Very long chain n-3 and n-6 polyunsaturated fatty acids bind strongly to liver fatty acid-binding protein.
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Synthesis of n-3 and n-6 very long chain-PUFAs (VLC-PUFAs) from 18-carbon essential fatty acids is differentially regulated. The predominant product arising from n-3 fatty acids is docosahexaenoic acid (22:6n-3), with the liver serving as the main site of production. The synthetic pathway requires movement of a 24-carbon intermediate from the endoplasmic reticulum to peroxisomes for retroconversion to 22:6n-3. The mechanism of this intra-organelle flux is unknown, but could be binding-protein facilitated. We thus investigated binding of a series of previously untested VLC-PUFAs to liver fatty acid-binding protein (L-FABP). Three fluorometric assays were employed, all of which showed strong binding (K(d)' approximately 10(-8) to 10(-7) M) of 20-, 22-, and 24-carbon n-3 PUFAs to L-FABP. In contrast, synthesis of the predominant n-6 PUFA product, arachidonic acid, does not require intra-organelle transport. However, we found that n-6 VLC-PUFAs bound to L-FABP with affinities (K(d)' approximately 10(-8) to 10(-7) M) comparable to their n-3 counterparts. Although these results raise the possibility that L-FABP may participate in the cytoplasmic processing of n-3 and n-6 VLC-PUFAs, there is no evidence on the basis of binding affinities that L-FABP accounts for differences in the predominant products formed by the n-3 and n-6 PUFA metabolic pathways. (+info)
Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods.
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The binding interactions of small molecules with carbonic anhydrase II were used as model systems to compare the reaction constants determined from surface- and solution-based biophysical methods. Interaction data were collected for two arylsulfonamide compounds, 4-carboxybenzenesulfonamide (CBS) and 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), binding to the enzyme using surface plasmon resonance, isothermal titration calorimetry, and stopped-flow fluorescence. We demonstrate that when the surface plasmon resonance biosensor experiments are performed with care, the equilibrium, thermodynamic, and kinetic constants determined from this surface-based technique match those acquired in solution. These results validate the use of biosensor technology to collect reliable data on small molecules binding to immobilized macromolecular targets. Binding kinetics were shown to provide more detailed information about complex formation than equilibrium constants alone. For example, although carbonic anhydrase II bound DNSA with twofold higher affinity than CBS, kinetic analysis revealed that CBS had a fourfold slower dissociation rate. Analysis of the binding and transition state thermodynamics also revealed significant differences in the enthalpy and entropy of complex formation. The lack of labeling requirements, high information content, and high throughput of surface plasmon resonance biosensors will make this technology an important tool for characterizing the interactions of small molecules with enzymes and receptors. (+info)
A bromine compound existing in blood.
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Since a bromine compound with REM-sleep-inducing and anti-choline esterase activities have been isolated from human cerebrospinal fluid, and was identified as 1-methylheptyl gamma-bromoacetoacetate, the compound was chemically synthesized. It was found that this compound was composed with three forms, i.e., a keto-form, an enol-form that changed gradually from keto-form by tautomerism, and a stable six-membered ring form (= cyclic r-Br) converted from enol form, when it was chemically synthesized. In addition, it was found that the six-membered ring form of this bromine compound was present in the human blood. However, in this case, the keto-form and the enol-form were not detected. When 14C-butyrate was injected to rats, it was incorporated into the bromine compound in the blood of the animal and the bromine compound formed was found to be present mainly as the six-membered ring form. From these results, the mechanism for the formation of bromine compounds in human and animal blood were deduced. (+info)
Aortic smooth muscle cell phenotypic modulation and fibrillar collagen deposition in angiotensin II-dependent hypertension.
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BACKGROUND: We investigated the effect of nifedipine, AT-1 and ET-1 receptor blockade on arterial smooth muscle cell phenotypes and collagen deposition in TGRen2 transgenic rat (TGR). METHODS: Four-week-old TGR were blood pressure (BP)-matched and allocated to receive a placebo (n=8), the calcium antagonist nifedipine (n=6), the AT-1 specific receptor antagonist irbesartan (n=6), the ET(A)/ET(B) antagonist bosentan (n=6) or the ET(A)-selective antagonist BMS-182874 (n=5). Sprague-Dawley normotensive rats served as controls (n=6). After 4 weeks of treatment animals were euthanized and the left ventricle (LV) and the structural changes in intracardiac arterioles and aorta were assessed histomorphometrically. Smooth muscle cell phenotypes and fibrillar collagen content of the aortic wall were evaluated by immunostaining, using differentiation markers-specific antibodies and Syrius red staining, respectively. The changes in ET(A) and ET(B) receptor density were also assessed with quantitative autoradiography. RESULTS: Compared to placebo, only irbesartan lowered BP (P<0.001) and prevented LV and small resistance artery hypertrophy. The aorta of placebo-treated TGR showed an increase in foetal-type smooth muscle cell content and fibrillar collagen staining, compared to controls. These changes were blunted by irbesartan, which increased ET(A) receptors in the arterial wall, enhanced by BMS-182874 and unaffected by bosentan. Nifedipine also blunted both the VSMC and collagen changes despite having no effect on BP and ET(A) receptors. CONCLUSIONS: In TGRen2, vascular hypertrophy entails both smooth muscle cell phenotypic modulation and collagen deposition. These alterations do not follow closely the BP changes and seem to imply the dihydropyridine-sensitive calcium channels. (+info)
Elevated mean arterial pressure in the ovariectomized rat was normalized by ET(A) receptor antagonist therapy: absence of cardiac hypertrophy and fibrosis.
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1. The influence of menopause on ventricular function and remodelling remains undefined. The following study examined the effect of ovariectomy on ventricular contractility, cardiac hypertrophy and extracellular matrix protein expression. 2. Elevated circulating levels of the vasoconstrictor endothelin-1 have been reported in post-menopausal women. Moreover, endothelin-1 has been shown to influence blood pressure, ventricular function and cardiac remodelling. In this regard, the potential pathophysiological role of endothelin-1 in the ovariectomized rat was assessed via the administration of the selective endothelin(A) receptor (ET(A)) antagonist BMS-182874. 3. In 3 and 6 week ovariectomized female Sprague - Dawley rats, uterus atrophy was associated with a significant increase in mean arterial pressure, and left ventricular systolic pressure, as compared to sham. By contrast, right ventricular contractile indices were normal in the ovariectomized rat. Despite increased systolic load, left ventricular hypertrophy was not evident, prepro-atrial natriuretic peptide (prepro-ANP) mRNA levels and collagen protein content were similar to sham. 4. The treatment of ovariectomized rats with BMS-182874 (60 mg kg(-1) per day) did not reverse uterus atrophy. However, BMS-182874 normalized mean arterial pressure, and left ventricular systolic pressure in the ovariectomized rat. 5. Thus, despite elevated blood pressure, ovariectomized rats were not associated with either cardiac hypertrophy or fibrosis. Lastly, endothelin-1, acting via the stimulation of the ET(A) receptor represents an integral mechanism implicated in the increase of mean arterial pressure following ovariectomy. (+info)
Early kinetic intermediate in the folding of acyl-CoA binding protein detected by fluorescence labeling and ultrarapid mixing.
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Early conformational events during folding of acyl-CoA binding protein (ACBP), an 86-residue alpha-helical protein, were explored by using a continuous-flow mixing apparatus with a dead time of 70 micros to measure changes in intrinsic tryptophan fluorescence and tryptophan-dansyl fluorescence energy transfer. Although the folding of ACBP was initially described as a concerted two-state process, the tryptophan fluorescence measurements revealed a previously unresolved phase with a time constant tau = 80 micros, indicating formation of an intermediate with only slightly enhanced fluorescence of Trp-55 and Trp-58 relative to the unfolded state. To amplify this phase, a dansyl fluorophore was introduced at the C terminus by labeling an I86C mutant of ACBP with 5-IAEDANS [5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid]. Continuous-flow refolding of guanidine HCl-denatured ACBP showed a major increase in tryptophan-dansyl fluorescence energy transfer, indicating formation of a partially collapsed ensemble of states on the 100-micros time scale. A subsequent decrease in dansyl fluorescence is attributed to intramolecular quenching of donor fluorescence on formation of the native state. The kinetic data are fully accounted for by three-state mechanisms with either on- or off-pathway intermediates. The intermediate accumulates to a maximum population of 40%, and its stability depends only weakly on denaturant concentration, which is consistent with a marginally stable ensemble of partially collapsed states with approximately 1/3 of the solvent-accessible surface buried. The findings indicate that ultrafast mixing methods combined with sensitive conformational probes can reveal transient accumulation of intermediate states in proteins with apparent two-state folding mechanisms. (+info)
Contribution of endothelin-1 to renal activator protein-1 activation and macrophage infiltration in aldosterone-induced hypertension.
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Aldosterone-induced hypertension is associated with renal damage that may be mediated by endothelin-1 (ET-1). We evaluated whether inflammatory cell infiltration and DNA-binding activity of the transcription factors nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) were increased in kidneys from aldosterone-infused rats. The role of ET-1 in these processes was evaluated by treating rats with the ET(A)-receptor blocker, BMS 182874. Rats were infused with aldosterone (0.75 microg/h) via a mini-osmotic pump and were given 1% NaCl in the drinking water in the absence and presence of BMS 182874 or of the aldosterone receptor blocker, spironolactone. Renal sections were used to assess inflammatory cell infiltration, which was identified immunocytochemically using monoclonal antibodies against macrophages (ED1+). Electrophoretic mobility shift assays evaluated the DNA-binding activity of NF-kappa B and AP-1 in renal tissue. Systolic blood pressure (BP) was increased in the aldosterone-infused group compared with controls (123+/-6 versus 110+/-10 mmHg, P<0.05). BMS 182874 and spironolactone significantly decreased BP (P<0.05). Macrophage infiltration was increased in the kidneys of aldosterone-infused rats compared with controls. Renal binding activity (arbitrary units) of AP-1, in contrast with that of NF-kappa B, increased in aldosterone-infused rats compared with control rats (AP-1, 4.2+/-0.3 versus 1.0+/-0.1, P<0.05; NF-kappa B, 1.6+/-0.5 versus 1.2+/-0.5). BMS 182874 and spironolactone decreased macrophage infiltration (by 70% and 50% respectively) and AP-1 binding activity (1.0+/-0.3 and 0.8+/-0.3 respectively). In conclusion, kidneys from aldosterone-infused rats exhibited macrophage infiltration and increased AP-1 DNA-binding activity. These processes were attenuated by BMS 182874. Our findings suggest that renal damage in aldosterone-dependent hypertension is associated with inflammatory processes that are mediated in part via ET-1. (+info)
On the activation of bovine plasma factor XIII. Amino acid sequence of the peptide released by thrombin and the terminal residues of the subunit polypeptides.
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A blood coagulation factor, Factor XIII, was highly purified from bovine fresh plasma by a method similar to those used for human plasma Factor XIII. The isolated Factor XIII consisted of two subunit polypeptides, a and b chains, with molecular weights of 79,000 +/- 2,000 and 75,000 +/- 2,000, respectively. In the conversion of Factor XIII to the active enzyme, Factor XIIIa, by bovine thrombin [EC 3.4.21.5], a peptide was liberated. This peptide, designated tentatively as "activation peptide," was isolated by gel-filtration on a Sephadex G-75 column. It contained a total of 37 amino acid residues with a masked N-terminal residue and C-terminal arginine. The whole amino acid sequence of "Activation peptide" was established by the dansyl-Edman method and standard enzymatic techniques, and the masked N-terminal residue was identified as N-acetylserine by using a rat liver acylamino acid-releasing enzyme. This enzyme specifically cleaved the N-acetylserylglutamyl peptide bond serine and the remaining peptide, which was now reactive to 1-dimethylamino-naphthalene-5-sulfonyl chloride. A comparison of the sequences of human and bovine "Activation peptide" revealed five amino acids replacements, Ser-3 to Thr; Gly-5 to Arg; Ile-14 to Val; Thr-18 to Asn, and Pro-26 to Leu. Another difference was the deletion of Leu-34 in the human peptide. Adsorption chromatography on a hydroxylapatite column in the presence of 0.1% sodium dodecyl sulfate was developed as a preparative procedure for the resolution of the two subunit polypeptides, a or a' chain and b chain, constituting the protein molecule of Factor XIII or Factor XIIIa. End group analyses on the isolated pure chains revealed that the structural change of Factor XIII during activation with thrombin occurs only in the N-terminal portion of the a chain, not in the N-terminal end of the b chain or in the C-terminal ends of the a and b chains. From these results, it was concluded that the activation of bovine plasma Factor XIII by thrombin must be accompanied by a limited proteolysis of the arginyl-glycyl bond located in the N-terminal region of the a chain, liberating the "Activation peptide." The possibility of activating Factor XII with other porteinases was examined using Factor Xa [EC 3.4.21.6], Factor XIIa, kallikreins [EC 3.4.21.8], urokinase [EC 3.4.99.26], trypsin [EC 3.4.21.4], ficin [EC 3.4.22.3], papain [EC 3.4.22.2], and bromelain [EC 3.4.22.4]. Among these enzymes, only bromelain and trypsin showed clear activating effects. (+info)