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PubMed1992

A sensitive sandwich enzyme immunoassay for gamma-seminoprotein and its application to sex discrimination of blood and bloodstains.

A sensitive sandwich enzyme immunoassay for gamma-seminoprotein (p30, prostate-specific antigen) is described for sex discrimination of blood and bloodstains. A polystyrene ball coated with rabbit anti-gamma-seminoprotein IgG was incubated with gamma-seminoprotein and, after washing, with affinity-purified rabbit anti-gamma-seminoprotein Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as hydrogen donor. The detection limit of gamma-seminoprotein was 0.15 pg per assay. Blood levels of gamma-seminoprotein, measured using 1-10 microliters of blood, were at least 3.3-fold higher in male adults than in female adults. The ratio of gamma-seminoprotein in terms of pg to hemoglobin in terms of mg was significantly higher in male adults than in female adults. Thus, the measurement of gamma-seminoprotein or both gamma-seminoprotein and hemoglobin was useful for the discrimination of blood and bloodstains of male and female adults, although with some limitations.

Adolescent↗
PubMed1993

Analysis of selenium metabolites in urine samples of minke whale (Balaenoptera Acutorostrata) using ion exchange chromatography.

1. To study the distribution of selenium metabolites in whale urine, an analytical methodology for separation and determination has been developed. 2. From whale urine, five selenium components, including trimethylselenonium ion were separated and determined by a combination of ion exchange chromatography and fluorometry. 3. The mean urinary selenium level of five minke whales was 1500 ng/ml, with a standard deviation of 400 ng/ml, i.e., about 30 times as high as that for humans.

Animals↗
PubMed1991

The use of europium (Eu3+) labelled primers in PCR amplification of specific target DNA.

The polymerase chain reaction (PCR) has many potential applications in the field of DNA probe diagnostics. Here we describe a method that utilizes PCR and time-resolved fluorometry (TRF) for the detection of specific target DNA. First the DNA segment to be detected is amplified according to standard procedures. Then a pair of europium (Eu3+) and biotin-labelled primers nested within the amplified fragment is incorporated in a few additional PCR cycles. Thus amplified DNA fragments are generated that contain an affinity label (biotin) and a detectable label (europium). The doubly-labelled amplified DNA fragments are collected onto streptavidin coated microtitration strips and the bound Eu3+ is measured in a time-resolved fluorometer. We show here the application of this method to the detection of HIV-1 DNA. As few as five copies of HIV-1 DNA could readily be detected using this assay. The method described here is sensitive, rapid and easy to employ. In addition it lends itself to automation.

Bacterial Proteins↗
PubMed1989

DNA damage and repair in female C57BL/10 mice of different ages injected with the carcinogen benzo[a]pyrene-trans-7,8-diol.

Female C57BL/10 mice 2 and 14 months of age were killed 3, 6, 9, 12, 18 and 24 h after injection with 0.4 mg of benzo[a]pyrene-trans-7,8-dihydrodiol. The amount of carcinogen bound to DNA isolated from liver and kidney of each mouse was determined as benzo[a]pyrene-7,8,9,10-tetrol liberated upon acid hydrolysis of the DNA and measured by synchronous scanning fluorometry. Considerable variability was observed and a subset of animals in the middle-aged group failed to sustain appreciable damage upon injection of the carcinogen. Nevertheless, repair of DNA-bound carcinogen from both the liver and kidney of 2-month-old animals was clearly evident. In the subset of 14-month-old animals who sustained damage, evidence for removal of DNA-bound carcinogen was marginal.

Aging↗
PubMed1993

Enhancement by captopril of bradykinin-induced calcium transients in cultured endothelial cells of the bovine aorta.

Using microscopic fluorometry and fura-2-loaded cultured bovine aortic endothelial cells, we determined the effects of captopril, an angiotensin converting enzyme (ACE) inhibitor, on bradykinin-induced Ca2+ transients in endothelial cells. In the presence of extracellular Ca2+, 10(-9) M bradykinin induced an early rise in the transients followed by sustained elevations of cytosolic calcium concentration ([Ca2+]i). Bradykinin concentration-dependently increased [Ca2+]i (EC50 6.7 x 10(-9) M). Captopril, 10(-5) M, enhanced and prolonged the bradykinin-induced Ca2+ transients and shifted the concentration-response curve to the left (EC50 8.5 x 10(-10) M). In porcine coronary aterial strips with intact endothelium, cumulative applications of bradykinin induced an endothelium-dependent relaxation during prostaglandin F2 alpha-induced contraction (EC50 = 2.0 x 10(-9) M). Treatment with 10(-5) M captopril enhanced the bradykinin-induced relaxation and shifted the concentration-response curve to the left (EC50 = 7.6 x 10(-10) M). Thus, captopril enhances the bradykinin-induced relaxation by mechanisms mainly dependent on the endothelium, namely the inhibition of ACE.

Animals↗
PubMed1993

Cholecystokinin increases intracellular Ca2+ concentration in the Human JURKAT T Lymphocyte cell line.

We have recently demonstrated the presence of specific high-affinity cholecystokinin binding sites of the central type on the Human JURKAT T Lymphocyte Cell line. In this paper, changes in intracellular Ca2+ concentration ([Ca2+]i) in response to CCK (cholecystokinin) peptides were measured in the Human JURKAT T Lymphocyte Cell line by fura-2 fluorometry. CCK-8 (the C-terminal octapeptide of CCK), the potent CCK analog Boc-[Nle28,31]CCK-7, stimulated ([Ca2+]i) mobilization in a dose-dependent manner in cells preloaded with fura-2 AM with an EC50 of 2.4 +/- 1 nM and 8 +/- 2 nM, respectively. The selective CCKB receptor agonists, namely Boc-Trp-Nle-Asp-Phe-NH2 and the cyclic analog JMV320, [formula: see text], were also potent in stimulating mobilization of [Ca2+]i with an EC50 of 32 +/- 10 nM and 25 +/- 10 nM, respectively. Compound JMV180, Boc-Tyr(SO3H)-Nle-Trp-Nle-Asp-2-phenylethyl ester, did not stimulate [Ca2+]i but inhibited the mobilization of [Ca2+]i elicited by 10 nM CCK-8 in a dose-dependent manner with an IC50 of 10 +/- 2 nM. The selective non-peptide CCKB receptor antagonist L-365,260 was more potent than the selective CCKA receptor antagonist MK-329 in inhibiting the [Ca2+]i mobilization elicited by 10 nM CCK-8 with IC50 values of 20 +/- 8 nM and 400 +/- 100 nM, respectively. These data indicated that CCK-8 and potent CCK analogs induced [Ca2+]i mobilization in the Human JURKAT T cell line through the CCKB/gastrin receptor type.

Amino Acid Sequence↗
PubMed1994

Myosin phosphorylation and Ca2+ sensitization in porcine coronary arterial smooth muscle stimulated with endothelin-1.

We examined the mechanism of endothelin-1-induced contraction in the porcine coronary artery by assessing the changes in myosin light chain phosphorylation and cytosolic Ca2+ concentration ([Ca2+]i) determined by fura-2 and front-surface fluorometry. Endothelin-1 induced a rapid phosphorylation of myosin both in the presence and in the absence of extracellular Ca2+. During the later phase of contraction (5-30 min), the tension and [Ca2+]i were maintained, while the phosphorylated myosin decreased in the presence of extracellular Ca2+. At a similar extent of [Ca2+]i elevation, endothelin-1 induced a greater tension development than did 118 mM K(+)-depolarization and this was not attributed to a greater degree of phosphorylation of myosin. In the Ca(2+)-depleted strips, endothelin-1 induced a slow and transient contraction without an increase in either [Ca2+]i or myosin phosphorylation. Therefore, in addition to the activation of mechanisms of [Ca2+]i-myosin phosphorylation, endothelin-1 may activate additional calcium regulatory mechanisms which may thus increase the Ca2+ sensitivity of contractile elements while also enhance and prolong the contractions in the porcine coronary artery.

Animals↗
PubMed1994

Characterization of YM022: its CCKB/gastrin receptor binding profile and antagonism to CCK-8-induced Ca2+ mobilization.

We investigated the antagonistic activity of (R)-1-[2,3-dihydro-1-(2'- methylphenacyl)-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl]-3- (3-methylphenyl) urea (YM022), a benzodiazepine derivative, at CCKB/gastrin receptors. This compound potently inhibited [125I]CCK-8 binding to rat brain CCKB/gastrin receptors with a Ki value of 0.26 nM, but it showed weak affinity for rat pancreas CCKA receptors (Ki = 270 nM). Selectivity for CCKB/gastrin receptors was 1000-fold greater than that for CCKA receptors. Changes in intracellular free Ca2+ concentration ([Ca2+]i) in response to CCK-8 were measured in a rat anterior pituitary cell line GH3 by fura-2 fluorometry. CCK-8 (1-100 nM) dose-dependently increased [Ca2+]i in these cells, whereas YM022 had no effect on baseline [Ca2+]i even at the highest concentration of 100 nM. YM022 inhibited the mobilization of [Ca2+]i elicited by 10 nM CCK-8 in a concentration-dependent manner with an IC50 value of 4 nM. In conclusion, YM022 is an extremely potent and highly selective antagonist of CCKB/gastrin receptors. This compound is therefore useful for studying the physiological and pharmacological roles of CCKB/gastrin receptors.

Animals↗
PubMed1995

Inhibitory effect of sevoflurane on nitric oxide release from cultured endothelial cells.

We investigated the effect of sevoflurane (fluoromethyl-2,2,2-trifluoro-1-(trifluoromethyl) ethylether) on intracellular calcium concentration ([Ca2+]i) and nitric oxide (NO) release from cultured porcine aortic endothelial cells using fura-2 fluorometry, and direct (ESR spectrometry with NO-trapping by 2-(4-carboxyphenyl)-4,4,5,5-tetramiethylimidazoline-1-oxyl 3-oxide) or indirect (nitrite accumulation measured by Greiss reaction) NO measurement. Sevoflurane alone did not change resting [Ca2+]i, but diminished bradykinin-induced transient increase in [Ca2+]i in a concentration-dependent manner. The inhibitory effect of sevoflurane on bradykinin-induced transient rise in [Ca2+]i was larger than that of a non-selective Ca2+ channel blocker (CO2+). Application of sevoflurane following bradykinin-evoked [Ca2+]i transient diminished [Ca2+]i significantly, while bradykinin B2 receptor antagonist (D-Arg-[Hyp3, Thi5,8, D-Phe7] bradykinin) or CO2+ abolished it. Sevoflurane impaired nitrite accumulation stimulated by bradykinin, and reduced the amount of NO released from endothelial cells. Our results indicate that the negative effect of sevoflurane appears to be due to the inhibition of bradykinin-induced Ca2+ efflux from endoplasmic stores and Ca2+ influx through membrane Ca2+ channels.

Analysis of Variance↗
PubMed1989

Decay time distribution analysis of Yt-base in benzene-methanol mixtures.

Frequency-domain fluorometry was used to measure intensity decays of synthetic Yt-base in mixtures of benzene-methanol at 20 degrees C. Multiexponential analysis shows that the decay of Yt-base fluorescence in benzene and methanol can be well fitted to a single-exponential model with tau = 9.67 ns and 6.25 ns respectively. In mixtures of benzene-methanol the decays became heterogeneous, and the maximum of heterogeneity observed was in a mixture containing 6% methanol. Since we expected a distribution of Yt-base solvation states in the solvent mixtures, and because the decay times of Yt-base are sensitive to solvent, we analyzed the data in terms of decay time distributions. The goodness-of-fit for the unimodal distribution model which has two floating parameters was equivalent to that found using the double exponential model with three floating parameters. The Lorentzian distribution model appears to provide a slightly superior fit relative to the Gaussian distribution model. These results suggest that the intensity decays of solvent-sensitive fluorophores in mixed solvents are described by a distribution of decay times.

Benzene↗
PubMed2004

Measurement of the affinity and cooperativity of annexin V-membrane binding under conditions of low membrane occupancy.

We developed a method for measuring the binding affinity of annexin V for phospholipid vesicles and cells at very low levels of membrane occupancy. The annexin V-117 mutant was labeled with fluorescein iodoacetamide on its single N-terminal cysteine residue; binding to phospholipid vesicles containing phosphatidylserine (PS) and 2% rhodamine-phosphatidylethanolamine was measured by fluorescence quenching due to resonance energy transfer; binding to cells with exposed PS was measured by fluorometry after elution of bound protein. The equilibrium constant was calculated as a function of the midpoint of the calcium titration curve, the Hill coefficient, and the concentration of membrane binding sites. Calcium titrations at very low ratios of protein to membrane revealed Hill coefficients of approximately 8 for both vesicles and cells, far higher than previously measured, but as the protein-membrane ratio was increased above 3% of maximum membrane occupancy, the value of the Hill coefficient progressively decreased to a limiting value of about 2. High Hill coefficients were also observed for measurements performed at different ionic strengths and with membrane PS content varied over the range from 20 to 50%. This method allows the accurate determination of the affinity and cooperativity of annexin V-membrane binding and will be useful for the evaluation of modified annexin V derivatives intended for diagnostic and therapeutic applications.

Annexin A5↗
PubMed2005

Chloroperoxidase-catalyzed oxidation of 4,6-dimethyldibenzothiophene as dimer complexes: evidence for kinetic cooperativity.

A sigmoidal kinetic behavior of chloroperoxidase for the oxidation of 4,6-dimethyldibenzothiophene (4,6-DMDBT) in water-miscible organic solvent is for the first time reported. Kinetics of 4,6-DMDBT oxidation showed a cooperative profile probably due to the capacity of chloroperoxidase to recognize a substrate dimer (pi-pi dimer) in its active site. Experimental evidence is given for dimer formation and its presence in the active site of chloroperoxidase. The kinetic data were adjusted for a binding site able to interact with either monomer or dimer substrates, producing a cooperative model describing a one-site binding of two related species. Determination of kinetics constants by iterative calculations of possible oxidation paths of 4,6-DMDBT suggests that kinetics oxidation of dimer substrate is preferred when compared to monomer oxidation. Steady-state fluorometry of substrate in the absence and presence of chloroperoxidase, described by the spectral center of mass, supports this last conclusion.

Catalysis↗
PubMed2006

Salivary proteinase activity: a potential biomarker for preterm premature rupture of the membranes.

OBJECTIVE: The objective of this study was to design a method to identify patients at risk for preterm premature rupture of the membranes using a simple assay of salivary proteinase activity. STUDY DESIGN: Saliva samples were collected from women in the following groups using Salivette: (1) nonpregnant control; (2) during the second trimester of pregnancy; (3) during active labor at term; (4) women with premature rupture of the membranes before preterm delivery; and (5) postpartum (within 3 hours after delivery at term). Total proteolytic activity in saliva samples was measured by fluorometry using the generic substrate DQ-gelatin in the presence of specific inhibitors to selectively detect matrix metalloproteinase activities. The concentrations of various matrix metalloproteinases in saliva samples were also measured by multiplex bead assay using the Luminex platform. RESULTS: All saliva samples exhibited detectable matrix metalloproteinase activity. Salivary matrix metalloproteinase activity is low in nonpregnant females (0.27 +/- 0.15) and increases in samples taken in the second trimester (0.5 +/- 0.5) and at term during active labor (1.03 +/- 1.2). Samples collected from women with premature rupture of the membranes before preterm delivery had the highest activity (2.5 +/- 3.7) followed by postpartum after normal term delivery (2.1 +/- 1.6). The matrix metalloproteinase activity was higher in premature rupture of the membranes before preterm delivery samples, compared with all other stages of pregnancy. Multiplex matrix metalloproteinase assay documented a significant increase in total matrix metalloproteinase-9 concentration in saliva from premature rupture of the membranes before preterm delivery, compared with any of the other groups. Similarly matrix metalloproteinase-9 activity was also significantly increased in premature rupture of the membranes before preterm delivery group, compared with all others. CONCLUSION: Herein we report a simple test to monitor proteolytic enzyme activity in saliva during pregnancy. The highest matrix metalloproteinase activity is seen in premature rupture of the membranes before preterm delivery and postpartum samples. Ongoing studies aim to further define salivary proteinase activity in patients at high risk for premature rupture of the membranes before preterm delivery and to evaluate its potential as a predictive test for premature rupture of the membranes before preterm delivery.

Biomarkers↗
PubMed2004

Correlation between lifetime heterogeneity and kinetics heterogeneity during chlorophyll fluorescence induction in leaves: 1. Mono-frequency phase and modulation analysis reveals a conformational change of a PSII pigment complex during the IP thermal phase.

The relationship between the fluorescence lifetime (tau) and yield (Phi) obtained in phase and modulation fluorometry at 54 MHz during the chlorophyll fluorescence induction in dark-adapted leaves under low actinic light has been investigated. Three typical phases have been identified: (i) linear during the OI photochemical rise, (ii) convex curvature during the subsequent IP thermal rise, and (iii) linear during the PS slow decay. A similar relationship has been obtained in the fluorescence induction for the fluorescence yield measured at 685 nm plotted versus the fluorescence yield measured at 735 nm. A spectrally resolved analysis shows that the curvature of the tau-Phi relationship is not due to chlorophyll fluorescence reabsorption effects. Several other hypotheses are discussed and we conclude that the curvature of the tau-Phi relationship is due to a variable and transitory nonphotochemical quenching. We tentatively propose that this quenching results from a conformational change of a pigment-protein complex of Photosystem II core antenna during the IP phase and could explain both spectral and temporal transitory changes of the fluorescence. A variable blue shift of the 685 nm peak of the fluorescence spectrum during the IP phase has been observed, supporting this hypothesis.

Adaptation, Physiological↗
PubMed2004

Correlation between lifetime heterogeneity and kinetics heterogeneity during chlorophyll fluorescence induction in leaves: 2. Multi-frequency phase and modulation analysis evidences a loosely connected PSII pigment-protein complex.

We report the first direct decomposition of the fluorescence lifetime heterogeneity during multiphasic fluorescence induction in dark-adapted leaves by multi-frequency phase and modulation fluorometry (PMF). A very fast component, assigned to photosystem I (PSI), was found to be constant in lifetime and yield, whereas the two slow components, which are strongly affected by the closure of the reaction centers by light, were assigned to PSII. Based on a modified "reversible radical pair" kinetic model with three compartments, we showed that a loosely connected pigment complex, which is assumed to be the CP47 complex, plays a specific role with respect to the structure and function of the PSII: (i) it explains the heterogeneity of PSII fluorescence lifetime as a compartmentation of excitation energy in the antenna, (ii) it is the site of a conformational change in the first second of illumination, and (iii) it is involved in the mechanisms of nonphotochemical quenching (NPQ). On the basis of the multi-frequency PMF analysis, we reconciled two apparently antagonistic aspects of chlorophyll a fluorescence in vivo: it is heterogeneous with respect to the kinetic structure (several lifetime components) and homogeneous with respect to average quantities (quasi-linear mean tau-Phi relationship).

Adaptation, Physiological↗
PubMed2004

Identification and characterization of novel endogenous proteolytic forms of the human angiogenesis inhibitors restin and endostatin.

Restin and endostatin are C-terminal fragments of the noncollagenous domains of collagen XV and collagen XVIII exhibiting high sequence homology. Both polypeptides are distinguished by strong anti-angiogenic activity in vivo restricting the growth of solid tumors and metastasis. They are therefore currently being tested in clinical trials as anti-cancer drugs. We present the identification of new endogenous variants of both angiogenesis inhibitors isolated from a human hemofiltrate peptide library. Using an immunological screening approach with time-resolved rare earth metal fluorometry, immunoreactive compounds were purified chromatographically and characterized by mass spectrometry. We discovered four novel proteolytic products of restin as well as four variants of endostatin. Two endostatin products were characterized as short internal fragments (R176-L215 and R176-S219) of the entire molecule containing the recently identified beta1 integrin receptor binding site, which plays a major role in endothelial cell migration and angiogenesis. Two additional forms contain mucin-type O-glycosylations. The O-glycosylated variants possess an oligosaccharide unit consisting of one N-acetylgalactosamine (GalNAc), one N-acetylneuraminic acid (NANA) and two galactose residues (Gal) occurring as sialo-(V117-S311-GalNAc-Gal2-NANA) and asialoglycopeptides (V117-S311-GalNAc-Gal2). The four restin variants (R(I)-R(IV)) were identified with identical C- but different N-termini and no posttranslational modification (R(I): P66-A254, R(II): P75-A254, R(III): Y81-A254 and R(IV): A89-A254). Following a differential peptide mass fingerprint approach by reflector mode MALDI-TOFMS, the disulfide patterns of these circulating restins were determined as Cys1-Cys4 and Cys2-Cys3. These endogenous circulating collagen fragments will help to understand the physiological processing of the therapeutic proteins.

Amino Acid Sequence↗
PubMed2005

Inhibition of the transcription factor Yin Yang 1 activity by S-nitrosation.

Treatment of several prostate cancer (CaP) cell lines (PC-3, CL-1, and DU-145) with the nitric oxide (NO) donor DETA/NONOate upregulated Fas expression and sensitized the CaP cells to the Fas ligand CH-11 agonist monoclonal antibody-induced apoptosis. Previous findings demonstrated that the transcription repressor Yin Yang 1 (YY1), which is inhibited by NO, negatively regulates Fas transcription [H.J. Garban, B. Bonavida, Nitric oxide inhibits the transcription repressor Yin-Yang 1 binding activity at the silencer region of the Fas promoter: a pivotal role for nitric oxide in the upregulation of Fas gene expression in human tumor cells, J. Immunol. 167 (2001) 75-81]. YY1 is a zinc finger protein and thus, we hypothesized that NO inhibits YY1 activity via S-nitrosation of critical cysteines residues coordinated by Zn2+. Treatment of PC-3 cells with DETA/NONOate inhibited the constitutive DNA-binding activity of YY1 as assessed by EMSA. Further, treatment with DETA/NONOate resulted in S-nitrosation of YY1 as detected by two different methods. The DAN-based method examined NO-treated tumor-derived cell lysates that were immunoprecipitated with an anti-YY1 specific antibody and the NO released was determined quantitatively by fluorometry. The second method consisted of immunoprecipitation of the tumor cell lysates by an anti-SNO cysteine antibody and the immunoprecipitate was immunoblotted with anti-YY1 antibody. Both methods revealed significant S-nitrosation of YY1 by DETA/NONOate treatment over control untreated cells. The S-nitrosation of YY1 was further corroborated by immunohistochemistry using dual color immunofluorescence. The direct role of YY1 in the negative regulation of Fas expression was demonstrated by transfection of cells with siRNA YY1. The transfectants exhibited upregulation of Fas expression in the absence of treatment with DETA/NONOate and were sensitized to CH-11-induced apoptosis. Altogether, these findings reveal that NO inhibits YY1 DNA-binding activity through S-nitrosation and consequently results in upregulation of Fas expression and tumor cell sensitization to Fas-induced apoptosis.

Apoptosis↗
PubMed2006

Identification of a novel inactivating R465Q mutation of the calcium-sensing receptor.

In this study, we describe a 52-year-old woman, who was diagnosed with familial benign hypocalciuric hypercalcemia (FBHH), a condition characterized by hypercalcemia, low urinary calcium excretion, and normal parathyroid hormone PTH levels, resulting from inactivating mutations of the calcium-sensing receptor (CaSR). In order to identify and characterize the underlying mutation in the CASR gene, direct sequence analysis of CASR exons 2-7 was performed, and functional activity was examined by transient transfection of human embryonic kidney (HEK-293) cells with wild-type and mutant CaSRs, followed by intracellular calcium measurement using fluorometry, and Western blot analysis. Sequence analysis demonstrated, in addition to the already described A986S polymorphism, a novel heterozygous G--> A substitution in CASR exon 5 that causes an arginine to glutamine substitution at codon 465 (R465Q). Functional analysis showed a rightward shift of the dose-response curve with a significant increase of the EC50 from 5.4 mM of the CaSR carrying the A986S polymorphism alone to 11.3 mM of the CaSR carrying the R465Q mutation in the presence of the A986S polymorphism. Western blot analysis of membrane protein revealed an even higher expression level of the R465Q mutant protein compared to wild-type CaSR. In conclusion, we identified a novel heterozygous loss-of-function R465Q mutation of the CASR gene, which is characterized by a blunted response to calcium stimulation, thereby causing FBHH.

Blotting, Western↗