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Biomedical subjects

E Carmona

Publications and source records attributed to E Carmona.

At least 19 recordsLinked to original sources
PubMed2001

Step-by-step uncoordination of the pyrazolyl rings of hydrotris(pyrazolyl)borate ligands in comlexes of Rh and RhIII.

Compounds of rhodium(I) and rhodium(III) that contain ancillary hydrotris(pyrazolyl)borate ligands (Tp') react with monodentate and bidentate tertiary phosphanes in a step-wise manner, with incorporation of P-donor atoms and concomitant replacement of the Tp' pyrazolyl rings. Accordingly, [Rh(kappa3-TpMe2)(C2H4)(PMe3)] (1b), converts initially into [Rh(kappa2-TpMe2)-(PMe3)2] (3), and then into [Rh(kappa1-TpMe2)-(PMe3)3] (2) upon interaction with PMe3 at room temperature, in a process which can be readily reversed under appropriate experimental conditions. Full disengagement of the Tp' ligand is feasible to give Tp' salts of rhodium(I) complex cations, for example, [Rh(CO)(dppp)2]-[TpMe2,4-Cl] (5; dppp = Ph2P(CH2)3PPh2), or [Rh(dppp)2][TpMe2,4-Cl] (6). Bis(hydride) derivatives of rhodium(III) exhibit similar substitution chemistry, for instance, the neutral complex [Rh(Tp)-(H)2(PMe3)] reacts at 20 degrees C with an excess of PMe3 to give [Rh(H)2-(PMe3)4][Tp] (9b). Single-crystal X-ray studies of 9b, conducted at 143 K, demonstrate the absence of bonding interactions between the [Rh(H)2(PMe3)4]+ and Tp ions, the closest Rh...N contact being at 4.627 A.

Journal Article↗
PubMed2001

Synthesis and insertion reactions of the cyclometalated palladium-alkyl complexes Pd(CH(2)CMe(2)-o-C(6)H(4))L(2). Observation of a pentacoordinated intermediate in the insertion of SO(2).

Palladacycles of the type 2, have been synthesized by treatment of the corresponding chloroalkylcomplexes Pd(CH(2)CMe(2)C(6)H(5))ClL(2) (L(2) = (PMe(3))(2), 2a, or 1,5-cyclooctadiene (cod), 2b) with suitable bases, or by simple ligand exchange reactions from the cyclooctadiene derivative 2b. The metallacycles 2 react with activated alkynes and with sulfur dioxide, giving rise to different insertion products.

Journal Article↗
PubMed2001

Cathepsin B activity regulation. Heparin-like glycosaminogylcans protect human cathepsin B from alkaline pH-induced inactivation.

It has been shown that lysosomal cysteine proteinases, specially cathepsin B, has been implicated in a variety of diseases involving tissue remodeling states, such as inflammation, parasite infection, and tumor metastasis, by degradation of extracellular matrix components. Recently, we have shown that heparin and heparan sulfate bind to papain specifically; this interaction induces an increase of its alpha-helix content and stabilizes the enzyme structure even at alkaline pH (Almeida, P. C., Nantes, I. L., Rizzi, C. C. A., Júdice, W. A. S., Chagas, J. R., Juliano, L., Nader, H. B., and Tersariol, I. L. S. (1999) J. Biol. Chem. 274, 30433-30438). In the present work, a combination of circular dichroism analysis, affinity chromatography, cathepsin B mutants, and fluorogenic substrate assays were used to characterize the interaction of human cathepsin B with glycosaminoglycans. The nature of the cathepsin B-glycosaminoglycans interaction was sensitive to the charge and type of polysaccharide. Like papain, heparin and heparan sulfate bind cathepsin B specifically, and this interaction reduces the loss of cathepsin B alpha-helix content at alkaline pH. Our data show that the coupling of cathepsin B with heparin or heparan sulfate can potentiate the endopeptidase activity of the cathepsin B, increasing 5-fold the half-life (t(12)) of the enzyme at alkaline pH. Most of these effects are related to the interaction of heparin and heparan sulfate with His(111) residue of the cathepsin B occluding loop. These results strongly suggest that heparan sulfate may be an important binding site for cathepsin B at cell surface, reporting a novel physiological role for heparan sulfate proteoglycans.

Amino Acid Substitution↗
PubMed2001

Sulfogalactosylglycerolipid is involved in human gamete interaction.

Recent results from our laboratory have revealed the role of sulfogalactosylglycerolipid (SGG) in mouse sperm-zona pellucida (ZP) binding. In this report, we demonstrated the presence of SGG in Percoll-gradient centrifuged (PGC) human sperm by high performance thin layer chromatography with orcinol and Azure A staining, specific for glycolipids and sulfolipids, respectively. SGG in human PGC sperm was quantified by its affinity to Azure A to be 12-15 mol% of sperm lipids. Indirect immunofluorescence revealed that SGG existed on both live and aldehyde fixed human sperm in the head region. Pretreatment of human PGC sperm with affinity purified antiSGG Fab markedly inhibited sperm binding to the ZP in a concentration dependent manner, without any changes in the spontaneous acrosome rate or sperm motility parameters. Fluorescently labeled SGG liposomes also bound uniformly to isolated human ZP, while fluorescently labeled galactosylglycerolipid (GG, SGG's parental lipid) or phosphatidylserine (PS, negatively charged like SGG) liposomes did not. All of these results suggested the role of human sperm SGG in ZP binding.

Acrosome Reaction↗
PubMed2001

Influence of age on clinical and immunological characteristics of newly diagnosed type 1 diabetic patients.

The aim of this study was to analyse the immunological and clinical characteristics of a group of patients at the onset of type 1 diabetes and to determine if these findings are age related. For this purpose, 68 newly diagnosed type 1 diabetes mellitus patients referred to our hospital between 1997 and 1999 were studied; 42 were adults (mean age 24+/-3.5 years) and 26 children (mean age 6.1+/-4 years). Autoantibody markers islet cell antibodies, glutamic acid decarboxylase antibodies (GADA) and tyrosine phosphatase antibodies (IA-2A), pancreatic reserve (glucagon test) and HbA1c were determined. Some clinical characteristics, such as mode of presentation and insulin requirements, were also analysed. Type 1 diabetes mellitus was found to be autoimmune in 83.8% of the patients and idiopathic in 16.2%, without significant differences between adults and children. In the whole autoimmune group, GADA was more prevalent in adults and IA-2A more frequent in children. On the other hand, adults showing autoimmune markers developed ketosis more frequently and needed higher insulin doses at diagnosis, while children did not exhibit clinically significant differences associated with the presence or absence of antibodies. In conclusion, in children the presence of autoimmune markers is not related to the mode of presentation or characteristics of type 1 diabetes. In adults, however, the autoimmune group presents with more-severe clinical disease than antibody negative patients. Age at onset seems to be an important parameter in the natural history of type 1 diabetes and must be taken into account in epidemiological or intervention studies.

Adult↗
PubMed2001

Characterization of a Paramyxovirus from a Fer de Lance viper (Bothrops jararaca): partial nucleotide sequence of the putative fusion protein.

During the generation of Expressed Sequence Tags (ESTs) from the Fer de Lance viper (Bothrops jararaca) venom glands, a partial cDNA (clone H8) coding for a protein with all the features of a paramyxovirus fusion protein was characterized. It has 920 bp and codes for a partial protein of 279 amino acids. Two potential N-glycosylation sites are present in the sequence which also possesses a typical membrane anchoring domain made of a stretch of hydrophobic amino acids. The polyadenylation signal sequence was identified. When compared to other fusion proteins, it showed the highest sequence similarity (37-39%) with those of human parainfluenza 3 and Sendai virus.

Amino Acid Sequence↗
PubMed2001

Expression and processing of recombinant sarafotoxins precursor in Pichia pastoris.

Sarafotoxins are peptides isolated from the Atractaspis snake venom, with strong constrictor effect on cardiac and smooth muscle. They are structurally and functionally related to endothelins. The sarafotoxins precursor cDNA predicts an unusual structure 'rosary-type', with 12 successive similar stretches of sarafotoxin (SRTX) and spacer. In the present work, the recombinant precursor of SRTXs was sub-cloned and expressed in the yeast Pichia pastoris, and secreted to the culture medium. Characterization by SDS-PAGE, immunoblot, mass spectrometry and biological activity, suggests that intact precursor was expressed but processing into mature toxins also occurred. Furthermore, our results indicate that the correct proportion of sarafotoxin types as contained in the precursor, is obtained in the yeast culture medium. Contractile effects of the expressed toxins, on rat and Bothrops jararaca isolated aorta, were equivalent to 5x10(-10)M and 5x10(-11)M of sarafotoxin b, respectively. The enzymes responsible for the complete maturation of sarafotoxins precursor are still unknown. Our results strongly suggest that the yeast Pichia pastoris is able to perform such a maturation process. Thus, the yeast Pichia pastoris may offer an alternative to snake venom gland to tentatively identify the molecular process responsible for SRTXs release.

Animals↗
PubMed2000

Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2' and P3' variations in extended substrates.

We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S(4) and S(3) subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S(2)' and S(3)'. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P(4), P(3), P(2)' and P(3)' were made. The S(4) to S(2)' subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S(3)', indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S(4), S(3), S(2)' and S(3)' of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P(2)' respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P(3)'. Basic residues at P(3) and P(4) were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P(2)' (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His(111) (H111A) and His(110) (H110A) of cathepsin B led to an increase in k(cat) values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.

Amino Acid Sequence↗
PubMed2000

Substitution and hydrogenation reactions on rhodium(I)-ethylene complexes of the hydrotris(pyrazolyl)borate ligands T (T = Tp, TpMe2).

The bis(ethylene) Rh species TpMe2Rh(C2H4)2(1*) (TpMe2 = tris(3,5-dimethyl-1-pyrazol-1-yl)hydroborate) has been obtained from [RhCl(C2H4)2]2 and KTpMe2. Complex 1* easily decomposes in solution to give mainly the butadiene species TpMe2Rh(eta74-C4H6). In the solid state its thermal decomposition follows a different course and the allyl TpMe2RhH(syn-C3H4Me) is cleanly obtained as a mixture of exo and endo isomers. The complexes Tp'Rh(C2H4)2 (Tp' = Tp, TpMe2) afford the monosubstituted species Tp'Rh(C2H4)(PR3) upon reaction with PR3 but react differently with L = CO or CNR: the Tp compound gives dinuclear [TpRh]2(mu-L)3 complexes, while, in the case of 1*, TpMe2Rh(C2H4)(L) species are obtained. The ethylene ligand of complexes TpMe2Rh(C2H4)(PR3) is labile, and several peroxo compounds of composition TpMe2Rh(O2)(PR3) have been isolated by their reaction with O2. All the mononuclear Rh(I) complexes are formulated as 18e- trigonal bipyramidal species on the basis of IR and NMR spectroscopic studies. A series of dihydride complexes of Rh(III) of formulation Tp'RhH2(PR3) have been prepared by the hydrogenation of the corresponding ethylene derivatives. Complexes [TpRh]2(mu-CNCy)3, TpMe2Rh(C2H4)(PEt3), and TpMe2Rh(O2)(PEt3) have been further characterized by X-ray diffraction studies.

Journal Article↗
PubMed2000

Denticity Changes of Hydrotris(pyrazolyl)borate Ligands in Rh(I) and Rh(III) Compounds: From kappa(3)- to Ionic "kappa(0)"-Tp'

Isolated hydrotris(pyrazolyl)borate anions Tp' were obtained as salts of metal complex cations (see picture) by the displacement of Rh-coordinated kappa(3)-N,N',N"-Tp' by PMe(3) (Tp'=Tp and Tp(Me2)). With [(kappa(3)-Tp(Me2))Rh(C(2)H(4))(2)], stepwise diplacement of the Tp(Me2) ligand allowed the isolation of complexes exhibiting the kappa(2)- Tp(Me2) and kappa(1)-Tp(Me2) coordination modes.

Journal Article↗
PubMed1998

Autocatalytic processing of recombinant human procathepsin L. Contribution of both intermolecular and unimolecular events in the processing of procathepsin L in vitro.

The autocatalytic processing of procathepsin L was investigated in vitro using purified recombinant proenzyme expressed in Pichia pastoris. Pure intermolecular processing was studied by incubating the mutant procathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L. The results clearly establish that, contrary to recent reports, intermolecular processing of procathepsin L is possible. The main cleavage sites are located at or near the N terminus of the mature enzyme, in an accessible portion of the proregion, which contains sequences corresponding to the known substrate specificity of cathepsin L. Contrary to procathepsins B, K, and S, autocatalytic processing of procathepsin L can generate the natural mature form of the enzyme. A continuous assay using the substrate benzyloxycarbonyl-Phe-Arg 4-methylcoumarinyl-7-amide hydrochloride has also been used to obtain information on the nature of the steps involved in the autocatalytic processing of wild-type procathepsin L. Processing is initiated by decreasing the pH from 8.0 to 5.3. The influence of proenzyme concentration on the rate of processing indicates the existence of both unimolecular and bimolecular steps in the mechanism of processing. The nature of the unimolecular event that triggers processing remains elusive. Circular dichroism and fluorescence measurements indicate the absence of large scale conformational change in the structure of procathepsin L on reduction of pH. However, the bimolecular reaction can be attributed to intermolecular processing of the zymogen.

Amino Acid Sequence↗
PubMed1998

Object-based perceptual grouping affects negative priming.

In 5 experiments the authors examine the role of object-based grouping on negative priming. The experiments used a letter-matching task with multiple letters presented in temporally separated prime and probe displays. On mismatch trials, distractor letters in primes were repeated as targets in probes, or distractor and target letters were completely different. Negative priming was shown by slowed responses when distractors were repeated as targets relative to when the stimuli differed. This occurred both when only letters were presented (Experiments 1 and 4) and when letters were surrounded by boxes (Experiment 5). Experiments 2, 3, and 4 showed that negative priming was affected by the grouping of target and distractor letters in prime displays. Negative priming was reduced when 1 of the distractor letters was placed in the target box and 1 was left outside the box; facilitatory priming was observed when both distractor letters appeared in the target box. The data were accounted for in terms of there being (a) object-based competition for visual selection, (b) inhibition of distractor objects that compete for selection with target objects, and (c) activation or inhibition of the identities of all component elements within target or distractor objects.

Adult↗
PubMed1998

[Significance of the determination of antineutrophil cytoplasmic antibodies (ANCA) in ulcerative colitis and Crohn's disease].

We have studied prospectively 126 consecutive patients recruited with a known diagnosis of ulcerative colitis (UC; n = 78) and Crohn's disease (CD; n = 48) for anti-neutrophil cytoplasmatic antibodies (ANCA) by indirect immunofluorescence (IFI). Forty-six percent of UC and 18% of CD patients were found positive. The sensitivity and specificity for UC diagnosis were 0.46 and 0.81, respectively. We evaluated the pattern of IFI exhibited (perinuclear: pANCA and cytoplasmatic: cANCA). cANCA was found in 77% of CD and in only 30% of UC patients (p = 0.01). Sera from all CD patients were positive at a 1:20 dilution (and not at higher dilution) and it occurred in only in 14 UC patients (30%). Positive sera were also tested to characterize the antigen specificity by enzyme-linked immunosorbent assay (ELISA) but the antigenic nature of ANCA could not be identified in most cases. No differences were found between ANCA positive and ANCA negative patients regarding colonic extension (UC) or colonic involvement (CD), activity and colectomy. We conclude that ANCA may be a helpful diagnostic test in UC patients but it not seems to be important as a marker of activity. ANCA positivity can reflect disease heterogeneity in UC patients, perhaps discriminating those with immunologic disturbances.

Adolescent↗
PubMed1997

Major increase in endopeptidase activity of human cathepsin B upon removal of occluding loop contacts.

The main feature distinguishing cathepsin B from other cysteine proteases of the papain family is the presence of a large insertion loop, termed the occluding loop, which occupies the S' subsites of the enzyme. The loop is held in place mainly by two contacts with the rest of the enzyme, involving residues His110 and Arg116 on the loop that form salt bridges with Asp22 and Asp224, respectively. The influence of this loop on the endopeptidase activity of cathepsin B has been investigated using site-directed mutagenesis and internally quenched fluorogenic (IQF) substrates. Wild-type cathepsin B displays poor activity against the substrates Abz-AFRSAAQ-EDDnp and Abz-QVVAGA-EDDnp as compared to cathepsin L and papain. Appreciable increases in kcat/KM were observed for cathepsin B containing the single mutations D22A, H110A, R116A, and D224A. The highest activity however is observed for mutants where both loop to enzyme contacts are disrupted. For the triple-mutant D22A/H110A/R116A, an optimum kcat/KM value of 12 x 10(5) M-1 s-1 was obtained for hydrolysis of Abz-AFRSAAQ-EDDnp, which corresponds to a 600-fold increase relative to wild-type cathepsin B and approaches the level of activity observed with cathepsin L or papain. By comparison, the mutations have little effect on the hydrolysis of Cbz-FR-MCA. The influence of the mutations on the pH dependency of activity also indicates that the complexity of pH activity profiles normally observed for cathepsin B is related to the presence of the occluding loop. The major increase in endopeptidase activity is attributed to an increase in loop "flexibility" and suggests that the occluding loop might move when an endopeptidase substrate binds to the enzyme. The possible contribution of these interactions in regulating endopeptidase activity and the implications for cathepsin B activity in physiological or pathological conditions are discussed.

Cathepsin B↗