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PubMed1975

Membrane potentials in pinched-off presynaptic nerve ternimals monitored with a fluorescent probe: evidence that synaptosomes have potassium diffusion potentials.

1. Some physiological properties of tissue fractions from rat brain homogenates have been examined. Of the three fractions studied (presynaptic nerve terminals, mitochondria and fragmented membranes), only the nerve terminals (synaptosomes) have the ability to accumulate 42K from physiological salt solutions. 2. The ability to accumulate and retain K is lost if synaptosomes are exposed to very hypotonic solutions. The K uptake and total K content is reduced by ouabain and by inhibitors of glycolysis and oxidative phosphorylation. 3. These results suggest that synaptosomes in physiological saline accumulate K against a concentration gradient, and may have K diffusion potentials across their surface membranes. The voltage-sensitive fluorescent probe, 3,3'-dipentyl 2,2'-oxacarbocyanine (CC5), was used to test this possibility. 4. In the squid axon, the fluorescent emission of CC5 is directly proportional to membrane potential; depolarization causes an increase in fluorescence. 5. The fluorescence of synaptosomes ('synaptosome fluorescence') treated with CC5 is increased when [K]o is increased or [K]o is reduced; replacement of external Na by Li or choline has little effect on the synaptosome fluorescence. In quantitative terms, synaptosome fluorescence is proportional to log ([K]o plus 0-05[Na]o). Rb is about as effective as K in enhancing synaptosome fluorescence; Cs is about 1/4 as effective. The effect of increased [K]o is reversible. 6. The fluorescence data provide corroborative evidence that there is normally a large K gradient ([K]o smaller than [I]i) across the synaptosome surface membrane. The data suggest the [K]i may be in excess of 100 mM. 7. Replacement of Cl- by methylsulphate did not significantly affect the relationship between synaptosome fluorescence and [K]o, nor did removal of external Ca. 8. The fluorescence of CC5-treated mitochondria, membrane fragmnets, or lysed synaptosomes is unaffected by changes in the K concentration of the medium. 9. Veratridine and gramicidin D, both of which enhance Na permeability (PNa) in some intact tissues, increase synaptosome fluorescence when added to the standard medium. The increment is greatly reduced or abolished when external Na is replaced by choline. 10. If synaptosomes are first Na-loaded (by pre-treatment with cyanide + iodoacetate), and then placed in a choline medium, addition of gramicidin D significantly decreases fluorescence. This effect could be explained if, with [Na]o smaller than [Na]i, the increase in PNa causes the synaptosomes to hyperpolarize. 11. The veratridine-induced increase in synaptosome fluorescence was prevented by 3 times 10- minus 7M tetrodotoxin, which also blocks the depolarizing effect of veratridine in intact neurones. 12. The main conclusion is that synaptosomes may retain resting membrane potentials and the ability to increase Na permeability.

Alkenes↗

PubMed2001

Quantification of 5-aminolevulinic acid induced fluorescence improves the specificity of bladder cancer detection.

PURPOSE: 5-Aminolevulinic acid induced fluorescence endoscopy has outstanding sensitivity for detecting early stage bladder cancer. Nevertheless, a third of the lesions that show specific fluorescence are histologically benign. We decreased the false-positive rate of 5-aminolevulinic acid induced fluorescence endoscopy by incorporating protoporphyrin IX fluorescence quantification into the standard cystoscopy procedure. MATERIALS AND METHODS: In 25 cases (53 biopsies) of a history of or suspicion for bladder cancer 5-aminolevulinic acid induced fluorescence endoscopy and fluorescence image quantification were performed. For fluorescence image quantification images obtained with a target integrating color charge-coupled device camera were digitized and stored in a personal computer. Red-to-blue ratios were calculated from fluorescence positive lesions and results were correlated with hematoxylin and eosin histology. RESULTS: Malignant fluorescence positive lesions showed significantly stronger fluorescence intensity than fluorescing lesions with benign histology. A threshold was established that decreased the false-positive rate by 30% without affecting sensitivity. CONCLUSIONS: Fluorescence image quantification is a new endoscopic method for objectively selecting multicolor fluorescence bladder lesion images for biopsy. It has the potential of eliminating human error by different surgeons with variable experience in fluorescence endoscopy.

Adult↗

PubMed2002

NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes.

During the development of neoplasia, epithelial tissues undergo biochemical and structural changes that can manifest in tissue fluorescence. There have been several reports on different in vivo fluorescence characteristics between normal and precancerous (dysplastic) tissues. However, it has been difficult to identify and quantify the origins of these changes, mainly because of distortions introduced in measured tissue fluorescence spectra by tissue scattering and absorption. Such distortions can be removed by combining information in simultaneously measured fluorescence and reflectance spectra. Thus, we can recover the intrinsic (undistorted) tissue fluorescence. In this report, we show that extraction of the intrinsic fluorescence allows us: (a) to determine the fluorescence spectra of NAD(P)H and collagen in an in vivo environment, and (b) to use these NAD(P)H and collagen spectra to describe, quantitatively, diagnostically significant biochemical changes between normal and dysplastic tissues. Specifically, by analyzing intrinsic fluorescence of human epithelial tissue as it becomes deoxygenated in vivo, we can resolve the fluorescence spectra of NAD(P)H and collagen, two of the major tissue fluorophores. This is important because fluorescence depends on the local environment of the chromophore. Then, we extract the intrinsic fluorescence spectra of sites from 35 patients with suspected cervical lesions and 7 patients with Barrett's esophagus and describe them accurately as a linear combination of NAD(P)H and collagen contributions. In both tissue cases, we find that low collagen and high NAD(P)H fluorescence characterizes the high-grade dysplastic lesions when compared with nondysplastic tissues. These data present evidence for the presence of detectable levels of NAD(P)H fluorescence in human epithelial tissues in an in vivo setting and demonstrate that NAD(P)H and collagen may be used as quantitative fluorescence biomarkers for in vivo detection of dysplasia in the cervix and the esophagus.

Barrett Esophagus↗

PubMed1992

Endocytosis of cigarette-smoke condensate by rabbit alveolar macrophages in vitro measured as fluorescence intensity.

It has previously been shown that alveolar macrophages (AMs) from smokers have increased fluorescence compared to AMs from nonsmokers. In the present investigation, AMs lavaged from rabbits were exposed to a cigarette-smoke condensate (CSC) in order to study its influence on the fluorescence of AMs. Flow cytofluorometry was used. Alveolar cells exposed to 0.01% of CSC showed a fluorescence which increased with higher concentrations of the CSC. Incubation with CSC for 5 min was enough to give a significant rise (p less than 0.001) in cell fluorescence compared to controls. After incubating fluorescent cells with untreated non-fluorescent AMs, fluorescent material was distributed to the non-fluorescent AMs within 30 min. Further more, fluorescence decreased when exposed cells were cultured in CSC-free medium. When incubating CSC with blood leucocytes, an almost threefold increase in fluorescence was observed among the phagocytic cells at 37 degrees C compared to 4 degrees C, whereas the fluorescence in the non-phagocytic cell population only showed a minor enhancement. Endocytosis of fluorescent material in CSC can explain the strong fluorescence seen in AMs from smokers. Exocytosis and re-endocytosis of the material by new AMs may explain the slow decline in the fluorescence of AMs observed after cessation of smoking.

Animals↗

PubMed2001

Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration.

Use of green fluorescent protein (GFP) as a molecular reporter is restricted by several environmental factors, such as its requirement for oxygen in the development of the fluorophore, and its poor fluorescence at low pH. There are conflicting data on these limitations, however, and systematic studies to assess the importance of these factors for growing bacterial cultures are lacking. In the present study, homogeneous expression of the gfpmut3* gene directed by a synthetic constitutive lactococcal promoter was demonstrated in batch cultures and in biofilms of Streptococcus gordonii DL1. A lower limit of oxygen concentration for maturation of the GFP fluorophore was determined: fluorescence was emitted at 0.1 p.p.m. dissolved oxygen (in conventionally prepared anaerobic media lacking reducing agents), whereas no fluorescence was detected in the presence of 0.025 p.p.m. dissolved oxygen (obtained by addition of L-cysteine as reducing agent). When an anaerobically grown (non-fluorescent) >50 microm thick biofilm was shifted to aerobic conditions, fluorescence could be detected within 4 min, reaching a maximum over the next 16 min. It was not possible to detect any fluorescence gradients (lateral or vertical) within the >50 microm thick biofilm, and fluorescence development after the shift to aerobic conditions occurred throughout the biofilm (even at the substratum). This suggests that oxygen gradients, which might result in reduced GFP fluorescence, did not exist in the >50 microm thick biofilm of this organism. Production of lactic acid and the subsequent acidification in batch cultures of S. gordonii DL1 led to a decrease in fluorescence intensity. However, severe pH reduction was prevented when the bacterium was grown as a biofilm in a flowcell, and a homogeneous distribution of a strong fluorescence signal was observed. These findings show that GFP can be applied to studies of oxygen-tolerant anaerobic bacteria, that densely packed, flowcell-grown biofilms of S. gordonii do not develop oxygen gradients inhibitory to GFP fluorescence development, and that the often transient nature of GFP fluorescence in acid-producing bacteria can be overcome in flowcells, probably by the elimination of metabolic by-product accumulation.

Anaerobiosis↗

PubMedDate not supplied

Fluorescence spectroscopy of aromatic species produced in rich premixed ethylene flames.

The fluorescence spectra of the condensed species (CS) collected in the soot inception region of a rich premixed laminar ethylene/oxygen flame have been measured by excitation in the UV at 266 and 355 nm excitation wavelength. The contribution of the most abundant polycyclic aromatic hydrocarbons (PAH) to the CS fluorescence has been evaluated in order to attribute the CS fluorescence at different emission wavelengths to specific aromatic structures. The fluorescence peaks detected in the UV region of the CS fluorescence spectrum was found to be mainly due to a typical PAH like fluorene, that is, the most fluorescent one among the PAH analyzed in the CS by chromatographic analysis. The CS exhibited the larger emission in the visible where the PAH contribution has been shown to be negligible and other fluorescing aromatic species, not identified by chromatographic analysis of the CS, have to be considered responsible for the visible fluorescence. Laser induced fluorescence (LIF) flame measurements excited at 266 nm and detected at two selected wavelengths (310 and 410 nm) have been performed along the flame axis and compared with the CS fluorescence intensity. The LIF and CS fluorescence signals show quite similar axial trends demonstrating that the LIF signals are related to CS fluorescence. In particular, the LIF fluorescence signals detected in the UV could be attributed to the PAH fluorescence whereas the unidentified species contained in the CS can be followed by LIF detection in the visible region.

Journal Article↗

PubMed2006

External electric field effects on fluorescence of pyrene butyric acid in a polymer film: concentration dependence and temperature dependence.

Fluorescence spectra and electrofluorescence spectra (plots of the electric field-induced change in fluorescence intensity as a function of wavelength) have been measured at different temperatures for pyrene butyric acid (PBA) in a PMMA film at different concentrations. At a low concentration of 0.5 mol % where fluorescence emitted from the locally excited state of PBA (LE fluorescence) is dominant, LE fluorescence spectra show only the Stark shift in the presence of an electric field (F), which results from the difference in molecular polarizability between the ground and emitting states. At a high concentration of 10 mol % where the so-called sandwich-type excimer fluorescence (EX(1)) is dominant, both EX(1) and LE fluorescence are quenched by F. Another fluorescence assigned to a partially overlapped excimer (EX(2)) also exists at room temperature, and this emission is enhanced by F. As the temperature decreases, three fluorescence emissions whose electric field effects are different from each other become clear besides EX(1) and LE fluorescence, indicating that at least five fluorescence components exist at high concentrations at low temperatures. At a medium concentration of 5 mol % where EX(1) is comparable in intensity to the LE fluorescence, the intensity of EX(1) is not affected by F at any temperature, but LE fluorescence and EX(2) are markedly influenced by F at room temperature, and four fluorescence emissions are confirmed at low temperatures.

Journal Article↗

PubMed1992

Fluorospectroscopic analysis of the fluorescent substances in peroxidized microsomes of rat liver.

The fluorescent substances formed in rat liver microsomes in the course of lipid peroxidation were investigated by fluorescence techniques. The fluorescence emitted from peroxidizing microsomes continuously increased as lipid peroxidation progressed, while the steady-state fluorescence anisotropy increased and then reached a plateau. A similar increase was observed in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene in peroxidizing microsomes. The fluorescence from peroxidized microsomes consisted of at least three species having short, middle or long fluorescence lifetimes. The lifetimes and relative amplitudes of fluorescence were unaffected by the extent of lipid peroxidation. Both fluorescence of the chromolipids extracted and the proteins isolated from peroxidized microsomes had the same characteristics in fluorescence lifetimes as the fluorescence from whole peroxidized microsomes. Thus, these lipids and proteins appear to be the major biological substances responsible for the fluorescence emanating from whole peroxidized microsomes. Furthermore, fluorescent substances formed in microsomes seem to increase in quantity rather than change in quality as lipid peroxidation progresses.

Animals↗

PubMed2005

A nonparametric method for analysis of fluorescence emission in combined time and wavelength dimensions.

We report a method for accurate recovery of tissue intrinsic fluorescence emission characteristics, including fluorescence lifetimes and spectral profiles, from complex two-dimensional (spectro-temporal) emission waveforms. Most algorithms for analysis of fluorescence data address separately the characteristics of either spectral emission or fluorescence relaxation time. We developed a novel nonparametric analytical method that allows for identification and estimation of the intrinsic Fluorescent Impulse Response Kernel (FIRK) simultaneously in time and wavelength dimensions. Modeling of FIRK was based on the characteristics of spectro-temporal fluorescence waveforms. Due to the decaying behavior of the fluorescence, a linear combination of discrete Laguerre functions was used to model the fluorescence response in time. To address the large variability of spectral profiles of distinct fluorophores, a discrete Fourier series expansion was used to model the variation of fluorescence intensity across wavelength. The proposed method was validated on synthetic fluorescence data and data measured from fluorescence lifetime standards and tissue endogenous fluorescent biomolecules. We determined that this method provides a direct recovery of the two-dimensional FIRK and accurate estimation (residual error < 6%) of a broad range of fluorescence lifetimes including the sub-nanosecond range. The FIRK retrieved using this method can further facilitate modeling and recognition of pathological and physiological conditions in tissues.

Algorithms↗

PubMed1995

Simultaneous measurement of [Ca2+]i and secretion-coupled membrane turnover, by single cell fluorescence microscopy.

Thyrotropin releasing hormone (TRH), which stimulates prolactin secretion, increases the fluorescence of cultured bovine anterior pituitary (bAP) cells in the presence of the non-permeant membrane indicator dye FM 1-43 [Stafford SJV. Shorte SL. Schofield JG. (1993) Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells. Biosci. Rep., 13, 9-17]. FM 1-43 is non-fluorescent in aqueous solution but becomes fluorescent when incorporated into the plasma membrane. The membrane area accessible to FM 1-43 dye, and therefore cell fluorescence, increases during exocytosis as secretory granules fuse with the plasma membrane, and endocytosis as vesicles formed at the plasma-membrane fuse with intracellular organelle membranes. We have here measured changes in FM 1-43 uptake and the intracellular calcium concentration ([Ca2+]i) concurrently in the same cells on exposure to TRH, phorbol myristate acetate (PMA) or NH4Cl. TRH (0.1-10 microM) caused a transient increase in [Ca2+]i in 70-90% of bAP cells and in 60-90% of the responding cells also caused a sustained increased FM 1-43 fluorescence. TRH increased [Ca2+]i but did not affect FM 1-43 fluorescence in GH3 rat pituitary cells, probably because they contain too few secretory granules to give a detectable increase. The dopamine D2-receptor agonist quinpirole (10 microM) had little effect on the TRH-induced [Ca2+]i rise in bAP cells, but abolished the increase in FM 1-43 fluorescence. The phorbol ester PMA (0.3-3 microM) caused a small, transient increase in [Ca2+]i followed by a fall to levels lower than original resting levels in 40-60% of bAP cells and increased FM 1-43 uptake in cells showing these changes. Extracellular NH4Cl, which mobilises calcium from an ionomycin-insensitive calcium store, caused a transient [Ca2+]i increase in over 90% of the bAP-cells and increased FM 1-43 uptake in a subpopulation (> 50%) of these. The Na+/H+ ionophore monensin prevented the increase in FM 1-43 fluorescence but not the [Ca2+]i rise induced by TRH, prevented the increases in both FM 1-43 fluorescence and [Ca2+]i caused by NH4Cl, and reduced the number of cells showing a rise in FM 1-43 fluorescence in response to PMA from 64% to 34%. The Ca(2+)-ATPase inhibitor thapsigargin reduced the number of bAP cells displaying TRH-induced increases in [Ca2+]i and membrane-turnover from 74% to 18%, but did not affect the changes in [Ca2+]i or FM 1-43 fluorescence caused by PMA or NH4Cl. We discuss the relationships between the secretogogue-induced increases in FM 1-43 fluorescence and changes in intracellular [Ca2+]i under these conditions.

Animals↗

PubMed2004

Hexaminolevulinate-induced fluorescence endoscopy in patients with rectal adenoma and cancer: a pilot study.

BACKGROUND: Fluorescence endoscopy is a promising new method for detection and treatment of premalignant and malignant lesions. The aim of this pilot study was to investigate the feasibility of hexaminolevulinate-based photodetection of rectal adenoma and cancer, including safety, dose finding, and efficacy. METHODS: Ten patients with known rectal adenoma or cancer were sensitized by instillation of 3.2 mM of hexaminolevulinate as an enema. Fluorescence endoscopy was performed after retention of the enema for 30 to 60 minutes, followed by a rest time of up to 30 minutes before endoscopy. Biopsy specimens were taken from fluorescent and non-fluorescent areas and fluorescence microscopy studies were performed to assess the distribution of protoporphyrin IX fluorescence in different tissue layers. Adverse events were reported by direct questioning of all patients; skin photosensitivity, changes in biochemical tests of liver function, blood pressure and heart rate, and the occurrence of GI symptoms (nausea, vomiting) were recorded for 5 patients. OBSERVATIONS: Hexaminolevulinate-induced fluorescence endoscopy produced selective fluorescence of all rectal adenomas with intraepithelial neoplasia. For rectal cancer, there was only weak fluorescence or none at all. No hexaminolevulinate-induced side effect was observed. In two patients, fluorescence differentiated adenomas and hyperplastic polyps. CONCLUSIONS: Hexaminolevulinate-based fluorescence endoscopy (3.2 mM administered as an enema) in patients with rectal cancer and adenoma was well tolerated and produced no significant skin sensitivity or other side effects. The optimal duration of application is 30 to 45 minutes, with a rest time of 30 minutes. Selective fluorescence of adenoma with intraepithelial neoplasia suggests that hexaminolevulinate-based fluorescence endoscopy may be useful for detection of premalignant lesions.

Adenoma↗

PubMed1998

Fluorescent labelling of abasic sites: a novel methodology to detect closely-spaced damage sites in DNA.

PURPOSE: To test the ability of a new fluorescent reagent to label abasic sites in DNA and to use fluorescent energy transfer as a measure of closely-spaced abasic sites in DNA. MATERIALS AND METHODS: A fluorescein-conjugated hydroxylamine derivative (FARP, 5-(((2-(carbohydrazino)-methyl)thio)acetyl)aminofluorescein, aminooxyacetyl hydrazide) that reacts covalently with open chain aldehydes in DNA has been synthesized. Upon depurination of plasmid DNA and reaction with FARP a stable oxime bond is formed between FARP and the generated open chain aldehydes. RESULTS: By independently quantitating the generated abasic sites, it is shown that most of the generated abasic sites under acidic conditions become fluorescently labelled. The limit of sensitivity with the fluorometer used is approximately 90 femtomole abasic sites, corresponding to 1 abasic site per 17000 base pairs. DNA can be fluorescently labelled over a wide range of FARP:base pair ratios following different extent of depurination, and fluorescent loadings of 1 FARP:20000 base pairs up to 1 FARP:10 base pairs are demonstrated. The heavily labelled samples display significant fluorescence quenching due to the proximity of abasic sites labelled with FARP, that undergo fluorescence energy transfer. Treatment of heavily labelled DNA samples with nuclease P1 results in an increase in fluorescence due to the release of the fluorescent labels in the solution. The relative increase in fluorescence is a quantitative measure of the proximity of labelled abasic sites. Furthermore it is shown that if only 1% of DNA contains abasic sites generated in close proximity (within 10-20 base pairs or less of each other) the resulting quenching is significant enough to detect, even if the rest of the DNA contains isolated abasic sites. CONCLUSIONS: The present approach constitutes a novel fluorescence-based method to detect abasic sites in nucleic acids and demonstrates the feasibility of detecting the presence of closely-spaced damage sites in DNA via fluorescence energy transfer. The technique also comprises a general and convenient method to fluorescently label nucleic acids without introducing strand breaks as a result of the labelling procedure.

Aldehydes↗

PubMed1985

Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane.

When monolayer cultures of Chinese hamster lung fibroblasts are briefly incubated at 2 degrees C with the fluorescent sphingolipid analogue, C6-NBD-ceramide (N- [7-(4-nitrobenzo-2-oxa-1,3-diazole)] aminocaproyl sphingosine), fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope occur. During further incubation at 37 degrees C, the Golgi apparatus, and later the plasma membrane, become intensely fluorescent. Within this period, the C6-NBD-ceramide is converted to equal amounts of fluorescent sphingomyelin and glucocerebroside (Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA., 80:2608-2612). In the present study, the intracellular translocation of these metabolites and their subsequent appearance at the plasma membrane were investigated by fluorescence microscopy, the addition of the ionophore monensin, and the technique of "back exchange," in which the amounts and types of fluorescent lipids present at the cell surface are identified after their transfer from the cell surface into recipient vesicles. In control cells, the amount of fluorescent glucocerebroside and sphingomyelin that could be removed from the cell surface by back exchange increased during incubation at 37 degrees C, correlating with the increased fluorescence of the plasma membrane observed by microscopy. In the presence of 10 microM monensin, visible labeling of the plasma membrane was greatly diminished, whereas the Golgi apparatus became highly fluorescent and distended. The ability to remove fluorescent metabolites from the cell surface by back exchange was significantly but reversibly inhibited by monensin. Monensin also increased the total amount of fluorescent sphingomyelin, but not the glucocerebroside found in cells. Subcellular fractions were assayed for their ability to convert radiolabeled and fluorescent ceramides to the corresponding sphingomyelins and glucocerebrosides. The activities of parallel fractions coincided, suggesting that the presence of the NBD moiety did not affect the cellular metabolism of ceramide. Furthermore, the major peak of sphingomyelin- and glucocerebroside-synthesizing activity appeared to coincide with an enriched Golgi fraction. These results strongly suggest that fluorescent sphingomyelin was not synthesized at the plasma membrane as has recently been suggested for endogenous sphingomyelin. Rather, both the sphingomyelin and glucocerebroside analogues were synthesized intracellularly from C6-NBD-ceramide and translocated through the Golgi apparatus to the cell surface.

Animals↗

PubMed1983

Microinjection of fluorescent tubulin into dividing sea urchin cells.

To follow the dynamics of microtubule (MT) assembly and disassembly during mitosis in living cells, tubulin has been covalently modified with the fluorochrome 5-(4,6-dichlorotriazin-2-yl)aminofluorescein and microinjected into fertilized eggs of the sea urchin Lytechinus variegatus. The changing distribution of the fluorescent protein probe is visualized in a fluorescence microscope coupled to an image intensification video system. Cells that have been injected with fluorescent tubulin show fluorescent linear polymers that assemble very rapidly and radiate from the spindle poles, coincident with the position of the astral fibers. No fluorescent polymer is apparent in other areas of the cytoplasm. When fluorescent tubulin is injected near the completion of anaphase, little incorporation of fluorescent tubulin into polymer is apparent, suggesting that new polymerization does not occur past a critical point in anaphase. These results demonstrate that MT polymerization is very rapid in vivo and that the assembly is both temporally and spatially regulated within the injected cells. Furthermore, the microinjected tubulin is stable within the sea urchin cytoplasm for at least 1 h since it can be reutilized in successive daughter cell spindles. Control experiments indicate that the observed fluorescence is dependent on MT assembly. The fluorescence is greatly diminished upon treatment of the cells with cold or colchicine agents known to cause the depolymerization of assembled MT. In addition, cells injected with fluorescent bovine serum albumin or assembly-incompetent fluorescent tubulin do not exhibit fluorescence localized in the spindle but rather appear diffusely fluorescent throughout the cytoplasm.

Anaphase↗

PubMed1985

Fusion between Sendai virus envelopes and biological membranes. The use of fluorescent probes for quantitative estimation of virus-membrane fusion.

The fluorescent probes, N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine and lissamine-rhodamine-B-sulfonylphosphatidylethanolamine, were inserted at the appropriate surface density into membranes of reconstituted Sendai virus envelopes, thus allowing transfer of energy between the fluorescent probes. In addition, only the fluorescent molecule N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine was inserted into the viral envelopes, resulting in self-quenching. Incubation of fluorescent, reconstituted Sendai virus envelopes with human erythrocyte ghosts resulted in either reduction in the efficiency of energy transfer or in fluorescence dequenching. No reduction in the efficiency of energy transfer or fluorescence dequenching was observed when fluorescent, reconstituted Sendai virus envelopes were incubated with glutaraldehyde-fixed or desialized human erythrocyte ghosts. Similarly, no change in the fluorescence value was observed when nonfusogenic, reconstituted Sendai virus envelopes were incubated with human erythrocyte ghosts. These results clearly show that reduction in the efficiency of energy transfer or dequenching is due to virus-membrane fusion and not to lipid-lipid exchange. Incubation of reconstituted Sendai virus envelopes, carrying inserted N-4-nitrobenzo-2-oxa-1,3-diazolephosphatidylethanolamine, with cultured cells also resulted in a significant and measurable dequenching. However, incubation of nonfusogenic, fluorescent reconstituted Sendai virus envelopes with hepatoma tissue culture cells also resulted in fluorescent dequenching, the degree of which was about 50% of that observed with fusogenic, fluorescent reconstituted viral envelopes. It is therefore possible that, in addition to virus-membrane fusion, endocytosis of fluorescent viral envelopes results in fluorescence dequenching as well.

Calcium↗

PubMed1994

ATP hydrolysis-driven structural changes in the gamma-subunit of Escherichia coli ATPase monitored by fluorescence from probes bound at introduced cysteine residues.

Four mutants of the Escherichia coli F1ATPase, gamma S8-C, gamma T106-C, gamma S179-C, and gamma V286-C, which have a cysteine introduced at different sites in the gamma-subunit by site-directed mutagenesis, were reacted with the fluorescent reagent N-(4-7-(diethylamino)4-methylcoumarin-3-yl)-maleimide (CM) under conditions that selectively label the introduced Cys residue. With each mutant the effect of nucleotide binding on the fluorescence of the probe has been monitored. The results obtained with the mutants gamma S8-C and gamma T106-C are similar. In both cases, there was a spectral shift and change in fluorescence intensity on adding AMP.PNP or ATP to enzyme emptied of nucleotide from catalytic sites, while no change in the fluorescence spectrum was observed upon adding ADP. The fluorescence spectral changes obtained with ATP were transient and involved an initial rapid fluorescence enhancement followed by a subsequent fluorescence quenching. The kinetics of these ATP-induced fluorescence changes and the kinetics of ATP hydrolysis as monitored by the rates of ATP binding and of Pi formation were the same under conditions of unisite catalysis, indicating that the conformational changes in the gamma-subunit being measured by the fluorescent probe are driven by ATP hydrolysis in catalytic sites. No nucleotide-dependent fluorescence changes were observed with CM bound at a Cys at position 179. Nucleotide-dependent changes in fluorescence were seen with CM bound at position 286, but these appear to reflect structural changes due to binding of ADP or ATP in noncatalytic sites. The fluorescence changes observed in mutants gamma S8-C and gamma T106-C were not seen in subunit epsilon-free E. coli F1ATPase, although such enzyme preparations are highly active ATPases. We conclude that the structural changes monitored by the fluorescent probe are a part of the conformational coupling, whereby catalytic site events are linked to proton channeling.

Adenosine Triphosphate↗

PubMed1997

Use of a fluorescent analog of CDP-DAG in human skin fibroblasts: characterization of metabolism, distribution, and application to studies of phosphatidylinositol turnover.

We studied the uptake metabolism, and distribution of a fluorescent analog of CDP-diacylglycerol [cytidine diphosphate-1, 2-oleoyl, (N-(4-nitrobenzo-2-oxa-1,3-diazole) aminocaproyl) diacylglycerol; CDP-NBD-DAG]. When cells were incubated with CDP-NBD-DAG for 60 min at 11 degrees C and washed, the fluorescent lipid was localized to the plasma membrane. However, upon warming to 37 degrees C, the fluorescent lipid redistributed into various intracellular membranes and was metabolized primarily to fluorescent analogs of DAG and phosphatidylcholine (PC), although small amounts of fluorescent phosphatidic acid and phosphatidylinositol (PI) were also formed. The incorporation of 32Pi into some of the fluorescent lipids was also determined in order to assess their turnover. Stimulation of cells with platelet-derived growth factor enhanced the synthesis of fluorescent PI relative to unstimulated cells by approximately 68%, while the synthesis of fluorescent PC was unaffected. In addition, the incorporation of 32Pi into fluorescent PI was enhanced. Stimulation of cells with interleukin-1 beta enhanced the synthesis of both fluorescent PI (approximately 88%) and PC (approximately 250%) compared to non-stimulated cells, but with less incorporation of 32Pi into fluorescent PI. Finally, incubation of CDP-NBD-DAG-treated cells with inhibitors of phosphatidic acid phosphohydrolase and DAG kinase resulted in a dramatic increase in the amount of fluorescent PI formed (approximately 64% of all the CDP-NBD-DAG metabolites). We conclude that CDP-NBD-DAG can be used for the de novo synthesis of fluorescent PI, and in combination with 32P labeling, provides a convenient method for studying PI turnover.

4-Chloro-7-nitrobenzofurazan↗

PubMed1998

In vivo analysis of hepatic NADH fluorescence. Methodological approach to exclude Ito-cell vitamin A-derived autofluorescence.

Assessment of hepatic surface NADH fluorescence is complicated by questions concerning the relative contribution of Ito cell-associated vitamin A autofluorescence, which cannot be distinguished from each other using ultraviolet epi-illuminated microfluorographs. However, as presented herein, vitamin A autofluorescence can easily be eliminated due to its rapid photobleaching property. In line with in vitro studies on liver perfusion reported by Suematsu and coworkers (1993b), we assessed NADH fluorescence in liver surface regions, which were epi-illuminated for elimination of vitamin A autofluorescence and were allowed to recover for 20 min prior to onset of the experiment. To assess whether this procedure is stringent for accurate estimation of NADH fluorescence, we studied NADH fluorescence in non prebleached surface regions after a 30 sec and 60 sec epi-illumination period. Epi-illumination of these hepatic surface regions for more than 20 sec guaranteed also complete elimination of vitamin A autofluorescence (Vollmar et al., 1996) and further allowed to calculate the subsequent loss of NADH fluorescence due to a potential photobleaching effect after 30 sec and 60 sec of continuous light exposure. Corresponding data of the present study reveal a constant loss of NADH fluorescence of approximately 20% due to its photobleaching property over the time period from 20-60 sec of epi-illumination. Linear regression analysis of the kinetics of NADH fluorescence upon continuous light exposure allowed the calculation of the initial NADH fluorescence, excluding the interference with the Ito cell vitamin A-derived autofluorescence. The concurrency of this mathematically assessed value for the initial (non-bleached) NADH fluorescence within normal hepatic livers with the recovered value of NADH fluorescence allows the conclusion that the procedure for elimination of vitamin A autofluorescence, including ultraviolet epi-illumination with a subsequent NADH recovery period of at least 20 min, is not a prerequisite for the reliable assessment of intraindividual changes of hepatic NADH fluorescence in vivo. Thus, valid assessment of hepatic NADH fluorescence in vivo can be performed by analyzing initial NADH fluorescence bleaching kinetics, and does not necessarily require the 20 min recovery period after bleaching of the Ito cell vitamin A-derived autofluorescence.

Animals↗