Pathobiology : journal of immunopathology, molecular and cellular biology, Jan 10, 2016
Soft tissue sarcomas (STSs) are heterogeneous tumors displaying multiple and complex molecular ab... more Soft tissue sarcomas (STSs) are heterogeneous tumors displaying multiple and complex molecular abnormalities with no specific pattern. Despite current therapeutic advances, the patients with STS still have a poor outcome, which makes it necessary to find out new prognostic markers. The Raf kinase inhibitory protein (RKIP) has been associated with prognosis in several human neoplasms; however, its role in STS is unknown. In the present study RKIP expression was assessed by immunohistochemistry in a series of 87 STSs, and its expression profile was associated with the patients' pathological parameters. We found that RKIP is expressed in the cytoplasm of the great majority of cases, and absent in only approximately 18% of cases (16/87). Importantly, we observed that loss of RKIP expression was associated with poor outcome, constituting an independent prognostic marker. This is the first study assessing RKIP expression levels in STS. We showed that loss of RKIP expression is present...
The expression of alpha 1-adrenoceptor subtypes in several tissues is regulated by gonadal hormon... more The expression of alpha 1-adrenoceptor subtypes in several tissues is regulated by gonadal hormones. In this study, we investigated whether castration regulates the alpha 1-adrenoceptor subtypes mediating the contractions of the aorta from male rats to noradrenaline. Noradrenaline induced similar concentration-dependent contractions in the aorta from control and castrated rats. Treatment of the aorta from both control and castrated rats with the alpha 1B/alpha 1D-adrenoceptor alkylating agent chloroethylclonidine resulted in approximately 1600-fold rightward shift in the concentration-response curves to noradrenaline. The pA2 values found for WB 4101, benoxathian (alpha 1A-selective) and BMY 7378 (alpha 1D-selective) indicate that alpha 1D-adrenoceptors are involved in the contractions of the aorta from control and castrated rats to noradrenaline. However, there was a 15-fold difference between the pKB estimated through the lowest effective concentrations of the alpha 1A-adrenoceptor selective antagonist 5-methyl-urapidil in the aorta from control and castrated rats. The pKB estimated in aorta from control rats is consistent with the interaction with alpha 1D-adrenoceptors (7.58 +/- 0.06), while that calculated in organs from control rats is consistent with alpha 1A-adrenoceptors (8.76 +/- 0.09). These results suggest that castration induces plasticity in the alpha 1-adrenoceptor subtypes involved in the contractions of the aorta to noradrenaline.
The expression of alpha 1-adrenoceptor subtypes in several tissues is regulated by gonadal hormon... more The expression of alpha 1-adrenoceptor subtypes in several tissues is regulated by gonadal hormones. In this study, we investigated whether castration regulates the alpha 1-adrenoceptor subtypes mediating the contractions of the aorta from male rats to noradrenaline. Noradrenaline induced similar concentration-dependent contractions in the aorta from control and castrated rats. Treatment of the aorta from both control and castrated rats with the alpha 1B/alpha 1D-adrenoceptor alkylating agent chloroethylclonidine resulted in approximately 1600-fold rightward shift in the concentration-response curves to noradrenaline. The pA2 values found for WB 4101, benoxathian (alpha 1A-selective) and BMY 7378 (alpha 1D-selective) indicate that alpha 1D-adrenoceptors are involved in the contractions of the aorta from control and castrated rats to noradrenaline. However, there was a 15-fold difference between the pKB estimated through the lowest effective concentrations of the alpha 1A-adrenoceptor selective antagonist 5-methyl-urapidil in the aorta from control and castrated rats. The pKB estimated in aorta from control rats is consistent with the interaction with alpha 1D-adrenoceptors (7.58 +/- 0.06), while that calculated in organs from control rats is consistent with alpha 1A-adrenoceptors (8.76 +/- 0.09). These results suggest that castration induces plasticity in the alpha 1-adrenoceptor subtypes involved in the contractions of the aorta to noradrenaline.
The Journal of Clinical Endocrinology & Metabolism, 2003
The PHEX gene that is mutated in patients with X-linked hypophosphatemia (XLH) encodes a protein ... more The PHEX gene that is mutated in patients with X-linked hypophosphatemia (XLH) encodes a protein homologous to the M13 family of zinc metallopeptidases. The present study was undertaken to assess the impact of nine PHEX missense mutations on cellular trafficking, endopeptidase activity, and protein conformation. Secreted forms of wild-type and mutant PHEX proteins were generated by PCR mutagenesis; these included C85R, D237G, Y317F, G579R, G579V, S711R, A720T, and F731Y identified in XLH patients, and E581V, which in neutral endopeptidase 24.11 abolishes catalytic activity but not plasma membrane localization. The wild-type and D237G, Y317F, E581V, and F731Y proteins were terminally glycosylated and secreted into the medium, whereas the C85R, G579R, G579V, S711R, and A720T proteins were trapped inside the transfected cells. Growing the cells at 26 C permitted the secretion of G579V, S711R, and A720T proteins, although the yield of rescued G579V was insufficient for further analysis....
The contractions of the rat vas deferens in response to noradrenaline are mediated through alpha(... more The contractions of the rat vas deferens in response to noradrenaline are mediated through alpha(1A)-adrenoceptors. We observed participation of alpha(1B)-adrenoceptors in these contractions after castration. We now investigated the time course of this plasticity and the effects of testosterone by determining the actions of competitive antagonists on noradrenaline-induced contractions after 7, 14, 21 and 30 days of castration. BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride) antagonised noradrenaline-induced contractions in control and castrated rats with low pA(2) values (approximately = 6.8). In control vas deferens, WB 4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) had a slope in the Schild plot no different from 1.0, while slopes lower than 1.0 (approximately 0.6) were observed for vas deferens from castrated rats. Chloroethylclonidine was ineffective in the control vas while it inhibited noradrenaline-induced contractions in vasa from castrated rats and converted the complex antagonism by WB 4101 into simple competitive antagonism. Treatment of castrated rats with testosterone prevented the effects of castration. The results suggest that alpha(1B)-adrenoceptors are detectable in vas deferens from at least the 7th through the 30th day after castration and that testosterone prevents this plasticity.
We examined the substrate specificity of the carboxydipeptidase activity of neprilysin (NEP) usin... more We examined the substrate specificity of the carboxydipeptidase activity of neprilysin (NEP) using fluorescence resonance energy transfer (FRET) peptides containing ortho-aminobenzoyl (Abz) and 2,4-dinitrophenyl (Dnp) as a donor/acceptor pair. Two peptide series with general sequences Abz-RXFK(Dnp)-OH and Abz-XRFK(Dnp)-OH (X denotes the position of the altered amino acid) were synthesized to study P1 (cleavage at the X-F bond) and P2 (cleavage at R-F bond) specificity, respectively. In these peptides a Phe residue was fixed in P1' to fulfill the well-known NEP S1' site requirement for a hydrophobic amino acid. In addition, we explored NEP capability to hydrolyze bradykinin (RPPGFSPFR) and its fluorescent derivative Abz-RPPGFSPFRQ-EDDnp (EDDnp=2,4-dinitrophenyl ethylenediamine). The enzyme acts upon bradykinin mainly as a carboxydipeptidase, preferentially cleaving Pro-Phe over the Gly-Phe bond in a 9:1 ratio, whereas Abz-RPPGFSPFRQ-EDDnp was hydrolyzed at the same bonds but at an inverted proportion of 1:9. The results show very efficient interaction of the substrates' C-terminal free carboxyl group with site S2' of NEP, confirming the enzyme's preference to act as carboxydipeptidase at substrates with a free carboxyl-terminus. Using data gathered from our study, we developed sensitive and selective NEP substrates…
The PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) e... more The PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) encodes a protein (PHEX) with structural homologies to members of the M13 family of zinc metallo-endopeptidases. Mutations in the PHEX gene are responsible for X-linked hypophosphataemia in humans. However, the mechanism by which loss of PHEX function results in the disease phenotype, and the endogenous PHEX substrate(s) remain unknown. In order to study PHEX substrate specificity, combinatorial fluorescent-quenched peptide libraries containing o-aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as the donor–acceptor pair were synthesized and tested as PHEX substrates. PHEX showed a strict requirement for acidic amino acid residues (aspartate or glutamate) in S1´ subsite, with a strong preference for aspartate. Subsites S2´, S1 and S2 exhibited less defined specificity requirements, but the presence of leucine, proline or glycine in P2´, or valine, isoleucine or histidine in P1 preclude...
Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were ... more Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-...
Pathobiology : journal of immunopathology, molecular and cellular biology, Jan 10, 2016
Soft tissue sarcomas (STSs) are heterogeneous tumors displaying multiple and complex molecular ab... more Soft tissue sarcomas (STSs) are heterogeneous tumors displaying multiple and complex molecular abnormalities with no specific pattern. Despite current therapeutic advances, the patients with STS still have a poor outcome, which makes it necessary to find out new prognostic markers. The Raf kinase inhibitory protein (RKIP) has been associated with prognosis in several human neoplasms; however, its role in STS is unknown. In the present study RKIP expression was assessed by immunohistochemistry in a series of 87 STSs, and its expression profile was associated with the patients' pathological parameters. We found that RKIP is expressed in the cytoplasm of the great majority of cases, and absent in only approximately 18% of cases (16/87). Importantly, we observed that loss of RKIP expression was associated with poor outcome, constituting an independent prognostic marker. This is the first study assessing RKIP expression levels in STS. We showed that loss of RKIP expression is present...
The expression of alpha 1-adrenoceptor subtypes in several tissues is regulated by gonadal hormon... more The expression of alpha 1-adrenoceptor subtypes in several tissues is regulated by gonadal hormones. In this study, we investigated whether castration regulates the alpha 1-adrenoceptor subtypes mediating the contractions of the aorta from male rats to noradrenaline. Noradrenaline induced similar concentration-dependent contractions in the aorta from control and castrated rats. Treatment of the aorta from both control and castrated rats with the alpha 1B/alpha 1D-adrenoceptor alkylating agent chloroethylclonidine resulted in approximately 1600-fold rightward shift in the concentration-response curves to noradrenaline. The pA2 values found for WB 4101, benoxathian (alpha 1A-selective) and BMY 7378 (alpha 1D-selective) indicate that alpha 1D-adrenoceptors are involved in the contractions of the aorta from control and castrated rats to noradrenaline. However, there was a 15-fold difference between the pKB estimated through the lowest effective concentrations of the alpha 1A-adrenoceptor selective antagonist 5-methyl-urapidil in the aorta from control and castrated rats. The pKB estimated in aorta from control rats is consistent with the interaction with alpha 1D-adrenoceptors (7.58 +/- 0.06), while that calculated in organs from control rats is consistent with alpha 1A-adrenoceptors (8.76 +/- 0.09). These results suggest that castration induces plasticity in the alpha 1-adrenoceptor subtypes involved in the contractions of the aorta to noradrenaline.
The expression of alpha 1-adrenoceptor subtypes in several tissues is regulated by gonadal hormon... more The expression of alpha 1-adrenoceptor subtypes in several tissues is regulated by gonadal hormones. In this study, we investigated whether castration regulates the alpha 1-adrenoceptor subtypes mediating the contractions of the aorta from male rats to noradrenaline. Noradrenaline induced similar concentration-dependent contractions in the aorta from control and castrated rats. Treatment of the aorta from both control and castrated rats with the alpha 1B/alpha 1D-adrenoceptor alkylating agent chloroethylclonidine resulted in approximately 1600-fold rightward shift in the concentration-response curves to noradrenaline. The pA2 values found for WB 4101, benoxathian (alpha 1A-selective) and BMY 7378 (alpha 1D-selective) indicate that alpha 1D-adrenoceptors are involved in the contractions of the aorta from control and castrated rats to noradrenaline. However, there was a 15-fold difference between the pKB estimated through the lowest effective concentrations of the alpha 1A-adrenoceptor selective antagonist 5-methyl-urapidil in the aorta from control and castrated rats. The pKB estimated in aorta from control rats is consistent with the interaction with alpha 1D-adrenoceptors (7.58 +/- 0.06), while that calculated in organs from control rats is consistent with alpha 1A-adrenoceptors (8.76 +/- 0.09). These results suggest that castration induces plasticity in the alpha 1-adrenoceptor subtypes involved in the contractions of the aorta to noradrenaline.
The Journal of Clinical Endocrinology & Metabolism, 2003
The PHEX gene that is mutated in patients with X-linked hypophosphatemia (XLH) encodes a protein ... more The PHEX gene that is mutated in patients with X-linked hypophosphatemia (XLH) encodes a protein homologous to the M13 family of zinc metallopeptidases. The present study was undertaken to assess the impact of nine PHEX missense mutations on cellular trafficking, endopeptidase activity, and protein conformation. Secreted forms of wild-type and mutant PHEX proteins were generated by PCR mutagenesis; these included C85R, D237G, Y317F, G579R, G579V, S711R, A720T, and F731Y identified in XLH patients, and E581V, which in neutral endopeptidase 24.11 abolishes catalytic activity but not plasma membrane localization. The wild-type and D237G, Y317F, E581V, and F731Y proteins were terminally glycosylated and secreted into the medium, whereas the C85R, G579R, G579V, S711R, and A720T proteins were trapped inside the transfected cells. Growing the cells at 26 C permitted the secretion of G579V, S711R, and A720T proteins, although the yield of rescued G579V was insufficient for further analysis....
The contractions of the rat vas deferens in response to noradrenaline are mediated through alpha(... more The contractions of the rat vas deferens in response to noradrenaline are mediated through alpha(1A)-adrenoceptors. We observed participation of alpha(1B)-adrenoceptors in these contractions after castration. We now investigated the time course of this plasticity and the effects of testosterone by determining the actions of competitive antagonists on noradrenaline-induced contractions after 7, 14, 21 and 30 days of castration. BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride) antagonised noradrenaline-induced contractions in control and castrated rats with low pA(2) values (approximately = 6.8). In control vas deferens, WB 4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) had a slope in the Schild plot no different from 1.0, while slopes lower than 1.0 (approximately 0.6) were observed for vas deferens from castrated rats. Chloroethylclonidine was ineffective in the control vas while it inhibited noradrenaline-induced contractions in vasa from castrated rats and converted the complex antagonism by WB 4101 into simple competitive antagonism. Treatment of castrated rats with testosterone prevented the effects of castration. The results suggest that alpha(1B)-adrenoceptors are detectable in vas deferens from at least the 7th through the 30th day after castration and that testosterone prevents this plasticity.
We examined the substrate specificity of the carboxydipeptidase activity of neprilysin (NEP) usin... more We examined the substrate specificity of the carboxydipeptidase activity of neprilysin (NEP) using fluorescence resonance energy transfer (FRET) peptides containing ortho-aminobenzoyl (Abz) and 2,4-dinitrophenyl (Dnp) as a donor/acceptor pair. Two peptide series with general sequences Abz-RXFK(Dnp)-OH and Abz-XRFK(Dnp)-OH (X denotes the position of the altered amino acid) were synthesized to study P1 (cleavage at the X-F bond) and P2 (cleavage at R-F bond) specificity, respectively. In these peptides a Phe residue was fixed in P1' to fulfill the well-known NEP S1' site requirement for a hydrophobic amino acid. In addition, we explored NEP capability to hydrolyze bradykinin (RPPGFSPFR) and its fluorescent derivative Abz-RPPGFSPFRQ-EDDnp (EDDnp=2,4-dinitrophenyl ethylenediamine). The enzyme acts upon bradykinin mainly as a carboxydipeptidase, preferentially cleaving Pro-Phe over the Gly-Phe bond in a 9:1 ratio, whereas Abz-RPPGFSPFRQ-EDDnp was hydrolyzed at the same bonds but at an inverted proportion of 1:9. The results show very efficient interaction of the substrates' C-terminal free carboxyl group with site S2' of NEP, confirming the enzyme's preference to act as carboxydipeptidase at substrates with a free carboxyl-terminus. Using data gathered from our study, we developed sensitive and selective NEP substrates…
The PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) e... more The PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) encodes a protein (PHEX) with structural homologies to members of the M13 family of zinc metallo-endopeptidases. Mutations in the PHEX gene are responsible for X-linked hypophosphataemia in humans. However, the mechanism by which loss of PHEX function results in the disease phenotype, and the endogenous PHEX substrate(s) remain unknown. In order to study PHEX substrate specificity, combinatorial fluorescent-quenched peptide libraries containing o-aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as the donor–acceptor pair were synthesized and tested as PHEX substrates. PHEX showed a strict requirement for acidic amino acid residues (aspartate or glutamate) in S1´ subsite, with a strong preference for aspartate. Subsites S2´, S1 and S2 exhibited less defined specificity requirements, but the presence of leucine, proline or glycine in P2´, or valine, isoleucine or histidine in P1 preclude...
Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were ... more Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-...
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