma (Hepa 1c1c7) cells and to physically select variants with either markedly elevated or markedly decreased ability to metabolize to low, nontoxic concentrations of BP (25 nM) and measuring the rate of disappearance of BP fluorescence. We show by several criteria that the disappearance of BP fluorescence is due to the metabolism of the carcinogen. Cells sorted according to their rae of loss of BP fluorescence exhibit the predicted phenotypes in BP-metabolizing activity, as measured by the aryl hydrocarbon hydroxylase assay.

Animals↗

PubMed1981

Fixation of porous titanium implants in cortical bone enhanced by electrical stimulation.

The effect of in vivo electrical stimulation on the interfacial strength between porous titanium implants and cortical bone was studied in mongrel dogs. The interfacial shear strength of the stimulated implants was consistently greater than that of controls. No difference could be observed in the quality of bone ingrowth. The data suggest that the rate and quantity of bone ingrowth were enhanced by electrical stimulation.

Animals↗

PubMed1980

Normal maturational changes in bone matrix, mineral, and crystal size in the rat.

Normal rat bone maturation has been studied using biochemical methods and hydrazine separation of matrix and mineral for X-ray diffraction. In the bone, the amount of mineral increases between 4 and 22 weeks of age, while in the matrix, the ratio of noncollagenous protein to collagen progressively decreases. In mineral, in the absence of serum ion changes, growth in mean crystal size appears to be the determinant of the changing ratios of calcium, magnesium, carbonate, and phosphorus, and of the increasing mineral density.

Aging↗