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PubMed2003

Cardiac fibrosis occurs early and involves endothelin and AT-1 receptors in hypertension due to endogenous angiotensin II.

OBJECTIVES: We investigated if endothelin (ET)-1 and the renin-angiotensin-aldosterone system play a role in cardiac fibrosis. BACKGROUND: Angiotensin II (Ang II) can induce cardiac fibrosis, but the underlying mechanisms are incompletely understood. METHODS: Four-week-old transgenic (mRen2)27 rat (TGRen2) received for four weeks a placebo, the mixed ET(A)/ET(B) endothelin receptor antagonist bosentan, the angiotensin II type I receptor (AT-1) antagonist irbesartan, the ET(A) endothelin receptor antagonist BMS-182874, and a combined treatment with irbesartan plus BMS-182874. We measured collagen density on Sirius red-stained serial sections of the left ventricle (LV) with a photomicroscope equipped with specific software and assessed the gene expression of procollagen alpha1(I), atrial natriuretic peptide (ANP), transforming growth factor-beta 1 (TGFbeta1), endothelin converting enzyme, and ET(B) receptor. RESULTS: In the placebo group, hypertension was associated with LV hypertrophy and cardiac fibrosis (LV weight: 4.0 +/- 0.3 mg/g body weight; collagen density: 2.21 +/- 0.16%), which were all prevented with irbesartan (2.3 +/- 0.1, 1.30 +/- 0.13, p < 0.001), but not with BMS-182874 (4.0 +/- 0.2, 2.41 +/- 0.22). Bosentan also prevented fibrosis (1.39 +/- 0.18) but not hypertension and LV hypertrophy (3.38 +/- 0.27). Combined irbesartan and BMS-182874 treatment prevented LV hypertrophy (2.9 +/- 0.1) but not fibrosis (2.52 +/- 0.16). Collagen density correlated (r = 0.414, p < 0.05) with plasma aldosterone levels. In TGRen2 with LV hypertrophy, the gene expression of ANP and ET(B) but not that of TGFbeta1 and procollagen alpha1(I) was increased. CONCLUSIONS: In Ang II-dependent hypertension, cardiac fibrosis was associated with LV hypertrophy and was hindered by both mixed ET(A)/ET(B) blockade and AT-1 blockade. Only the latter treatment prevented both hypertension and LV hypertrophy. Thus, there is a dissociation between the mechanisms of cardiac fibrosis and hypertension, which do and do not entail ET-1, respectively.

Angiotensin II↗
PubMed1985

Thiol-specific probes indicate that the beta-chain of platelet glycoprotein Ib is a transmembrane protein with a reactive endofacial sulfhydryl group.

We used two membrane-permeable fluorescent reagents, monobromobimane and N-[[5-(dimethylamino)-1-naphthalenyl]sulfonyl]aziridine (N-dansylaziridine), and one membrane-impermeable fluorescent probe, monobromo(trimethylammonio)bimane, all three of which react selectively with protein thiols, to assess the presence of reactive sulfhydryls in the platelet glycoprotein Ib (GPIb) molecule and establish the topology of any GPIb-reactive thiols in the platelet membrane. Intact platelets were reacted with 1-10 mM monobromobimane or monobromo(trimethylammonio)bimane or 50-100 microM N-dansylaziridine for 30-60 min at 37 degree C. The platelets were then washed, solubilized in 1% Triton X-100, and analyzed by nonreduced-reduced polyacrylamide gel electrophoresis either directly or indirectly after immunopurification of GPIb. Monobromobimane and N-dansylaziridine labeled GPIb beta but not GPIb alpha in intact platelets. This labeling could be inhibited by pretreating the platelets with either N-ethylmalemide or p-(chloromercuri)benzenesulfonic acid, confirming the specificity of these probes for thiol groups. Monobromo(trimethylammonio)bimane, the membrane-impermeable reagent, did not label GPIb beta in intact platelets. However, it did label GPIb beta in sonicated platelets, indicating that the thiol group of GPIb beta occupies an intracellular location. Since the carbohydrate moiety of GPIb beta can be labeled from the outside of intact platelets with membrane-impermeable reagents, we conclude that GPIb beta has a transmembrane orientation.

Aziridines↗
PubMed1992

Isolation of transglutaminase-reactive sequences from complex biological systems: a prominent lysine donor sequence in bovine lens.

The transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13)-catalyzed cross-linking of proteins in biological systems can often be inhibited by inclusion of small primary amines or glutamine-containing peptides, which act as site-specific blockers of the relevant acceptor (i.e., glutamine) and donor (i.e., lysine) functionalities of the natural substrates. Compounds such as dansylcadaverine and dansyl-epsilon-aminocaproyl-Gln-Gln-Ile-Val are particularly useful in sorting out acceptor-donor relationships among lens crystallins. Apart from its fluorescent properties, the dansyl hapten offered special advantages as a "handle" for the rapid isolation of transglutaminase targets even in the complex system of lens cortical homogenate. The dansylated peptide was incorporated into bovine lens proteins under the influence of the Ca(2+)-activated intrinsic transglutaminase and, after digestion by endoproteinase Glu-C, the tracer-containing fragments were isolated by affinity chromatography on an anti-dansyl antibody column. The major fluorescent peak was isolated by HPLC and sequenced by Edman degradation, which yielded phenylthiohydantoin amino acid derivatives for the first 10 cycles, EKPAVTAAPK, and none for the next 2. The sequence, corresponding to residues 165-174 of alpha B-crystallin, unambiguously identifies the known carboxyl-terminal domain, EK-PAVTAAPKK, as the prominent lysine-donating fragment in bovine lens.

Amino Acid Sequence↗
PubMed1995

Subcutaneous injection of an analog of neuropeptide FF prevents naloxone-precipitated morphine abstinence syndrome.

There is evidence that neuropeptide FF (NPFF) has antiopiate activity and may play a role in opiate dependence and subsequent abstinence syndrome. A fragment of NPFF was modified at the C-terminal in an effort to convert it to an NPFF antagonist. It was also dansylated at the N-terminal in an effort to render it more lipophilic and increase its penetration of the blood-brain barrier. Third ventricle administration of the resulting compound, dansyl-PQRamide (0.75 microgram and 1 microgram), dose-dependently antagonized the quasi-morphine abstinence activity of NPFF (10 micrograms) in opiate-naive rats. Subcutaneous injection of dansyl-PQRamide (13 mg/kg) in chronically morphine-infused rats attenuated opiate dependence as indicated by prevention of naloxone-precipitated abstinence syndrome. Dansyl-PQRamide displaced radiolabelled ligand from NPFF receptors in a concentration-dependent manner with a Ki of 13 microM, and had a half-life over 300 times longer than NPFF under aminopeptidase digestion.

Animals↗
PubMed1994

Determination of conjugated and unconjugated serum 3 alpha-OH bile acids by high-performance liquid chromatography.

A simple reverse-phase liquid chromatographic method has been developed for the quantitative measurement of 11 3 alpha-OH bile acids in paediatric serum samples. Bile acids are enzymatically reduced to the corresponding 3-keto compound and then derivatized with the fluorophore dansyl hydrazine. Preliminary fractionation of bile acids is not required. The limit of detection is 0.1 mumol/L using a sample size of 200 microL. One hundred and twenty-three serum samples were analysed by the high-performance liquid chromatography (HPLC) method and the results compared with a commercial kit method (Enzabile) presently used in many laboratories for the measurement of total bile acids. There was a good correlation between the two methods (r = 0.96). The method is suitable for use in a clinical laboratory for the further investigation of those patients with abnormally high total bile acid levels where quantification of bile acid fractions is required.

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

A chromogenic enzymatic assay capable of detecting prourokinase-like material in plasma.

We report a functional assay capable of quantifying prourokinase (ProUK)- like material in plasma where urokinase (UK) is also present. The assay involves inactivation of urokinase with a specific, active site directed irreversible inhibitor, dansyl-glutamyl glycyl arginine chloromethylketone (dansyl- GGACK). Excess inhibitor is subsequently quenched with dithiothreitol (DTT). The ProUK-like material in plasma is then converted to active urokinase with thermolysin, a proteolytic enzyme of bacterial origin. Alpha 2-macroglobulin in plasma inhibits thermolysin; however alpha 2-macroglobulin is inactivated with methylamine. The assay can detect as little as 20 ng of ProUK and is linear from 20 to 120 ng. The assay was applied to quantify the amount of ProUK-like material in plasma obtained from dog at various times after i.v. administration of 100,000 or 75,000 U/kg, of pro-urokinase.

Amino Acid Chloromethyl Ketones↗
PubMed2004

Estimation of helix-helix association free energy from partial unfolding of bacterioopsin.

To obtain thermodynamic information about interactions between transmembrane helices in integral membrane proteins, partial unfolding of bacterioopsin in ethanol/water mixtures was studied by Förster-type resonance energy transfer (FRET) from tryptophan to a dansyl group on Lys 41. Tryptophan to dansyl FRET was detected by measuring sensitized emission at 490-500 nm from 285 nm excitation. FRET was observed in dansylbacterioopsin in apomembranes and in detergent micelles but not in 90% ethanol/water or in the chymotrypsin fragment C2 (residues 1-71). The main fluorescence donors are Trp 86 and Trp 182. Increase of FRET from C2 with added chymotrypsin fragment C1 (residues 72-248) provides an estimate of the C1-C2 association constant as 7.7 x 10(6) M(-1). With increasing ethanol concentration, the FRET signal from dansylbacterioopsin in detergent micelles disappeared with a sharp transition above 60% ethanol. No transition occurred in Trp fluorescence from bacterioopsin lacking the dansyl acceptor, nor did dansyl model compounds undergo a similar transition. Light scattering measurements show that the detergent micelles dissipate below 50% ethanol. Thus the observed transition is likely to be a partial unfolding of bacterioopsin. Assuming a two-state unfolding model, the free energy of unfolding was obtained by extrapolation as 9.0 kcal/mol. The slope of the transition (m-value) was -0.8 kcal mol(-1) M(-1). The unfolding process probably involves dissociation of several helices. The rate of association was measured by stopped-flow fluorometry. Two first-order kinetic processes were observed, having approximately equal weights, with rate constants of 2.32 s (-1) and 0.185 s(-1).

Bacteriorhodopsins↗
PubMed1975

Distribution of end-to-end distances of oligopeptides in solution as estimated by energy transfer.

A homologous series of oligopeptides each containing at its ends a donor and an acceptor of electronic excitation energy was synthesized by the solid-phase method. N-5-(2-Hydroxyethyl)-L-glutamine was the repeating unit, and peptides containing 4, 5, 6, 7, 8, and 9 of these amino-acid residues were prepared. The chromophores naphthalene and dansyl, which were used as donor and acceptor, respectively, fulfil the conditions necessary for energy transfer according to the Förster mechanism. A distance corresponding to 50% efficiency of energy transfer, tro = 22 plus or minus 1 A, was calculated. The kinetics of fluorescence decay of an oligomer containing the naphthalene chromophore only could be described precisely by a monoexponential function. In contrast, the kinetics of the decay curves of the fluorescence of the donor of all of the oligomers containing both donor and acceptor, as measured in viscous solution, deviated markedly from monoexponential behavior. The deviation was interpreted in terms of the great number of different conformations that the various molecules of each of the oligomers attain in solution, leading to characteristic end-to-end distribution functions between the donor and acceptor. Numerical adjustment of the parameters of some of the previously proposed expressions to describe the end-to-end distribution enabled the reconstruction of the kinetics of the fluorescence decay of the donor with great precision. The end-to-end distribution functions for the various oligopeptides were thus evaluated.

Alanine↗
PubMed1998

Membrane type-1 matrix metalloprotease and stromelysin-3 cleave more efficiently synthetic substrates containing unusual amino acids in their P1' positions.

The influence of the substrate P1' position on the specificity of two zinc matrix metalloproteases, membrane type-1 matrix metalloprotease (MT1-MMP) and stromelysin-3 (ST3), was evaluated by synthesizing a series of fluorogenic substrates of general formula dansyl-Pro-Leu-Ala-Xaa-Trp-Ala-Arg-NH2, where Xaa in the P1' position represents unusual amino acids containing either long arylalkyl or alkyl side chains. Our data demonstrate that both MT1-MMP and ST3 cleave substrates containing in their P1' position unusual amino acids with extremely long side chains more efficiently than the corresponding substrates with natural phenylalanine or leucine amino acids. In this series of substrates, the replacement of leucine by S-para-methoxybenzyl cysteine increased the kcat/Km ratio by a factor of 37 for MT1-MMP and 9 for ST3. The substrate with a S-para-methoxybenzyl cysteine residue in the P1' position displayed a kcat/Km value of 1.59 10(6) M-1 s-1 and 1.67 10(4) M-1 s-1, when assayed with MT1-MMP and ST3, respectively. This substrate is thus one of the most rapidly hydrolyzed substrates so far reported for matrixins, and is the first synthetic peptide efficiently cleaved by ST3. These unexpected results for these two matrixins suggest that extracellular proteins may be cleaved by matrixins at sites containing amino acids with unusual long side chains, like those generated in vivo by some post-translational modifications.

Amino Acids↗
PubMed1997

Antithrombotic efficacy of inactivated active site recombinant factor VIIa is shear dependent in human blood.

Several studies have indicated a profound role for factor VII(a) [FVII(a)] in venous and arterial thrombogenesis. In the present study, we quantified the inhibitory efficacy of dansyl-glutamyl-glycyl-arginyl-recombinant FVIIa (DEGR-rFVIIa) on acute thrombus formation. Thrombus formation was elicited by immobilized tissue factor (TF) in a parallel-plate perfusion chamber device at blood flow conditions characterized by wall shear rates of 100 S-1 (veins) and 650 S-1 (medium-sized healthy arteries). Native human blood was drawn directly from an antecubital vein by a pump into a heparin-coated mixing device in which DEGR-rFVIIa (0.09 to 880 nmol/L final plasma concentration) or buffer was mixed homogeneously with flowing blood. Subsequently, the blood was passed over a plastic coverslip coated with TF and phospholipids in the parallel-plate perfusion chamber. Fibrin deposition, platelet-fibrin adhesion, and platelet thrombus volume triggered by this surface were measured by morphometry. DEGR-rFVIIa inhibited thrombus formation in a dose-dependent manner, but the efficacy was shear rate dependent. At a wall shear rate of 100 S-1, the IC50 (50% inhibition) was 30 nmol/L, whereas at 650 S-1, the IC50 was 0.6 nmol/L. Binding studies to immobilized TF under flow conditions using surface plasmon resonance revealed a significantly higher on-rate for DEGR-rFVIIa and FVIIa than for FVII, 2.8 x 10(5), 2.6 x 10(5), and 1.8 x 10(5) M-1 S-1, respectively. This indicates that a contributing factor to the shear-dependent efficacy may be a differential importance of on-rates at arterial and venous blood flow conditions.

Binding Sites↗
PubMed1981

Fluorescence and N.M.R. studies of the binding of cholinergic fluorescent probes to horse serum cholinesterase.

The interaction of the cholinergic fluorescent probes, 1-(5-dimethyl-aminoaphthalene-1-sulfonamido) ethane-2-trimethylammonium perchlorate, 1-(5-dimethylaminoaphthalene-1-sulfonamido) pentane-5-trimethylammonium tartarate and 1-(5-dimethylaminonaphthalene-1-sulfonamido) decane-10- trimethylammonium tartarate with horse serum cholinesterase has been examined by fluorescence and n.m.r. methods. Fluorescence titrations show binding of the decane derivative to two sites on the protein whereas the lower homologs bind largely to one site. Active site inhibitors like carbamylcholine and decamethonium abolish binding of the decane derivative to the high affinity site. The inhibitors are largely without effect on the binding of the lower homologs. N.m.r. studies clearly establish immobilization of both ends of the molecule on binding in the case of the decane derivative, whereas in the lower homologs the dimethylamino group on the naphthalene ring is significantly more affected in the presence of enzyme. The probes are effective inhibitors of the enzyme with the decane derivative being two orders of magnitude more effective than its lower homologs. Based on the n.m.r., fluorescence and inhibition studies, a model for probe binding to the enzyme is advanced. It appears that the decane derivative binds with high affinity to the catalytic anionic site while the lower affinity site is assigned to a peripheral anionic site. The lower homologs probe only the peripheral site. A comparison of fluorescence, n.m.r. and inhibition studies with acetylcholinesterases from electric eel and bovine erythrocytes is presented.

Animals↗
PubMed2000

Blockade of angiotensin II type 1 receptor and not of endothelin receptor prevents hypertension and cardiovascular disease in transgenic (mREN2)27 rats via adrenocortical steroid-independent mechanisms.

We investigated the role of angiotensin II (Ang II) and endothelin-1 (ET-1) in transgenic (mREN2)27 rats, a model of the monogenic renin-dependent form of severe hypertension and cardiovascular disease. Four-week-old heterozygous male transgenic (mREN2)27 rats (n=24) were matched according to body weight (BW) and blood pressure (BP) and randomly allocated to receive a placebo (group P), the mixed endothelin type A and B receptor antagonist bosentan (100 mg/kg BW PO, group B), the Ang II type 1-specific receptor antagonist irbesartan (50 mg/kg BW PO, group I), or the endothelin type A-selective antagonist BMS-182874 (52 mg/kg BW PO, group BMS). After 4 weeks of treatment, during which BW and BP were measured weekly, animals were euthanized, and the heart, left ventricle, right ventricle, adrenal gland, brain, and kidney were weighed. The plasma levels of adrenocortical steroids were measured by high-performance liquid chromatography. The tension responses of ET-free segments of the thoracic aorta to 5 x 10(-6) mmol/L phenylephrine, 60 mmol/L KCl, and cumulative doses of ET-1 were assessed. The density of ET-1 receptor subtypes in the aorta and vascular structural changes in the mesenteric arterioles (100 to 200 microm ID) were also measured with autoradiography and myography, respectively. Compared with all other groups, group I rats showed significantly (P<0.001) lower systolic BP (group I, 161+/-8 mm Hg; group P, 269+/-23 mm Hg; group B, 275+/-17 mm Hg; and group BMS, 254+/-21 mm Hg), left ventricular weight (2.28+/-0.15 versus 3. 71+/-0.26, 3.38+/-0.27, and 3.96+/-0.51 mg/g BW, respectively), tension responses to vasoconstrictors, and normalized media thickness of the mesenteric arterioles (22.3+/-0.6 versus 25.3+/-0.5, 25.5+/-0.7, and 24.1+/-1.5 microm, respectively). Compared with levels in group P (78+/-25 pmol/mL), plasma aldosterone levels were significantly decreased in group B (51+/-11 pmol/mL) and group I (40+/-16 pmol/mL). Thus, endogenous ET-1 and Ang II contribute to the regulation of aldosterone, but only Ang II is crucial for the development of hypertension and related target organ damage via the Ang II type 1 receptor. Endogenous Ang II does not appear to enhance cardiovascular production of ET-1 in this model of hypertension within the time span of our experiment.

Adrenal Cortex Hormones↗
PubMed1976

Influence of various fluorescent and non-fluorescent labels on the kinetics of the complex formation of alpha-chymotrypsin with basic pancreatic trypsin inhibitor (Kunitz).

The association of alpha-chymotrypsin with basic pancreatic trypsin inhibitor was studied using extrinsic signals produced by fluorescent and nonfluorescent labels. The reactive dyes were covalently bound to the proteins in the complexed state, in which the binding region was protected. The signals were sufficiently large to measure the complex formation at protein concentrations of 10(-9)M by fluorescence and down to 10(-6)M by absorption. Therefore, the association and dissociation could be followed over a broad range of concentration. Good correspondence was observed between data which were obtained with different labels and with published values for the unlabeled proteins. Existing differences could be explained by different buffer conditions used by the different authors. Also the pH dependence of the dissociation rate constants was essentially unaltered by the introduction of the labels. The large signals allowed a direct measurement of the equilibrium constants of dissociation, even at high pH, at which they are in the range of 10(-8)M. The experimentally determined binding constants were in agreement with those calculated from the rate constants. The temperature dependence of the binding constants revealed a small positive and pH-dependent enthalpy change [deltaHo = 4.0 kcal/mol (16.7 kJ) at H 7.0[. The results prove that the labeling can be performed in such a way that the equilibrium and kinetic parameters of the system studied are not significantly influenced.

Affinity Labels↗
PubMedDate not supplied

Fluorescent labeling of drugs and simple organic compounds containing amine functional groups, utilizing dansyl chloride in Na(2)CO(3) buffer.

Fluorescent labeling of amine functional groups using dansyl chloride (DNS-Cl), and in sodium carbonate buffer, allowed the detection of 1 microg amounts of analytes. The methodology presented allows dansylation of primary, secondary, and tertiary amine groups at a temperature of 25 degrees C. The dansylation of tertiary amines involves a chemical reaction which removes one substituent (or branch) of the amine group. A one molar working concentration of Na(2)CO(3) is used, and is at pH 11.0. Compounds such as isopropylamine, dipropylamine, diethylamine, triethylamine, triisooctylamine, and N,N-dimethylaniline were labeled by use of DNS-Cl from samples obtained from a complex mixture of alkanes. The compound p-chloroaniline contains a primary amine group and is a solid at 25 degrees, quickly dissolves in the one molar sodium carbonate buffer and is dansylated in 15 min. Heroin, which contains a tertiary amine group, was extracted into ethyl acetate from an aqueous solution, then reacted with DNS-Cl. Benzocaine, a local anesthetic, was dansylated in 15 min. Tertiary amine groups incorporated in a rigid ring system, such as for caffeine, strychnine, and the ionic salt form of cocaine hydrochloride did not react with DNS-Cl under these conditions. The reaction time for tertiary amines was 2 h or less, and 15 min for compounds having primary and secondary amine groups. Separation of the dansylated compounds from unreacted DNS-Cl was accomplished by diethyl ether extraction of the aqueous reaction solution, followed by thin layer chromatography using various organic solvents such as acetone and methylene chloride.

Amines↗
PubMedDate not supplied

Development of a chemiluminescence detection system using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate for the sensitive determination of the fluorescent compounds separated with an acidic mobile phase.

A rotating flow mixing device having three directional inlets was developed for a high performance liquid chromatographic peroxyoxalate chemiluminescence detection system for the sensitive detection of fluorescent compounds separated with acidic mobile phases, such as those containing trifluoroacetic acid. The diameters of the three inlets were 0.3, 0.3 and 0.5 mm i.d., for an acidic column eluate, chemiluminogenic reagents (bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl) phenyl] oxalate and hydrogen peroxide) and imidazole solution (for neutralization and as a catalyst), respectively. The three mixed solutions flowed out through an outlet (0.5 mm i.d.) at the centre of the top of the vessel. The reaction conditions to give the appropriate signal-to-noise ratio (S/N) were investigated using dipyridamole (an antiplatelet aggregation drug) as a typical fluorescent compound. The system was applied to the detection of dipyridamole and dansylated amino acids with a mobile phase containing trifluoroacetic acid. The detection limits for dipyridamole and each dansylated amino acid were 10 amol and sub-fmol (S/N = 2), respectively.

Amino Acids↗