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1.
Oscillatory chloride efflux at the pollen tube apex has a role in growth and cell volume regulation and is targeted by inositol 3,4,5,6-tetrakisphosphate 总被引:12,自引:0,他引:12
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Oscillatory growth of pollen tubes has been correlated with oscillatory influxes of the cations Ca(2+), H(+), and K(+). Using an ion-specific vibrating probe, a new circuit was identified that involves oscillatory efflux of the anion Cl(-) at the apex and steady influx along the tube starting at 12 microm distal to the tip. This spatial coupling of influx and efflux sites predicts that a vectorial flux of Cl(-) ion traverses the apical region. The Cl(-) channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid completely inhibited tobacco pollen tube growth at 80 and 20 microM, respectively. Cl(-) channel blockers also induced increases in apical cell volume. The apical 50 micro m of untreated pollen tubes had a mean cell volume of 3905 +/- 75 microm(3). DIDS at 80 microM caused a rapid and lethal cell volume increase to 6206 +/- 171 microm(3), which is at the point of cell bursting at the apex. DIDS was further demonstrated to disrupt Cl(-) efflux from the apex, indicating that Cl(-) flux correlates with pollen tube growth and cell volume status. The signal encoded by inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P(4)] antagonized pollen tube growth, induced cell volume increases, and disrupted Cl(-) efflux. Ins(3,4,5,6)P(4) decreased the mean growth rate by 85%, increased the cell volume to 5997 +/- 148 microm(3), and disrupted normal Cl(-) efflux oscillations. These effects were specific for Ins(3,4,5,6)P(4) and were not mimicked by either Ins(1,3,4,5)P(4) or Ins(1,3,4,5,6)P(5). Growth correlation analysis demonstrated that cycles of Cl(-) efflux were coupled to and temporally in phase with cycles of growth. A role for Cl(-) flux in the dynamic cellular events during growth is assessed. Differential interference contrast microscopy and kymographic analysis of individual growth cycles revealed that vesicles can advance transiently to within 2 to 4 microm of the apex during the phase of maximally increasing Cl(-) efflux, which temporally overlaps the phase of cell elongation during the growth cycle. In summary, these investigations indicate that Cl(-) ion dynamics are an important component in the network of events that regulate pollen tube homeostasis and growth. 相似文献
2.
Cerovic Zoran G. Langrand Emmanuelle Latouche Gwendal Morales Fermin Moya Ismaël 《Photosynthesis research》1998,56(3):291-301
In the present communication we report a spectral analysis of the blue-green fluorescence related to changes in NAD(P) redox state in chloroplasts and leaves. To assess the contribution of reabsorption and the inner filter effect, we compared transmission and fluorescence at different chloroplast concentrations, and showed that reabsorption by the photosynthetic pigments (chlorophylls and carotenoids) was at the origin of the two peaks in the emission spectrum in vivo. The absence of potential green-emitting fluorophores in chloroplasts was determined by measuring variable and time-resolved fluorescence at different wavelengths. We defined the conditions which optimize the UV-excited blue-green fluorescence signal dependent on NAD(P)H, and we present an example of monitoring of NAD(P)H fluorescence in intact leaves. 相似文献
3.
Nirmal Mazumder Rodney K. Lyn Ragunath Singaravelu Andrew Ridsdale Douglas J. Moffatt Chih-Wei Hu Han-Ruei Tsai John McLauchlan Albert Stolow Fu-Jen Kao John Paul Pezacki 《PloS one》2013,8(6)
Hepatitis C virus (HCV) co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs) that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS) microscopy, that expression of domain 2 of the HCV core protein (D2) fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs) in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(P)H], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor’s abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(P)H fluorescence and an increase in the ratio of free to bound NAD(P)H. This is consistent with an approximate 10 fold increase in cellular NAD(P)H levels. Furthermore, the lifetimes of bound and free NAD(P)H were both significantly reduced – indicating viral protein-induced alterations in the cofactors’ binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(P)H fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic metabolism is tightly linked to alterations in NAD(P)H functional states. 相似文献
4.
P Viallet E Kohen D O Schachtschabel A Marty J M Salmon C Kohen H B Leising B Thorell 《Histochemistry》1978,57(3):189-201
Coenzyme fluorescence spectra of single living cells are due to free pyridine nucleotides (folded configuration), bound pyridine nucleotides (unfolded configuration) and a third component, possibly a mixture or flavins. Such spectra can be used to recognize possible differences in coenzyme composition between cell lines or changes of metabolic pathways due to chemicals acting at levels below or above cytotoxicity, by high resolution spectrofluorometry. A study of spectra recorded from cultured Ehrlich ascites (EL2), and Harding Passey melanoma cells (HPM-67 and HPM-73 line) grown under comparable conditions, shows that free NAD(P)H predominates in HPM-67 and EL2, while this coenzyme is bound in HPM-73. The free/bound ratio may be profoundly modifed by chemicals, e.g. in the HPM-73 increase of free and decrease of bound NAD(P)H occurred upon treatment with 10(-6) oligomycin. When atebrine at levels (10(-6) M) below cytotoxicity was added, there was a decrease of the free NAD(P)H spectrum possibly through energy transfer from NAD(P)H to atebrine. Consideration of long range energy transfer i.e., excitation of atebrine by fluorescence of NAD(P)H vs. short range transfer of excitation energy from free NAD(P)H to atebrine, favors the latter mechanism. A transient (reversible) increase in atebrine fluorescence is seen following intracellular microinjection of substrate (e.g. glucose-6-P) leading to an increase in free NAD(P)H. At cytotoxic levels of atebrine (e.g 2 x 10(-5) M) an irreversible increase of atebrine fluorescence is seen. The microspectrofluorometric technique appears therefore well suited to study physiological processes at the level of intracellular coenzymes, as well as possible processes of intermolecular energy transfer in the microenvironment. 相似文献
5.
P. Viallet E. Kohen D. O. Schachtschabel A. Marty J. M. Salmon C. Kohen H. B. Leising B. Thorell 《Histochemistry and cell biology》1978,57(3):189-201
Summary Coenzyme fluorescence spectra of single living cells are due to free pyridine nucleotides (folded configuration), bound pyridine nucleotides (unfolded configuration) and a third component, possibly a mixture of flavins. Such spectra can be used to recognize possible differences in coenzyme composition between cell lines or changes of metabolic pathways due to chemicals acting at levels below or above cytotoxicity, by high resolution spectrofluorometry.A study of spectra recorded from cultured Ehrlich ascites (EL2), and Harding Passey melanom a cells (HPM-67 and HPM-73 line) grown under comparable conditions, shows that free NAD(P)H predominates in HPM-67 and EL2, while this coenzyme is bound in HPM-73. The free/bound ratio may be profoundly modified by chemicals, e.g. in the HPM-73 increase of free and decrease of bound NAD(P)H occurred upon treatment with 10–6 oligomycin.When atebrine at levels (10–6 M) below cytotoxicity was added, there was a decrease of the free NAD(P)H spectrum possibly through energy transfer from NAD(P)H to atebrine. Consideration of long range energy transfer i.e., excitation of atebrine by fluorescence of NAD(P)H vs. short range transfer of excitation energy from free NAD(P)H to atebrine, favors the latter mechanism. A transient (reversible) increase in atebrine fluorescence is seen following intracellular microinjection of substrate (e.g. glucose-6-P) leading to an increase in free NAD(P)H. At cytotoxic levels of atebrine (e.g. 2×10–5 M) an irreversible increase of atebrine fluorescence is seen.The microspectrofluorometric technique appears therefore well suited to study physiological processes at the level of intracellular coenzymes, as well as possible processes of intermolecular energy transfer in the microenvironment. 相似文献
6.
A multiwavelength fluorescence probe is proposed for in situ monitoring of Eschscholtzia californica and Catharanthus roseus plant cell cultures. The potential of the probe as a tool for real-time estimation of biomass and production in secondary metabolites has been studied. The probe excitation range is 270-550 nm and the emission range is 310-590 nm, with a step of 20 nm for both excitation and emission filters. Many endogenous fluorophores such as NAD(P)H, riboflavins (riboflavin and derivatives such as FMN, FAD), tryptamine and tryptophan, and fluorescent secondary metabolites were analyzed simultaneously. NAD(P)H fluorescence signal (350/450 nm) showed to be an adequate signal for estimating cells activity. Riboflavins fluorescence signal (450/530 nm) followed C. roseus cell concentration both for the growth phase and after elicitation with jasmonic acid. Fluorescence from the alkaloids interfered with NAD(P)H signal during the production phase. For C. roseus, tryptophan, tryptamine, ajmalicine and serpentine were monitored by the probe. For E. californica, fluorescence from alkaloids overlapped with riboflavins preventing from using the probe to follow cell growth but global alkaloids production could be followed using the probe. 相似文献
7.
In this study we examined the utility of NAD(P)H fluorescence for monitoring aerobic fermentations of the threonine auxotroph Corynebacterium glutamicum ATCC 14296. Instead of attempting complicated mathematical corrections for inner-filter effects, we found that it is possible to use the information contained in the on-line NAD(P)H fluorescence signal to assess culture metabolic activities during fermentation. The first derivative of the filtered fluorescence signal, which approximates the turnover rate of the NAD(P)H pool, can be used to precisely identify the temporal points of threonine and glucose exhaustion. 相似文献
8.
Intracellular NAD(P)H oscillations exhibited by polarized neutrophils display congruent with 20 s periods, which are halved to congruent with 10 s upon stimulation with chemotactic peptides such as FNLPNTL (N-formyl-nle-leu-phe-nle-tyr-lys). By monitoring this frequency change, we have measured accurately the time interval between stimulus and metabolic frequency changes. A microscope flow chamber was designed to allow rapid delivery of FNLPNTL to adherent cells. Using fluorescein as a marker, we found delivery to be complete and stable throughout the chamber within approximately 400 ms. Peptides were injected into the chamber at concentrations ranging from 10(-6) to 10(-9) M. Injections also varied with respect to the relative phase of a cell's NAD(P)H oscillations. The time interval between injection of 10(-6) M FNLPNTL and the acquisition of congruent with 10 s period metabolic oscillations was found to be 12.2+/-3.3 s when injections occurred at the NAD(P)H oscillation peak whereas the lag time was 22.5+/-4.8 s when coinciding with a trough. At 10(-8) M FNLPNTL, lag times were found to be 26.1+/-5.2 and 30.5+/-7.3 s for injections at NAD(P)H peaks and troughs, respectively. FNLPNTL at 10(-9) M had no effect on metabolic oscillations, consistent with previous studies. Our experiments show that the kinetics of transmembrane signal processing, in contrast to a simple transmembrane chemical reaction, can depend upon both ligand dose and its temporal relationship with intracellular metabolic oscillations. 相似文献
9.
Jin-Suk Han Yoshitsugu Kosugi Hiroyasu Ishida Kazuhiko Ishikawa 《European journal of biochemistry》2002,269(3):969-976
A gene having high sequence homology (45-49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94-96 degrees C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H-dependent dihydroxyacetone phosphate reduction and NAD(+)-dependent glycerol-1-phosphate (Gro1P) oxidation. NADP(+)-dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)(+) acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi-bi mechanism. 相似文献
10.
Four key cellular metabolic fluorophores--tryptophan, pyridoxine, NAD(P)H, and riboflavin--were monitored on-line by a multiple excitation fluorometric system (MEFS) and a modified SLM 8000C scanning spectrofluorometer in three model yeast fermentation systems--bakers' yeast growing on glucose, Candida utilis growing on ethanol, and Saccharomyces cerevisiae RTY110/pRB58 growing on glucose. The measured fluorescence signals were compared with cell concentration, protein concentration, and cellular activity. The results indicate that the behavior and fluorescence intensity of various fluorophores differ in the various fermentation systems. Tryptophan fluorescence is the best signal for the monitoring of cell concentration in bakers' yeast and C. utilis fermentations. Pyridoxine fluoresce is the best signal for the monitoring of cell concentration in the S. cerevisiae RTY110/pRB58 fermentation. In bakers' yeast fermentations the pyridoxine fluorescence signal can be used to monitor cellular activity. The NAD(P)H fluorescence signal is a good indicator of cellular activity in the C. utilis fermentation. For this fermentation NAD(P)H fluorescence can be used to control ethanol feeding in a fed-batch process. 相似文献
11.
Various kinetic approaches were carried out to investigate kinetic attributes for the dual coenzyme activities of mitochondrial aldehyde dehydrogenase from rat liver. The enzyme catalyses NAD(+)- and NADP(+)-dependent oxidations of ethanal by an ordered bi-bi mechanism with NAD(P)+ as the first reactant bound and NAD(P)H as the last product released. The two coenzymes presumably interact with the kinetically identical site. NAD+ forms the dynamic binary complex with the enzyme, while the enzyme-NAD(P)H complex formation is associated with conformation change(s). A stopped-flow burst of NAD(P)H formation, followed by a slower steady-state turnover, suggests that either the deacylation or the release of NAD(P)H is rate limiting. Although NADP+ is reduced by a faster burst rate, NAD+ is slightly favored as the coenzyme by virtue of its marginally faster turnover rate. 相似文献
12.
On-line monitoring of NAD(P)H fluorescence and 2D fluorescence spectroscopy was performed with Enterobacter aerogenes, a bacterium sensitive to oxygen availability. The organism was grown in a reactor under low and high dissolved oxygen concentrations
and circulated through a bypass attached to the reactor. Under low dissolved oxygen concentration in the reactor, NAD(P)H
fluorescence in the reactor and the bypass showed a deviation, but not when the dissolved oxygen level in the reactor was
high. The pattern of growth curves was identical under low and high oxygen levels. This indicates a difference in the metabolic
activity of E. aerogenes in response to oxygen. The difference spectrum of the 2D fluorescence shows that growing E. aerogenes under high dissolved oxygen levels increases the NAD(P)H content of the cells.
Received: 2 March 1999 / Received revision: 25 May 1999 / Accepted: 28 May 1999 相似文献
13.
Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters 总被引:2,自引:0,他引:2
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Two-photon excitation fluorescence microscopy (TPEFM) permits the investigation of the topology of intercellular events within living animals. TPEFM was used to monitor the distribution of mitochondrial reduced nicotinamide adenine dinucleotide (NAD(P)H) in murine skeletal muscle in vivo. NAD(P)H fluorescence emission was monitored (~460 nm) using 710–720 nm excitation. High-resolution TPEFM images were collected up to a depth of 150 μm from the surface of the tibialis anterior muscle. The NAD(P)H fluorescence images revealed subcellular structures consistent with subsarcolemmal, perivascular, intersarcomeric, and paranuclear mitochondria. In vivo fiber typing between IIB and IIA/D fibers was possible using the distribution and content of mitochondria from the NAD(P)H fluorescence signal. The intersarcomeric mitochondria concentrated at the Z-line in the IIB fiber types resulting in a periodic pattern with a spacing of one sarcomere (2.34 ± 0.17 μm). The primary inner filter effects were nearly equivalent to water, however, the secondary inner filter effects were highly significant and dynamically affected the observed emission frequency and amplitude of the NAD(P)H fluorescence signal. These data demonstrate the feasibility, and highlight the complexity, of using NAD(P)H TPEFM in skeletal muscle to characterize the topology and metabolic function of mitochondria within the living mouse. 相似文献
14.
This work reports on the interaction of the fluorescent nicotinamide 1,N6-ethenoadenine dinucleotide (epsilonNAD+) with horse liver alcohol dehydrogenase, octopine dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase from different sources (yeast, lobster muscle, and rabbit muscle). The coenzyme fluorescence is enhanced by a factor of 10-13 in all systems investigated. It is shown that this enhancement cannot be due to changes in the polarity of the environment upon binding, and that it must be rather ascribed to structural properties of the bound coenzyme. Although dynamic factors could also be important for inducing changes in the quantum yield of epsilonNAD+ fluorescence, the close similarity of the fluorescence enhancement factor in all cases investigated indicates that the conformation of bound coenzyme is rather invariant in the different enzyme systems and overwhelmingly shifted toward an open form. Dissociation constants for epsilonNAD+-dehydrogenases complexes can be determined by monitoring the coenzyme fluorescence enhancement or the protein fluorescence quenching. In the case of yeast glyceraldehyde-3-phosphate dehydrogenase at pH 7.0 and t = 20 degrees the binding plots obtained by the two methods are coincident, and show no cooperativity. The affinity of epsilonNAD+ is generally lower than that of NAD+, although epsilonNAD+ maintains most of the binding characteristics of NAD+. For example, it forms a tight complex with horse liver alcohol dehydrogenase and pyrazole, and with octopine dehydrogenase saturated by L-arginine and pyruvate. One major difference in the binding behavior of NAD+ and epsilonNAD+ seems to be present in the muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, no difference was found for epsilon NAD+ between the affinities of the third and fourth binding sites. The results and implications of this work are compared with those obtained recently by other authors. 相似文献
15.
Summary Real-time on-line fluorescence measurements of the coenzymes NAD(P)H and F420 were evaluated as indicators of stability in a glucose-fed anaerobic methanogenic digester. A probe designed forin situ fluorimetric measurement of NAD(P)H provided an assessment of activity of the total microbial community, while the response of a fluorescence probe designed to measure coenzyme F420 correlated well with methanogenic activity. The two fluorescence-monitoring probes responded directly to fermentation imbalance during periods of substrate overloading and corresponded to traditional offline measurements, suggesting that the probes may be suitable candidates for inclusion in an on-line process control system for anaerobic digestion.Florida Agricultural Experimental Station, Journal Series no. R-00326 相似文献
16.
The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed. 相似文献
17.
Rapid microspectrofluorometry has been used to evaluate 1-pyrene-butyric acid as an oxygen probe in single living EL2 ascites tissue culture cells. Despite instrumental conditions preventing detection of the pyrene butyric acid maxima at 380 and 400 nm, the probe having penetrated the cell can be easily identified (maximum around 440 nm in unconnected spectra) from the fluorescence emission spectrum, as compared with NAD(P)H emission in controls (maximum around 460 nm). Fluorescence changes during gradually increasing anaerobiosis under nitrogen flow, are compatible with a linear relationship between the reciprocal of the fluorescence intensity and the intracellular oxygen concentration (increase in 430, 434, 442/461 nm ratios at anaerobiosis). The cells having absorbed the probe continue to catabolize glycolytic substrate, but some inhibition is noticeable (e.g. from the amplitude of the NAD(P)H fluorescence increase spectrum due to intracellular addition of glucose-6-P). In principle rapid microspectrofluorometry allows a multiprobe (e.g. 1-pyrene-butyric acid for oxygen, vs NAD(P)H for metabolism) exploration of the living cell. 相似文献
18.
McNally JS Davis ME Giddens DP Saha A Hwang J Dikalov S Jo H Harrison DG 《American journal of physiology. Heart and circulatory physiology》2003,285(6):H2290-H2297
Oscillatory shear stress occurs at sites of the circulation that are vulnerable to atherosclerosis. Because oxidative stress contributes to atherosclerosis, we sought to determine whether oscillatory shear stress increases endothelial production of reactive oxygen species and to define the enzymes responsible for this phenomenon. Bovine aortic endothelial cells were exposed to static, laminar (15 dyn/cm2), and oscillatory shear stress (+/-15 dyn/cm2). Oscillatory shear increased superoxide (O2.-) production by more than threefold over static and laminar conditions as detected using electron spin resonance (ESR). This increase in O2*- was inhibited by oxypurinol and culture of endothelial cells with tungsten but not by inhibitors of other enzymatic sources. Oxypurinol also prevented H2O2 production in response to oscillatory shear stress as measured by dichlorofluorescin diacetate and Amplex Red fluorescence. Xanthine-dependent O2*- production was increased in homogenates of endothelial cells exposed to oscillatory shear stress. This was associated with decreased xanthine dehydrogenase (XDH) protein levels and enzymatic activity resulting in an elevated ratio of xanthine oxidase (XO) to XDH. We also studied endothelial cells lacking the p47phox subunit of the NAD(P)H oxidase. These cells exhibited dramatically depressed O2*- production and had minimal XO protein and activity. Transfection of these cells with p47phox restored XO protein levels. Finally, in bovine aortic endothelial cells, prolonged inhibition of the NAD(P)H oxidase with apocynin decreased XO protein levels and prevented endothelial cell stimulation of O2*- production in response to oscillatory shear stress. These data suggest that the NAD(P)H oxidase maintains endothelial cell XO levels and that XO is responsible for increased reactive oxygen species production in response to oscillatory shear stress. 相似文献
19.
Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin 总被引:2,自引:0,他引:2
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Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells. 相似文献
20.
Fluorescence intensified/enhanced microscopy has been used to study the metabolic activation of living human neutrophils in time-lapse sequences. The autofluorescence associated with NAD(P)H's emission band was studied within individual quiescent and stimulated cells. Excitation of NAD(P)H-associated autofluorescence was provided by a high-intensity Hg-vapor lamp. The background-subtracted autofluorescence signals were computer enhanced. In some cases the ratio image of NAD(P)H-associated autofluorescence to tetramethyl-rhodamine methyl ester (TRME) fluorescence, which was found to be uniformly distributed within neutrophils, was calculated to normalize autofluorescence intensities for cell thickness. Activation of the NADPH oxidase by phorbol myristate acetate, F-, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or tumor necrosis factor (TNF) dramatically reduced autofluorescence levels. Membrane solubilization with sodium dodecyl sulfate eliminated autofluorescence. Thus, control experiments indicated that most or all of the detectable NAD(P)H-associated autofluorescence was due to NAD(P)H, consistent with previous non-microscopic studies. To understand the metabolic events surrounding the internalization and oxidative destruction of targets, we have imaged the NAD(P)H-associated autofluorescence of neutrophils and the Soret band of antibody coated target erythrocytes during cell-mediated cytotoxicity. Absorption contrast microscopy of the erythrocyte's Soret band is an especially sensitive indicator of the entry of reactive oxygen metabolites into this target's cytosol. Thus, it is possible to spectroscopically dissect and image the substrate (NADPH) and product (O2-) reactions of the NADPH oxidase in living unlabeled neutrophils. During real-time experiments at 37 degrees C, the level of NAD(P)H-associated autofluorescence surrounding phagosomes greatly increases before the disappearance of the target's Soret band. NAD(P)H-associated autofluorescence in the vicinity of phagocytosed erythrocytes is greatly diminished after target oxidation. This suggests that NAD(P)H is translocated to the vicinity of phagosomes prior to the oxidation of targets. The apparent cytosolic redistribution of NAD(P)H was confirmed by ratio imaging microscopy to control for cell thickness. We suggest that NADPH including its sources and/or carriers accumulate near phagosomes prior to target oxidation and that local NADPH molecules are consumed during target oxidation. 相似文献