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Aims The aim of this work was to evaluate the effects of treatment of hypertension on the autoantibodies to apolipoprotein B-derived peptides (anti-ApoB-D peptide Abs) response, inflammation markers and vascular function. Main methods Eighty-eight patients with hypertension (stage 1 or 2) were recruited and advised to receive perindopril (4 mg), hydrochlorothiazide (25 mg), or indapamide (1.5 mg) for 12 weeks in a blinded fashion. Office and 24-h ambulatory blood pressure monitoring (24 h ABPM), flow-mediated dilatation (FMD), nitrate-induced dilatation (NID), titers of IgG and IgM anti-ApoB-D peptide Abs, hsCRP, and interleukins (IL-8 and IL-10) were evaluated at baseline and 12 weeks after therapies. Key findings All treatments reduced office BP, and improved FMD ( P vs . baseline). The NID was improved only in the perindopril arm ( P vs . baseline). The 24 h-ABPM was reduced with perindopril and hydrochlorothiazide therapies ( P vs . baseline), but not with indapamide, and this effect was followed by increase in titers of IgM Anti-ApoB-D peptide Abs ( P vs . baseline), without modifications in titers IgG Anti-ApoB-D peptide Abs and interleukins. Multivariable regression analysis has shown that change in the titers of IgM anti-ApoB-D peptide was associated with the changes in FMD (β − 0.347; P Significance These findings shed light to a possible modulator effect of the antihypertensive therapy on the natural immunity responses and vascular function.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Laboratorio de Investigacao Medica 38 (LIM 38) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Instituto do Milenio de Fluidos Complexos Institute of Cardiology Dante Pazzanese Levels of autoantibodies to oxidized low-density lipoprotein (oxLDL) have been correlated to atherosclerosis; however, contradictory results have been shown. To better understand the role of autoantibodies to oxLDL in atherogenesis, and their potential to predict risk of developing coronary artery disease we investigated the antibody response of unstable angina (UA) patients and healthy controls against chromatographic separated fractions of oxLDL. Five major peaks were detected after chromatographic separation of oxLDL and 10 fractions were collected. Surprisingly, when the response to high molecular weight fractions was analysed, we observed a significant increase in the levels of autoantibodies in controls compared to UA. in contrast, when the autoantibody response to intermediate and low molecular weight fractions was analysed, we observed that the UA group showed consistently higher levels compared with controls. Our data demonstrates that within oxLDL there are major fractions that can be recognized by autoantibodies from either UA patients or healthy individuals, and that the use of total oxLDL as an antigen pool may mask the presence of some antigenic molecules and their corresponding antibodies. Further studies are needed, but the analysis of antibody profiles may indeed open up a novel approach for evaluation and prevention against atherosclerosis. Univ São Paulo, Dept Immunol, Inst Biomed Sci, São Paulo, Brazil Karolinska Univ Hosp, Expt Cardiovasc Res Unit, Ctr Mol Med, S-17176 Stockholm, Sweden Universidade Federal de São Paulo, Dept Biophys, São Paulo, Brazil Inst Cardiol Dante Pazzanese, São Paulo, Brazil Universidade Federal de São Paulo, Dept Biophys, São Paulo, Brazil Web of Science

Chylomicrons play a role in atherosclerosis, however, because the mechanisms involved in the cell uptake of these particles are not fully understood, investigations were carried out using a radioactively labeled protein-free triacylglycerol-rich emulsion incubated with peritoneal macrophages obtained from normal and apoE-knockout mice. Experiments were done in the presence of substances that inhibit several endocytic processes: EDTA for low density lipoprotein receptor, fucoidan for scavenger receptor, cytochalasin B for phagocytosis, and a lipopolysaccharide for lipoprotein lipase. In addition, triacylglycerol-rich emulsions were also prepared in the presence of native or modified radioactively labeled low density lipoprotein particles that are known to accumulate in the arterial intima. Probucol was also used to prevent the possible role played by an antioxidant in triacylglycerol-rich emulsion uptake. We have shown that triacylglycerol-rich emulsion alone is taken up by a coated-pit-dependent mechanism, mediated by macrophage secretion of apolipoprotein E. Furthermore, native, aggregated, acetylated, and moderately macrophage-oxidized low density lipoprotein stimulate the uptake of a triacylglycerol-rich emulsion through several mechanisms such as an actin-dependent pathway, scavenger receptors, and lipolysis mediated by lipoprotein lipase. On the other hand, in spite of the interaction of low density lipoprotein forms with a triacylglycerol-rich emulsion, the cellular triacylglycerol-rich emulsion uptake is impaired by copper-oxidized low density lipoprotein, possibly due to its diminished affinity towards lipoprotein lipase. We have also shown that macrophages take up aggregated low density lipoprotein better than the acetylated or oxidized forms of low density lipoprotein.

The ortho-aminobenzoic acid (Abz) group is widely employed as a fluorescent marker for peptides used as substrates for the study of proteolytic enzyme activity. In fact, a direct correlation has been observed between fluorescence intensity and enzyme activity. An unusual behavior of the fluorescence properties of this group, which would lead to erroneous evaluation of the enzyme activity, was observed when it is bound directly to proline. Here we report a systematic NMR, fluorescence and X-ray diffraction study of the compounds obtained from Boc-Abz-Pro-NH2, Boc-Abz-Pro-OH, as well as from various other Boc-Abz-Pro-X derivatives, after treatment with HCl or TFA under anhydrous conditions. We verified that, as recently reported, even under these synthetic conditions, deprotection of Boc-Abz-Pro-NH2 or Boc-Abz-Pro-OH leads to the formation of the same product: pyrrolobenzodiazepine-5,11-dione. However, the formation of this compound was not detected with Abz-Pro-N(CH3)2, Abz-Pro-Leu-Gly-NH2 or Abz-pyrrolidine. For all these compounds we observed an unusual behavior for the fluorescence quantum yield of Abz that can be explained as the consequence of a non-radiative deactivation process produced, specifically, by the amidation of the Abz carboxyl group with proline or a similar secondary amine such as pyrrolidine. In conclusion, these results indicate that Abz cannot be used as an internal fluorescence marker for proteolytic enzyme activity when bound directly to proline.

The thermal nonlinear optical response of human normal and copper‐oxidized low‐density lipoproteins (LDLs) were investigated by using the Z‐scan technique, as a function of temperature and concentration of LDL particles. The results show that the Z‐scan signals from normal LDL are characteristic of a sample with negative thermo‐optical coefficient. The amplitude of the Z‐scan signal increases linearly with the LDL concentration, following the usual Beer–Lambert law. On the other hand, oxidized LDLs do not show thermal nonlinear optical response. This behaviour can be attributed to an absorbing element that is modified by the oxidative process. On the other hand, changes in the physical state of the cores and conformation of the ApoB100 protein, due to an increase in temperature, seems to enhance the native LDL thermal nonlinear optical response.

Six intramolecularly quenched fluorogenic peptides related to the sequences Phe 8 to His 13 , His 6 to His 13 , and Tyr 4 to His 13 of the human angiotensinogen, containing o -aminobenzoyl (Abz) and ethylenediamine dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acids residues, were synthesized by classical solution methods. The Leu-Val is the only bond of all obtained peptides that was hydrolyzed by human renin with different degrees of purity and was resistant to hydrolysis by pig renin and cathepsin D. The hydrolysis of Abz-His-Pro-Phe-His-Leu-Val-Ile-His-EDDnp by human renin was inhibited by a highly specific transition-state analog of angiotensinogen (IC 50 = 7.8 × 10 −9 m ), described by K. Iizuka et al. (1990, J. Med. Chem. 33, 2707–2714). Therefore, specific and sensitive substrates for the continuous assay of human renin in which as little as 70 μGU of human renin could be detected by Abz-Phe-His-Leu-Val-Ile-His-EDDnp were described. The optimal pHs of hydrolysis of the substrates were in the range 4 to 6.

For better evaluation of the potential of benzhydrylamine resin (BHAR), used as a solid support for peptide synthesis, as a novel anion-exchange resin, the pKa of its amine group was estimated by microscopic measurement of the sizes of the dry and swollen beads. Using the volume of the bead occupied by the solvent (as a percentage) as the swelling parameter, a plot of the degree of swelling of BHAR loaded with 2.4 mmol g−1 amine groups against the pH of the medium produced a decreasing sigmoidal-type curve with increasing pH. By considering the point of inflection of the curve, a pKa value of approximately 7.5 was estimated for the amine group of the BHAR. The same approach was also applied to the lysyl derivative of the BHAR (Lys-BHAR) and pKa values around 6.5 and 10.0 were obtained for the α and e amine groups, respectively.

The nonlinear optical response of human normal and oxidized by Cu2+ low-density lipoproteins particles (LDL), were investigated by the Z-scan technique as a function of temperature and concentration of LDL particles. The Z-scan signals increase linearly with concentration of normal LDL particles, following the usual Beer–Lambert law in a broad range of concentrations. The oxidized LDL particles do not show nonlinear optical response. On the other hand, normal LDL increases its nonlinear optical response as a function of temperature. These behaviors can be attributed to an absorbing element that is modified by the oxidative process. Contrarily, changes in the physical state of the cores and conformation of the ApoB100 protein due to an increase in temperature seems to enhance their nonlinear optical properties. This tendency is not due to aggregation of particles. The main contribution to the nonlinear optical response of normal LDL particles comes from the phospholipid fraction of the particles.

Hormone replacement therapy (HRT) reduces cardiovascular risks, although the initiation of therapy may be associated with transient adverse ischemic and thrombotic events. Antibodies against heat shock protein (Hsp) and oxidized low density lipoprotein (LDL) have been found in atherosclerotic lesions and plasma of patients with coronary artery disease and may play an important role in the pathogenesis of atherosclerosis. The aim of the present study was to assess the effects of HRT on the immune response by measuring plasma levels of antibodies against Hsp 65 and LDL with a low and high degree of copper-mediated oxidative modification of 20 postmenopausal women before and 90 days after receiving orally 0.625 mg equine conjugate estrogen plus 2.5 mg medroxyprogesterone acetate per day. HRT significantly increased antibodies against Hsp 65 (0.316 ± 0.03 vs 0.558 ± 0.11) and against LDL with a low degree of oxidative modification (0.100 ± 0.01 vs 0.217 ± 0.02) (P

OBJECTIVE To examine the metabolism of kinins and angiotensin I in the pulmonary circulation of spontaneously hypertensive rats (SHR) and normotensive Wistar rats (NWR). METHODS Bradykinin inactivation was estimated in vivo by comparison of the hypotensive effect of intra-arterial and intravenous injections, and in the in situ perfused lung by analysing the breakdown products using high-performance liquid chromatography. RESULTS In vivo pulmonary degradation of bradykinin, but not that of higher homologues of this peptide, was shown to be significantly greater in SHR. Angiotensin I converting activity was found to be increased in lungs of SHR. The recovery of bradykinin and homologues from perfused SHR lung was decreased relative to NWR. Des-(Phe-Arg) fragments of all kinin analogues were identified in the pulmonary perfusates. When bradykinin and des-Arg9-bradykinin were injected in the perfused lungs, the respective fragments 4-9 and 4-8 were also identified in the perfusates. When kininase II was inhibited with enalaprilat, the recovery of bradykinin increased from 10 to 43% in SHR and from 23 to 58% in NWR, whereas about 90% of the higher bradykinin homologues were recovered in both SHR and NWR. Aminopeptidase P and dipeptidylaminopeptidase IV, as measured by the recovery of fragment 4-9 under kininase II inhibition, accounted for about 40% of the total pulmonary kininase activity in the SHR lungs and 25% of that of the NWR lungs. CONCLUSIONS The results show that SHR have increased kininase and angiotensin converting activity compared with NWR, and that kinins as well as angiotensin may contribute to the pathogenesis of hypertension. Aminopeptidase P and dipeptidylaminopeptidase IV may contribute to the increased in vivo degradation of bradykinin observed in the SHR.

The N-terminal heptadecapeptide of human angiotensinogen (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn-Glu-Ser-Thr-NH2 ), with the C-terminal carboxyl group amidated, was synthesized in order to study the role of Asn-Glu-Ser, a putative carbohydrate binding site, on the hydrolysis by human renin. The synthesis was performed by fragment condensation using the Honzl and Rudinger azide procedure. In our conditions for azide segment condensation, histidine racemization was demonstrated to be negligible for most of the condensation reactions. Human renin liberates angiotensin I from h-angiotensinogen (1-17)-NH2 with a Km value of 3.4 x 10(-5) M, at pH 7.3 and 37 degrees being similar to h-angiotensinogen (1-13), an analog without the carbohydrate binding site. However, the Vmax value of 4.1 x 10(-9) mol/G.U. min is one order of magnitude higher. Porcine pepsin was demonstrated to cleave preferentially Leu10-Val11 bond and, surprisingly, His9-Leu10 as well.

A study of the kinetics of tonin action on several synthetic homologs of angiotensinogen showed that the minimal peptide substrate for that enzyme is the (5–10)-hexapeptide. The C-terminal tetrapeptide segment of the angiotensinogen-(1–14)-peptide affects tonin specificity mainly through an effect on the catalytic step, and not through changes in enzyme-substrate affinity. When the three N-terminal residues of the (1–14)-peptide were removed, a poorer tonin substrate was obtained, mostly because of a sharp increase in Km. Bradykinin was not attacked by tonin, while substance P was hydrolyzed only with enzyme concentrations much higher than those effective with angiotensinogen fragments as substrates.

The study of the reaction of p-nitrophenyl acetate (PNPA) with histidine and certain derivatives showed that the species in which the amino group is unprotonated (R(NH2)Im) react with second-order rate constants ( k 2 am ) that are higher than predicted by a Bronsted relation for a series of neutral amino acids. The reason for this behavior was investigated through an analysis of the kinetics of the reaction of PNPA with these compounds in order to assess the reactivities of the amino and imidazole groups in the two species R(NH 3 + ) Im and R(NH 2 ) Im . The rate constant for the reaction with the imidazole group ( k 2 im ) of Nπ-methyl histidine agrees with the value predicted by a Bronsted relation obtained from a series of model imidazole compounds. Nτ-Methyl histidine, however, is unreactive, indicating that Nτ is the reactive nitrogen in the imidazole ring of histidine. The k 2 im values found for histidine, histidine methyl ester, and Nα-dimethyl histidine are lower than predicted by the Bronsted relation. This behavior was found to be due to low reactivity of the R(NH 3 + ) Im species, in contrast with the normal reactivity of R(NH 2 ) Im . The evidence presented suggests that the lower reactivity of R(NH 3 + ) Im is due to an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring, which displaces the tautomeric equilibrium toward the unreactive Nτ-H form. The higher reactivity of the imidazole group in the species R(NH2)Im, relative to that in R(NH 3 + ) Im , is responsible for the observed high k 2 am values for histidine, for histidine methyl ester, for Nτ-methyl histidine, and for Nα-dimethyl histidine, in contrast with the normal k 2 am value found for Nτ-methyl histidine. The conclusions from this study of histidine and its derivatives support the proposal of an interaction between the protonated N-terminal amino group and the imidazole ring of His6 in the octapeptide hormone angiotensin.

The pKa values for the proton dissociation of carboxyl, imidazolium, and ammonium groups for histidine and ten of its derivatives were determined electrometrically at seven temperatures in the range 10–40°C. The ΔH and ΔS values were estimated from the temperature dependence of the dissociation constants of histidine and its derivatives. These results and the pKa values compared in terms of inductive effect suggest an ion-dipole interaction between the protonated amino group and the unprotonated imidazole ring. The charge and the solvation effects of the neighboring groups are the main factors that determine the imidazole group pKa in histidine and its studied derivatives. The Nτ-H tautomer is favored over the Nπ-H by 1.6 kcal/mol, indicating that the inductive substituent effect at position 4 of the imidazole ring is the major component in determining this tautomeric preference.

Low doses of bradykinin (below 10 nM), as well as of K+ (below 10 mM) induced relaxation, whereas higher doses caused contraction of the rat duodenum. The relaxant responses induced by bradykinin and K+ were not affected by ouabain (1 microM), but pre-incubation with 5.9 mM K+ abolished the responses to that ion but not those to bradykinin. The contractile and relaxant components of the response to bradykinin (but not those to K+) increased with the time elapsed after mounting of the preparation, and this was due to stretching by the load of the recording system. Specific and reversible desensitization (tachyphylaxis) was observed with the contractile response (but not the relaxation) induced by bradykinin. Des-Arg9-bradykinin, an analogue specific for B1-receptors, was much less active than bradykinin, and elicited only a contractile response. Among four bradykinin potentiating peptides that were tested, potentiator C enhanced the relaxation only, whereas BPP5a and captopril potentiated only the contraction and BPP9a potentiated both types of response to bradykinin. Our results support the hypothesis that the relaxant and contractile components of the rat duodenum's response to bradykinin are due to actions at different receptor sites, which can be distinguished by their properties (desensitization) and their different apparent affinities for agonists and for potentiating peptides.

Objective and Design: Oxidized low-density lipoproteins (oxLDL) and protein fractions obtained by size exclusion chromatography of oxLDL were tested for vascu- lar permeability effects on topical application to the hamster cheek pouch. Materials: The hamster cheek pouch was prepared for intrav- ital microscopy observations of macromolecular leakage at post capillary venules (= leaks) with FITC-dextran as tracer. Treatment: OxLDL (0.1 mg/ml), PAF (platelet activation fac- tor, 50-100 nM) and protein fractions of oxLDL (10 mg/ml) were applied topically to hamster cheek pouches. Results: Application of oxLDL and PAF resulted in reversible increases in the number of leaks. The PAF-antago- nist WEB 2170, L-NAME and a b2-adrenoceptor agonist inhibited (P < 0.01) almost completely the macromolecular leakage induced with oxLDL or PAF. Protein fractions were found to be more effective than unfractionated oxLDL in inducing plasma leakage as calculated on mg/ml-basis. Conclusion: Hamster oxLDL is a potent inducer of macro- molecular leakage increase in the hamster cheek pouch microcirculation. The principal effect is mediated by PAF- like structures produced by the oxidation of the LDL-particle but oxLDL also contains low molecular weight proteins that could contribute to the overall vascular permeability increas- ing effect of ox LDL.