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PubMed1988

Histamine formation by enterococci isolated from home-made goat cheeses.

A survey was made of the histamine-producing capability of enterococci isolated from goat cheeses. The strains, 130 Streptococcus faecium and 106 S. faecalis, were grown in Trypticase Soy Broth Histidine medium (TSBH) at 35 degrees C for 24 h and the histamine formed was determined by fluorometry. Forty-one (31.5%) of the S. faecium strains and 2 (1.9%) of the S. faecalis strains produced histamine. The largest amount detected was 4.0 micrograms histamine/ml TSBH. Compared with the amounts of histamine produced by some Gram-negative bacteria, the histamine production by enterococci seems to be low. Under the present conditions the enterococci seem to have no relevance from a histamine intoxication point of view.

Animals↗
PubMed1992

Determination of cytotoxic T-lymphocyte precursor frequencies using europium labeling as a nonradioactive alternative to labeling with chromium-51.

We report on the use of europium (Eu) as a suitable nonradioactive alternative for target cell labeling in limiting dilution analysis (LDA) assays set up to determine cytotoxic T-lymphocyte precursor (CTLp) frequencies. A nonradioactive alternative to the commonly used chromium-51 (51Cr) release assay seems desirable because working with radioisotopes has some major disadvantages concerning possible health risks, environmental load, costs of facilities necessary for working with radioisotopes, and shelf life. Some groups have successfully applied the Eu release assay based on detection by time-resolved fluorometry, to tests in which NK- or LAK-cell activity or cytotoxic T-lymphocyte reactions were measured. This led to the investigation whether this method could also be applicable to the more specific determination of CTLp frequencies in LDA assays. After optimal labeling conditions had been established, the sensitivity of the Eu release assay was determined by performing several LDA assays in which the target cells were labeled with either Eu or radioactive 51Cr. When CTLp frequencies were compared, it was shown that the Eu release assay is at least as sensitive and specific as the 51Cr release assay. Moreover, although the labeling procedure takes longer, sample processing is much faster: only 1 second per sample. The fact that the Eu release assay is not radioactive enables the assay to be performed at any laboratory and even--because the frequency of CTLps may have implications for organ graft survival and for donor selection in bone marrow transplantation--to do so on a routine basis.

Cell Count↗
PubMed1991

Phagolysosomal pH and location of particles in alveolar macrophages.

Fluorescein-labeled silica particles (FSP) were instilled into the tracheae of rabbits. Groups of four rabbits were killed after 24 hr, 1 week, 1 month, or 3 months and their lungs were lavaged. Phagolysosomal pH in the alveolar macrophages (AM) was measured using microscope fluorometry with FSP as a probe. Due to the marked decline of the fluorescence intensities from the FSP between 1 and 3 months after instillation, it was not possible to measure pH at 3 months, but the values from 24 hr, 1 week, and 1 month were quite similar, with group means of 4.8 and 4.9, respectively. Phagolysosomal pH in AM which phagocytized the FSP in vitro showed values about half a pH unit higher. AM from rabbits lavaged at 1 week had more lysosomes in contact with the FSP-containing phagolysosomes and a higher degree of vacuolization between the FSP and the phagolysosomal membrane than AM lavaged at 1 day. The location of the FSP in the AM appeared to be similar in rabbits lavaged after 1 week and 3 months. In histologic sections from the lungs the large majority of the FSP were within cells at all time points.

Animals↗
PubMed1996

Status and progress in on-line spectrometric monitoring and control of plant nutrient solutions.

Biotronics has been involved, under NASA sponsorship, in a wide ranging research and development program for instrumentation used in the monitoring and control of controlled environment agriculture. This program has embraced both chemical monitoring of plant nutrient solutions as well as microbiological monitoring of bacteria and fungi in these same solutions. This paper emphasizes the microbiological monitoring aspects of this program. In contrast to traditional methods of microbiological analysis based on culturing, staining and microscopic observation, the development described here is based on spectroscopic measurements, more specifically spectral fluorometry. The rationale, objectives, analytical methods and new instrumentation employed in the development of an on-line microbiological analyzer (MBA) are presented in some detail. Finally, the signal processing/pattern recognition methods used to evaluate the spectral data and produce estimates of microbial populations are described along with experimental test results to conclude the paper.

Ecological Systems, Closed↗
PubMedDate not supplied

The detection, identification and measurement of indole, tryptamine and 2-phenethylamine in putrefying human tissue.

Indole, tryptamine and 2-phenethylamine are putrefactive products which may be found in decaying human tissue. They may be identified by the data given found in decaying human tissue. They may be identified by the data given for infrared and ultraviolet spectrometry, fluorometry, thin layer and gas chromatography and mass spectrometry. Quantitative studies may be made using the gas chromatography method described. The rate of formation is affected by temperature and preservatives and may be prevented if necessary by the use of sodium fluoride. No relationship between the production of these materials (and alcohol) and time since the post-mortem examination could be established. The post-mortem examinations took place within 24 hours of death.

Ethanol↗
PubMed1982

Theory of phase-modulation fluorescence spectroscopy for excited-state processes.

Theory is presented for the analysis of excited-state reactions by fluorescence phase shift and demodulation methods. Initially, a two-state model with spectral overlap is considered to illustrate most simply the effects of excited-state reactions on the expected phase and modulation values. Secondly, a multistate model is described to illustrate the probable effects of a fluorophore interacting with several solvent molecules. We note the following unique features of phase-modulation data expected from a fluorophore whose emission spectrum shifts during the lifetime of the excited state: (1) The modulation frequency dependence of the apparent phase (tau p) and modulation (tau m lifetimes of the reacted species is opposite to that of a heterogeneous population of fluorophores. (2) For the reacted species tau p greater than tau m. For a heterogeneous sample tau p less than tau m. (3) The phase angle of the reacted species can exceed 90 degrees. For a heterogeneous sample phase angles are always less than 90 degrees. Thus, phase and modulation measurements can distinguish between time-dependent processes and spectral heterogeneity by observation of any feature described above. Additionally: (4) The lifetime of the product species can be measured directly. (5) Reverse relaxation can be identified, and the reverse relaxation rates calculated. (6) The wavelength-dependent phase and modulation data can be used to resolve the individual spectra from a two-state reaction. (7) And finally, under favorable conditions, a two-state excited-state process can be distinguished from a continuous multiple-state process. In each instance, model calculations are presented to illustrate the unique potentials of phase-modulation fluorometry for investigations of excited-state processes.

Kinetics↗
PubMed1982

Differential-wavelength deconvolution of time-resolved fluorescence intensities. A new method for the analysis of excited-state processes.

We describe a new procedure for the analysis of time-resolved decays of fluorescence intensity observed for excited-state reactions. This procedure is particularly valuable because it simplifies both determination of the rate constants of excited-state reactions and calculation of the emission spectra of the unreacted and the reacted species. These advantages are obtained without additional data acquisition, by the further analysis of data which would already be collected. As is usual for an excited-state process, time-resolved decays of fluorescence intensity are collected at wavelengths across the emission spectrum. Generally, one derives analytical expressions for the impulse response by deconvolution using the time profile of the exciting pulse. We suggest that in addition to the procedure just described, the response observed at longer wavelengths be deconvolved using the response observed on the blue side of the emission. Typically, this emission is from the initially excited state of the fluorophore (F). Then, the derived decay time is the decay rate of the reacted species (R). In addition, spectral overlap of the F and R states is revealed quantitatively as an apparent zero-decay-time component in the derived impulse response function. This is because the population of the initially excited state is considered to be the excitation function. This spectral overlap component is easily quantified, and allows the emission spectra of the F and R states to be calculated. This deconvolution procedure requires one favorable circumstance, which is the ability to choose a wavelength at which only the initially excited state is emitting. We tested our procedure on the excited state protonation of acridine and the excited-state deprotonation of 2-naphthol. The former reaction is essentially irreversible, whereas depending upon pH the dissociation of 2-naphthol can be reversible. We demonstrated that this procedure, which we call differential-wavelength deconvolution, revealed the individual spectra and simplified determination of the kinetic constants from the time-resolved decays. Without differential-wavelength deconvolution considerably more complex methods are required. We expect this procedure to greatly facilitate the use of pulse fluorometry methods in the analysis of excited-state processes.

Acridines↗
PubMed1988

Frequency-domain measurements of the rotational dynamics of the tyrosine groups of calmodulin.

We used frequency-domain fluorometry to determine the intensity and anisotropy decay kinetics of tyrosine residues in calmodulin and its fragments. Excitation was provided by a continuous ultraviolet laser source, a frequency-doubled rhodamine 6G ring dye laser, whose output was externally modulated to 200 MHz. Both the intensity and anisotropy decays were found to be multiexponential and dependent upon temperature and solution conditions. By examination of calmodulin fragments we determined that energy transfer between the two tyrosine residues reduces the steady-state anisotropy values by about 20%. Additionally, the frequency-domain anisotropy decays indicate local torsional motions of the tyrosine residues, as well as significant individual motions of the two domains of calmodulin.

Calmodulin↗
PubMed1988

Pressure dependence of fluorescence quenching reactions in proteins.

The effect of hydrostatic pressure (0-2.6 kbar) on the acrylamide quenching of the fluorescence of indole derivatives and several single-tryptophan-containing proteins has been studied using phase fluorometry at 25 degrees C. For the model system, N-acetyl-L-tryptophanamide in water, there is essentially no pressure dependence of the quenching rate constant, kappa q. For the internal Trp residue of ribonuclease T1 and cod parvalbumin, there also is essentially no pressure dependence of the apparent kappa q at low pressure. Thus, the activation volume, delta V not equal to, for these quenching processes is approximately zero. Such small delta V not equal to values are expected for diffusion-limited reactions in water at this temperature. The low, apparent delta V not equal to values for the globular proteins characterize these quenching processes as involving very small amplitude fluctuations in the protein structures. Only for the poised tetramer in equilibrium monomer equilibrium of melittin were we able to observe a significant effect of pressure on kappa q and this is due to the pressure-induced shift in the equilibrium position.

Fluorescence↗
PubMed1995

Influence of ligands on the fluorescence polarisation anisotropy of ethidium bound to DNA.

The decay of the fluorescence polarisation anisotropy (FPA) of the ethidium-DNA complex has been measured by multifrequency phase fluorometry, in order to study the perturbations induced by the presence of different ligands on the torsional dynamics of DNA, a moderately flexible polymer that undergoes bending (flexure of the helix axis) and torsional (twisting of base pairs) motions. Two probes have been used together with ethidium: an intercalator, chloroquine, and a minor groove binding dye: hoechst 33258. Chloroquine is found to substantially modify the DNA torsional dynamics both in linear and in circularly closed DNAs only at high binding ratios, in agreement with previous reports [Wu et al. Biochem. 27 (1988) 8128]. The effective elastic constant becomes approximately three times larger when the dye/base pairs binding ratio is higher than 0.14. The minor groove ligand hoechst 33258, on the other hand, greatly increases the effective elastic constant to the point that at dye/base pairs ratios larger than 0.5, the effective elastic constant becomes stiffer by several orders of magnitude, suggesting a progressive hindering of internal motions. The results reported here show that DNA torsions are more effectively influenced by groove-binding molecules than by intercalators and it is expected that the large perturbation of the former ligand may be useful when describing the change in the dynamical properties induced by DNA binding proteins. FPA in the frequency domain, the technique adopted throughout this work, has proved to be very sensitive to changes of the elastic constant that describes DNA torsional dynamics. Several computer simulations performed in order to predict the FPA time decay of intercalated ethidium have led to good agreement with the observed results.

Animals↗
PubMed1990

Plasma membrane properties of cultured local LM cell tumors and metastases from athymic (nude) mice.

Some cell surface membrane properties of tumors may depend upon the host tissue site. Therefore, local tumors and lung metastases were excised from athymic (nude) mice injected subcutaneously with LM fibroblasts. The excised local tumors and lung metastases were cultured in chemically-defined, serum-free medium to characterize their plasma membrane properties without complicating factors such as site of growth, presence of host inflammatory cells, and variation in nutrition. Plasma membranes were isolated from the cultured local tumor cells and cultured metastatic cells. The specific activities of (N+,K+)-ATPase and 5'-nucleotidase were elevated and decreased, respectively, in plasma membranes from metastatic cells as compared to local tumor cells, a finding consistent with data from directly excised local tumors and lung metastases. Plasma membranes of metastatic cells had a lower ratio of sterol/phospholipid, higher ratio of phosphatidylcholine/phosphatidylethanolamine, and no differences in phospholipid unsaturated/saturated fatty acid ratio compared to plasma membranes from the locally-derived tumor cells. Plasma membranes of metastatic cells were more fluid (lower limiting anisotropy) than those of local tumor cells as indicated by multifrequency phase and modulation fluorometry and the fluorescence probe molecule, 1,6-diphenyl-1,3,5-hexatriene. Thus, the higher fluidity of metastatic as compared to local tumor plasma membranes was not due to differences in site of growth, host cell contamination, and/or nutrition.

5'-Nucleotidase↗
PubMed1996

Formation of etheno and oxoethyl adducts in liver DNA from rats exposed subchronically to urethane in drinking water and ethanol.

Exposure of Fisher-344 male rats to 10 000 ppm of urethane in drinking water for up to 90 days or in 5% ethanol for up to 14 days caused the formation of 7-[2'-oxoethyl]guanine (OEG) and 1,N(6)-ethenoadenine (epsilon A) in liver DNA. Mild-acid DNA hydrolysates were analyzed by high-performance liquid chromatography with photodiode array detection and fluorometry. The identification of OEG and epsilon A was confirmed by coelution with the authentic standards. Forty and 67% of rats showed OEG and epsilon A adducts at 2 and 90 days of treatment with urethane in drinking water, respectively. In comparison, only 0 and 10% of rats showed adducts at 2 and 14 days of treatment with urethane in 5% ethanol, respectively. Neither OEG nor epsilon A was observed in control rats receiving water or 5% ethanol. Although these data are still preliminary, they appear to suggest that ethanol may inhibit formation of DNA adducts by urethane. Studies designed to produce more conclusive information about the role of ethanol in modifying DNA damage induced by urethane in vivo are in progress.

Adenosine↗
PubMed1988

Stimulation of muscarinic receptor in hippocampal neuron induces characteristic increase in cytosolic free Ca2+ concentration.

Changes in cytosolic free Ca2+ concentration ([Ca2+]i) in response to acetylcholine (ACh) were examined by fura-2 fluorometry in cultured rat hippocampal neurons. ACh (greater than or equal to 10(-5) M) induced an increase in [Ca2+]i composed of fast transient and slow long-lasting phases. Atropine (10(-8) M) abolished the fast component and greatly reduced the slow component. The slow component was selectively blocked by pirenzepine (10(-6) M). The effect of ACh remained partially in a Ca2+-deficient medium where effects of L-glutamate and KCl (50 mM) were abolished. Present results suggest that ACh elevates [Ca2+]i by activation of muscarinic receptor subtypes, one of which is coupled with ion channels and the other of which transduces the ACh binding to mobilization of intracellularly stored Ca2+.

Acetylcholine↗
PubMed1991

Nerve growth factor-induced intracellular calcium ion release in chick dorsal root ganglion neurons.

Nerve growth factor (NGF) rapidly infused into the extracellular medium near cell bodies of cultured chick dorsal root ganglion neurons caused transient increases in intracellular calcium ions, which was detected by fura-2 fluorometry. Similar increases were also observed in a calcium-free medium. There was typically a delay of 20 min between NGF infusion and the release of calcium ions, and this delay was longer when NGF was applied at neurite tips. This is probably because NGF must be transported from neurites to the cell body, where calcium ions are released from intracellular stores.

Animals↗
PubMed1991

Cyanide induces Ca(2+)-dependent and -independent release of glutamate from mouse brain slices.

The effect of cyanide-induced histotoxic hypoxia on endogenous glutamate release from mouse cortical, cerebellar and hippocampal slices was studied. Incubation of slices with cyanide over a 30 min period resulted in extracellular accumulation of glutamate which was decreased in the absence of Ca2+ in the incubation media. When glutamate release was continuously monitored by fluorometry, cyanide initiated a rapid release of glutamate. This initial release was found to be independent of extracellular calcium. Depolarizing concentrations of potassium chloride produced a predominantly Ca(2+)-dependent release. It is concluded that cyanide exposure induced a rapid release of endogenous glutamate mediated by both Ca(2+)-dependent and Ca(2+)-independent mechanisms.

Animals↗
PubMed1978

Preparation and properties of biologically active fluorescent heparins.

Hog mucosal heparin (N-sulfate, 0.84 mol; O-sulfate, 1.55 mol; N-acetyl, 0.12 mol; anticoagulant activity assayed by the method of U.S. Pharmacopeia, 161 USP units/mg) or its partially N-desulfated heparin (N-sulfate, 0.71 mol; O-sulfate, 1.47 mol; N-acetyl, 0.12 mol; anticoagulant activity, 117 USP units/mg/ was reacted with 5-isothiocyanatofluorescein in 0.5 M carbonate buffer (pH 8.5) at 35 degrees C for 6 h to yield the corresponding N-fluoresceinylthiocarbamoyl heparins (lambdaem 516 nm, lambdaex 491 nm; degree of substitution 0.006 and 0.013, respectively, anticoagulant activity, 174 and 140 USP units/mg, respectively). The fluorescent heparin (degree of substitution, 0.006; 174 USP units/mg) was injected into rabbits intravenously. The half-life of the fluorescent heparin determined by fluorometry was 24 min, that determined by the clotting time assay was 39 min. The time-course of concentration and the half-life of the fluorescent heparin and of the starting heparin obtained by the clotting time assay were virtually identical.

Animals↗
PubMed1993

Highly sensitive determination of imidapril, a new angiotensin I-converting enzyme inhibitor, and its active metabolite in human plasma and urine using high-performance liquid chromatography with fluorescent labelling reagent.

A method for the simultaneous determination of imidapril and its active metabolite in human plasma and urine has been developed using high-performance liquid chromatography with a fluorescent labelling reagent, 9-anthryldiazomethane. Imidapril and its active metabolite were extracted from human plasma and urine using a solid-phase extraction cartridge (Bond Elut C18). Two compounds in the eluate were derivatized with 9-anthryldiazomethane and purified with a solid-phase extraction cartridge (Bond Elut SI). The derivatives were analysed using high-performance liquid chromatography with fluorometry. The detection limits of imidapril and its active metabolite were 0.2 ng/ml in plasma and 10 ng/ml in urine. This method could be applied to the pharmacokinetic study of imidapril.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1995

Simultaneous determination of N2-(3-aminopropyl)biopterin (oncopterin), biopterin and neopterin by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic method is described for the simultaneous determination of N2-(3-aminopropyl)biopterin (oncopterin, a newly found natural pteridine in urine from cancer patients), biopterin, and neopterin in urine. For the detection and quantification of the compounds, fluorometry was used. Using Develosil ODS K-5 and Develosil ODS HG-5 reversed-phase columns and a Nucleosil 100-SSA strong cation-exchange column, oncopterin, biopterin, and neopterin in urine were completely separated and assayed simultaneously by fluorescence detection. Similar values of oncopterin were obtained using each of the three columns, and the Develosil ODS K-5 reversed-phase column gave the most satisfactory separation. The sensitivity was high enough to measure 1 pmol of each pteridine. The HPLC method was highly reproducible. Our preliminary results indicate that oncopterin could be a most sensitive marker for cancer.

Biomarkers, Tumor↗