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N-(9-Acridinyl)maleimide (NAM) reacts with sulfite in wine and gives strong fluorescent derivatives that lead to highly sensitive fluorometry of both total and bound sulfite in wine. Values of free and bound sulfite in wine determined by the NAM method and the modified Rankine method agreed. Sulfite was determined in < 200 microL wine within 2 h.
A competitive enzyme-linked immunosorbent assay (ELISA), the most frequently used immunoassay for the determination of polycyclic aromatic hydrocarbon-DNA adducts in human tissues, has been modified to achieve approximately a 6-fold increase in sensitivity. The new assay, a competitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) has utilized the same rabbit antiserum as the ELISA, antiserum elicited against DNA modified with benzo[a]pyrene. However, the alkaline phosphatase conjugate has been replaced with a biotin-europium-labeled streptavidin signal amplification system, and the release of europium into the solution forms a highly fluorescent chelate complex that is measured by time-resolved fluorometry. The DELFIA has achieved a 5- to 6-fold increase in sensitivity for measurement of DNA samples modified in vitro with benzo[a]pyrene, for cultured cells exposed to radiolabeled benzo[a]pyrene, and for human samples from occupationally exposed workers. The assay has been validated by comparison of adduct levels determined by DELFIA, ELISA, and radioactivity in DNA from mouse keratinocytes exposed to radiolabeled benzo[a]pyrene. Human lymphocyte DNA samples from 104 Hungarian aluminum plant workers were assayed by ELISA and compared to blood cell DNA samples from 69 Italian coke oven workers assayed by DELFIA. The standard curves demonstrated that the limit of detection of 4.0 adducts in 10(8) nucleotides for polycyclic aromatic hydrocarbon-DNA adducts by ELISA, using 35 micrograms of DNA/microtiter plate well, has been decreased to 1.3 adducts in 10(8) nucleotides by DELFIA, using 20 micrograms of DNA/microtiter well. If 35 micrograms of DNA were used in the DELFIA, the calculated detection limit would be 0.7 adducts in 10(8) nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)
We describe a novel assay for detection of point mutations. The method combines the specificity and sensitivity of the polymerase chain reaction (PCR) and allele-specific oligonucleotides (ASO) with highly sensitive time-resolved fluorometry. ASO probes differing by a single base substitution and labeled with europium (Eu) chelates were hybridized in solution simultaneously with a biotinylated oligomer to a PCR-amplified nucleic acid fragment. The hybrids formed were then collected onto streptavidin-coated microtitration wells. Subsequently, the hybrids were washed under stringent conditions and the remaining ASO probe was measured in a time-resolved fluorometer. We discuss the strategy underlying the design of the Eu-labeled ASO probes for the solution hybridization assay. The method was applied to the detection of the Z-mutation in the alpha 1-antitrypsin gene. Evaluation of whole-blood samples spotted on Guthrie cards demonstrated successful accuracy of the method.
A new experimental model of a vascular carrier to prefabricate a "secondary" island flap, the popliteal musculovascular pedicle, was developed in the rat. Using quantitative skin-surface fluorometry 30 minutes after sodium fluorescein injection and a flap survival area in the prefabricated 8 x 2.5-cm abdominal composite island flap, we compared the revascularization ability of our muscular carrier to nonrevascularized controls: the skeletonized arteriovenous pedicle and the fasciovascular pedicle. The free composite graft with no vascular carrier exhibited near-total necrosis. The skeletonized vascular pedicle demonstrated 15.2% +/- 7.8% perfusion of normal skin on dye fluorescence index measurements and 50% flap survival. The fasciovascular pedicle exhibited better revascularization, with a dye fluorescence index of 36.2 +/- 15.5 (p < 0.01) and 90% +/- 10% flap survival (p < 0.001). India ink injection study and histological examination of our model provided visual evidence of revascularization from the musculovascular pedicle, along with preservation of the carrier's muscular architecture. The musculovascular pedicle is a reliable carrier for making new, vascularized composite flaps.
Fluorometry of histamine and serotonin in cadaveric blood revealed a threefold increase of their concentrations in comparison with those in whole blood of live subjects. This indicates liberation of amines in the course of death and after it when factors inactivating them cease to function. However, the authors consider it possible to validate their conclusions about changes in the content of histamine and serotonin in cadaveric tissues in different diseases, provided certain conditions of selecting the material are adhered to. The distribution of amines in the viscera is not sex-dependent, little depends on the blood content of tissues, and is determined by the organ-specific features of metabolism of these bioactive substances. To illustrate this, the authors consider the content of histamine and serotonin in the organs of patients dead from hemorrhagic fever with the renal syndrome.
PURPOSE: To study the process of nonenzymatic glycosolation of vitreous collagen in vitro to determine the contributions of the classic Maillard pathway and the oxidative pathway, as well as to evaluate possible inhibitors of both pathways. METHODS: Bovine vitreous collagen was extracted and then incubated with hexoses in vitro. The amount of advanced glycosylation end (AGE) products was measured by fluorometry under varying conditions in the presence and absence of glycosolation inhibitors. Oxygen consumption studies and electron spin resonance spectroscopy with and without free-radical inhibitors were performed to differentiate oxidative from nonoxidative glycosolation. RESULTS: Vitreous collagen undergoes nonenzymatic glycosolation in the presence of glucose or galactose in vitro. Oxygen consumption data show that oxygen is consumed in glucose and galactose solutions. Oxygen consumption is decreased by known free-radical inhibitors and rutin but not aminoguanidine. Electron spin resonance spectroscopy demonstrated the presence of a carbon-centered radical, and known free-radical inhibitors decreased the carbon-centered signal. CONCLUSIONS: Nonenzymatic glycosolation of vitreous collagen can occur not only by the classic nonoxidative pathway, but also by a second oxidative pathway that is susceptible to a number of inhibitors.
OBJECTIVE: To determine whether the respiratory burst of neutrophils from bovine blood and milk can be analyzed by use of a fluorometric resazurin reduction assay. SAMPLE POPULATION: Neutrophils were obtained from EDTA-anticoagulated blood of 7 dairy cows. Neutrophils also were isolated from milk samples of a cow intramammarily challenge exposed with Escherichia coli lipopolysaccharide. PROCEDURE: The respiratory burst of neutrophils was analyzed in parallel, using the conventional luminol-enhanced luminometric procedure and a novel fluorometric procedure with resazurin as the fluorogenic substrate. Opsonized zymosan and phorbol myristate acetate were used as stimulants. The mechanism of the fluorescent response was analyzed, using metabolic inhibitors to various cell functions. Luminometry and fluorometry were carried out in parallel, using microtitration tray-reading instruments. RESULTS: Stimulation of neutrophils induced resazurin reduction to resorufin and a fluorescent response. The luminescent response was transient, but the fluorescent response (build-up of fluorescent resorufin) was cumulative. Therefore, a single end-point measurement can be used for the fluorometric assay. CONCLUSIONS: The proposed fluorometric microtitration tray technology is simple and has a high throughput capacity. The fluorometric and luminometric assays seem to have similar potential in the analysis of phagocyte functions.
Transcapillary metabolism of the retina and effects of triglycerides on the transcapillary function of the retina in diabetes mellitus were examined in 79 patients (mean age 45.4 +/- 3 years) with various stages of diabetic retinopathy (DR) by fluorometry of the vitreous body. The filtration in the blood-retina direction was 1.2-1.3 times higher than in the control as early as during the functional and preclinical stages of DR, and the coefficient of fluorescein withdrawal from the vitreous body was 4.5 to 6.2% decreased. As DR progressed, the permeability of the blood-retinal barrier in the blood-retina direction increased and the discharge from the retina to the blood was decelerated, which was confirmed by a progressive prolongation of diffusion of 0.1 ml of 1% fluorescein solution injected subconjunctivally: from 1.3 +/- 0.2 and 1.4 +/- 0.5 h in the functional and preclinical stages, respectively, to 2.7 +/- 0.3 h in the proliferative stage of DR versus 1.05 +/- 0.01 h in the control. The level of serum triglycerides and coefficients of fluorescein entry in and withdrawal from the vitreous body, time of papule diffusion, and vascular changes in the microcirculatory vessels reliably correlated.
We report a quantitative analytical methodology for prostate-specific antigen (PSA) mRNA, which is based on the coamplification of the target with a recombinant RNA internal standard (IS) using reverse transcriptase-polymerase chain reaction. PSA mRNA and the RNA IS contain the same primer recognition sites and generate amplification products that have identical sizes but differ in a 24-bp sequence located in the center of the molecule. Amplified sequences are labeled with biotin using a biotinylated upstream primer. The products are captured on streptavidin-coated microtiter wells and hybridized to specific probes labeled with the hapten digoxigenin. The hybrids are determined using alkaline phosphatase-labeled anti-digoxigenin antibody and time-resolved fluorometry. The ratio of the fluorescence values obtained for the PSA mRNA and the RNA IS is a linear function of the amount of PSA mRNA present in the sample. Samples containing total RNA from PSA-expressing cells (LNCaP cells) in addition to 1 microg of RNA from healthy cells give fluorescence ratios related linearly to the number of cells in the range of 4 to 3000 cells.
PURPOSE: The release of adenosine by the ischemic retina may be an initial signal in the development of ischemic macular edema and neovascularization. The levels of adenosine have never been quantified in ocular fluids. In this study, a technique was developed for in vivo measurement of the concentration of adenosine in aqueous and vitreous. METHODS: Aqueous and vitreous samples were obtained from bovine eyes after death and from live porcine eyes with the subject under general anesthesia. Samples from live eyes were immediately incubated in the sampling syringe with pentoxifylline, erythro-9-(2-hydroxy-3-nonyl) adenine, and dipyridamole to prevent synthesis or degradation of adenosine during the collection procedure, filtered, and flash-frozen in liquid nitrogen. All samples were then filtered and purified on phenylboronate agarose columns and incubated with chloroacetaldehyde to convert the adenosine present in the sample to the fluorescent derivative 1,N6-ethenoadenosine. The 1,N6-ethenoadenosine was separated by high-pressure liquid chromatography and then measured by fluorometry. RESULTS: Levels of adenosine as low as 0.5 pmole could be detected with this procedure, compared with 20 pmoles by UV detection. By using this technique to measure adenosine levels in the eyes of normal weanling domestic pigs, it was determined that the adenosine concentration in the aqueous was 321.3 +/- 164.9 nM and in the vitreous was 210.8 +/- 41.5 nM. CONCLUSIONS: The conversion of adenine-containing compounds to fluorescent 1,N6-etheno derivatives offers analytical advantages of selectivity and sensitivity for the quantitative determination of these compounds, with the fluorometric detection providing substantially greater sensitivity than direct detection by UV absorption. The levels obtained in vivo from anesthetized but otherwise healthy pigs presumably reflected basal aqueous and vitreous adenosine levels under the described conditions. This method should be useful in investigating more directly the role of adenosine in models of retinal or ocular ischemia in vivo and in measuring adenosine levels in vitreous or aqueous samples from human patients.
According to electron-microscopic data, various cells in the M. smegmatis ATCC607 population interact differently with phage MTPH11. Fluorometric studies of phage-host interactions were performed using a membranotropic fluorescent probe, 8-anilino-1-naphthalene sulfonate (ANS). Changes in the electric characteristics of mycobacterial cells infected with the phage were studied by electro-orientational (EO) spectroscopy. The problem of the employment of fluorometry and EO spectroscopy for rapid phage typing of mycobacteria is discussed.
Twenty-six samples of CSF were assayed for their putrescine content by three analytical techniques: total and free putrescine levels were assayed by fluorometry after separation by column chromatography and compared with putrescine levels as assayed by the enzymatic-isotopic method. Putrescine levels obtained by the enzymatic-isotopic method correlated relatively well with free putrescine levels, but grossly underestimated the results obtained for total putrescine values. The difference between total and free putrescine values is mostly due to metabolites of putrescine, such as monoacetylputrescine, which account for about 40% of the total putrescine value. The importance of the free putrescine level as a marker of CNS malignant neoplasms in comparison to that of the total putrescine level deserves investigation.
With the method of corneal redox fluorometry, the autofluorescence of reduced pyridine nucleotides (PN) and oxidized flavoproteins (Fp) in rabbit corneal endothelium was measured as a function of storage time in McCarey-Kaufman (MK) medium and K-Sol medium. Measurements were started immediately after preparation of the corneal button, and the corneas were followed up for up to three weeks of storage. In both media, the PN/Fp ratio of the endothelium initially increased slightly in the first or second day and then began to decrease toward a level lower than baseline. This initial increase is possibly a result of an adaptive mechanism. The PN/Fp ratio maintained itself in the range of baseline values up to one week in MK medium but not in K-Sol medium. With scanning electron microscopy, the surface of the membrane and cell border were maintained for a three-week preservation period, and no apparent differences were found between the corneas stored in the two media. With transmission electron microscopy, the intracellular organelles appeared almost normal through one week of preservation in corneas stored in either media. During weeks 2 and 3, however, intracellular edema with increased endothelial thickness became prominent in the corneas stored in both media. Although no visual difference in the morphological features of the endothelium was apparent between corneas stored in either medium, computer-assisted morphometric analysis showed a statistically significant increase in the coefficient of variation of mean cell area for the corneas preserved in K-Sol medium but not for those preserved in MK medium.
BACKGROUND: Vocal fold paralysis is a common cause of neonatal stridor. Although it is usually classified as idiopathic or iatrogenic in origin, a small subset of patients have a family history of this disorder, indicating a possible genetic cause. OBJECTIVE: To identify the genetic locus of the gene that causes familial laryngeal abductor paralysis. DESIGN: A standard nonorganic protocol was used to extract DNA from whole-blood samples. The DNA samples were quantified by DNA fluorometry, and the concentration of all samples was standardized at 40 ng/microL. A pooled DNA strategy was used to facilitate rapid polymerase chain reaction screening of markers in the Weber v8.0 genome screening set. Polymerase chain reaction screening of individual DNA samples was performed using possible linked markers initially identified as having an allele that appeared with a higher incidence in the affected DNA pools. Statistical analysis of possible linkage was performed using the LINKAGE 5.1 set of linkage analysis computer programs. SUBJECTS: A family in which a form of familial laryngeal abductor paralysis segregates was ascertained. Whole blood samples were drawn from 40 participating individuals within this family after the subjects' fully informed consent was obtained. RESULTS: Initial screening of the pooled DNA specimens revealed a band pattern for D6S1021 on chromosome 6q16, indicating an allele with a higher incidence in the affected vs the nonaffected pool. Two-point analysis of individual allele patterns confirmed linkage to D6S1021 with an lod score of 3.86 (straight theta = 0.0) at a penetrance value of 0.8. Haplotype analysis with flanking markers defined a 5-centiray critical region between D6S283 and AFMA047YG1. CONCLUSION: An autosomal dominant form of familial laryngeal abductor paralysis is linked to a 5-centiray region on chromosome 6q16 surrounding D6S1021.
Investigations of the effect of dimethyl sulfoxide (DMSO) on skin flap survival have generated mixed results. In addition, to our knowledge, the effect of systemic DMSO on skin flap blood perfusion has not been previously studied. For this study, 48 rats were divided into three groups: (1) a control group, (2) a group injected with DMSO postoperatively only (for seven days), and (3) a preoperatively injected group (for three days preoperatively and seven days postoperatively). The DMSO was given intraperitoneally at a dose of 1.5 g/kg. On each rat, an abdominal island flap (3 X 6 cm) was raised and restored to its original site. Laser Doppler velocimetry and perfusion fluorometry were used to monitor flap perfusion immediately following surgery (day 0) and on postoperative day 3. Flap survival was significantly greater in the DMSO-treated groups when compared with the control group. Significant increases in blood perfusion were noted in the treated flaps on day 3.
OBJECTIVES: To investigate the effects of aortic clamping and prostaglandin E1 on systemic hemodynamics and renal cortical and medullary blood flow by means of continuous intraparenchymal laser Doppler fluorometry. DESIGN: Experimental animal study in a porcine model. With the animal under general anesthesia after hemodynamic monitoring was instituted, surgical exposure was obtained through a small left retroperitoneal incision. The kidney was left undisturbed. Intraparenchymal laser Doppler probes (0.44 mm in diameter) were inserted in the renal cortex and medulla. In the first group of six animals, systemic hemodynamic variables, urine output and renal cortical and medullary flow were measured at baseline after 60 minutes of equilibration, and after 15 minutes of aortic clamping and unclamping. Data are given as mean +/- SE. INTERVENTION: In another six animals, prostaglandin E1 (20-micrograms intravenous bolus given over 1 minute) was given before clamping, and the same variables were recorded. RESULTS: In the first group, aortic clamping caused no change in cardiac output or filling pressures. Cortical blood flow decreased from 40.4 +/- 3.7 to 33.3 +/- 2.7 mL/100 g per minute (P < .0004) after clamping, and to 27 +/- 2.3 mL/100 g per minute (P < .0001) after unclamping, and was associated with a decrease in urine output from 3.2 +/- 0.5 to 2 +/- 0.2 mL/min (P < .0013). Medullary flow remained the same at 9.2 +/- 0.8, 10 +/- 0.3, and 9.8 +/- 0.6 mL/100 g per minute, respectively. These adverse effects were prevented when prostaglandin E1 was given before clamping. There was an initial drop in blood pressure (100 +/- 4 to 89 +/- 5 mm Hg, P < .0004), but cardiac output (43.3 +/- 5.8 L/min) and filling pressures (6 +/- 1 mm Hg) were unchanged. Cortical flow was preserved during the entire period of clamping and unclamping (43.3 +/- 5.8 mL/100 g per minute). Medullary flow remained unchanged (10 +/- 0.8 mL/100 g per minute). Urine output increased from 2 +/- 0.3 to 3.4 +/- 0.6 mL/min (P < .006). CONCLUSIONS: In this animal model, infrarenal aortic clamping causes a significant decrease in renal cortical flow and urine output with no significant changes in filling pressures, cardiac output, or medullary blood flow. These adverse effects are prevented by pretreatment with prostaglandin E1, which prevents cortical ischemia and maintains brisk diuresis.
The esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus is a monomeric protein with a molecular weight of about 35.5 kDa. The enzyme is barely active at room temperature, displaying the maximal enzyme activity at about 80 degrees C. We have investigated the effect of the temperature on the protein structure by Fourier-transform infrared spectroscopy. The data show that between 20 degrees C and 60 degrees C a small but significant decrease of the beta-sheet bands occurred, indicating a partial loss of beta-sheets. This finding may be surprising for a thermophilic protein and suggests the presence of a temperature-sensitive beta-sheet. The increase in temperature from 60 degrees C to 98 degrees C induced a decrease of alpha-helix and beta-sheet bands which, however, are still easily detected at 98 degrees C indicating that at this temperature some secondary structure elements of the protein remain intact. The conformational dynamics of the esterase were investigated by frequency-domain fluorometry and anisotropy decays. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. Remarkably, the tryptophanyl fluorescence emission reveals that the indolic residues remained shielded from the solvent up to 80 degrees C, as shown from the emission spectra and by acrylamide quenching experiments. The relationship between enzyme activity and protein structure is discussed.
The effects of hyaluronic acid (HA) on the proliferation and chondroitin sulfate (CS) synthesis of chondrocytes embedded in collagen gels were examined. Articular cartilage was isolated from the humerus, femur, and tibia of 21 10-week-old Japanese white rabbits. Chondrocytes isolated by collagenase digestion were embedded in type I collagen gels and cultured in Dulbecco's modified Eagle's medium (DMEM) with various doses of HA for 4 weeks. Histological and biochemical evaluations were performed at postculture weeks 1, 2, 3, and 4. For biochemical evaluations, isomers such as chondroitin 6-sulfate (delta(di)-6S) and chondroitin 4-sulfate (delta(di)-4S) synthesized by cultured chondrocytes were determined by high performance liquid chromatography (HPLC) combined with fluorometry. Morphological and histological studies demonstrated that HA-treated chondrocytes in collagen gel proliferated profusely while maintaining their phenotype. At postculture week 4, 0.1 mg/ml of HA induced an eightfold increase in cell counts compared with HA pretreatment values, or 1.5-fold more than control group. Synthesis of delta(di)-6S (delta(di)-6S content/cell) in groups treated with 0.01 and 0.1 mg/ml of HA significantly increased, while gel accumulation rates in groups treated with 0.1 and 1.0 mg/ml of HA scored significantly higher values than other groups. In collagen gel culture, HA enhanced the proliferation and delta(di)-6S synthesis of chondrocytes while maintaining their phenotype. In clinical application, since the supply of autologous chondrocytes for transplantation is not unlimited, the HA-treated culture method may be useful for increasing the number of chondrocytes and thus improving the quality of implants.