Anjos L. Macedo

A randomized trial for the study of the elective surgical treatment of portal hypertension in mansonic schistosomiasis.

From 1977 to 1983, 94 patients with esophageal varices and gastrointestinal bleeding secondary to mansonic schistosomiasis were entered into a prospective randomized trial comparing the three operations mainly used in Brazil: esophagogastric devascularization associated with splenectomy (EGDS, 32 patients), classical splenorenal shunt (SRS, 32 patients), and distal splenorenal shunt (DSRS, 30 patients). The randomization was interrupted because of a significant incidence of portosystemic encephalopathy (PSE) in the SRS group (26%), as compared to the DSRS (7%) and EGDS (0%) groups. The rate of rebleeding was the same in the three groups, but the rate of failure, as defined by the presence of technical problems, postoperative complications, or death, was significantly higher in the SRS group. This 2-year follow-up shows that SRS should be abandoned in hepatosplenic schistosomiasis and that a comparison between DSRS and EGDS with a longer follow-up is urgently needed.

Gla-Rich Protein Acts as a Calcification Inhibitor in the Human Cardiovascular System

Objective—Vascular and valvular calcifications are pathological processes regulated by resident cells, and depending on a complex interplay between calcification promoters and inhibitors, resembling skeletal metabolism. Here, we study the role of the vitamin K–dependent Gla-rich protein (GRP) in vascular and valvular calcification processes. Approach and Results—Immunohistochemistry and quantitative polymerase chain reaction showed that GRP expression and accumulation are upregulated with calcification simultaneously with osteocalcin and matrix Gla protein (MGP). Using conformation-specific antibodies, both &ggr;-carboxylated GRP and undercarboxylated GRP species were found accumulated at the sites of mineral deposits, whereas undercarboxylated GRP was predominant in calcified aortic valve disease valvular interstitial cells. Mineral-bound GRP, MGP, and fetuin-A were identified by mass spectrometry. Using an ex vivo model of vascular calcification, &ggr;-carboxylated GRP but not undercarboxylated GRP was shown to inhibit calcification and osteochondrogenic differentiation through &agr;-smooth muscle actin upregulation and osteopontin downregulation. Immunoprecipitation assays showed that GRP is part of an MGP–fetuin-A complex at the sites of valvular calcification. Moreover, extracellular vesicles released from normal vascular smooth muscle cells are loaded with GRP, MGP, and fetuin-A, whereas under calcifying conditions, released extracellular vesicles show increased calcium loading and GRP and MGP depletion. Conclusions—GRP is an inhibitor of vascular and valvular calcification involved in calcium homeostasis. Its function might be associated with prevention of calcium-induced signaling pathways and direct mineral binding to inhibit crystal formation/maturation. Our data show that GRP is a new player in mineralization competence of extracellular vesicles possibly associated with the fetuin-A–MGP calcification inhibitory system. GRP activity was found to be dependent on its &ggr;-carboxylation status, with potential clinical relevance.

Molecular determinants of ligand specificity in family 11 carbohydrate binding modules – an NMR, X‐ray crystallography and computational chemistry approach

The direct conversion of plant cell wall polysaccharides into soluble sugars is one of the most important reactions on earth, and is performed by certain microorganisms such as Clostridium thermocellum (Ct). These organisms produce extracellular multi‐subunit complexes (i.e. cellulosomes) comprising a consortium of enzymes, which contain noncatalytic carbohydrate‐binding modules (CBM) that increase the activity of the catalytic module. In the present study, we describe a combined approach by X‐ray crystallography, NMR and computational chemistry that aimed to gain further insight into the binding mode of different carbohydrates (cellobiose, cellotetraose and cellohexaose) to the binding pocket of the family 11 CBM. The crystal structure of C. thermocellum CBM11 has been resolved to 1.98 Å in the apo form. Since the structure with a bound substrate could not be obtained, computational studies with cellobiose, cellotetraose and cellohexaose were carried out to determine the molecular recognition of glucose polymers by CtCBM11. These studies revealed a specificity area at the CtCBM11 binding cleft, which is lined with several aspartate residues. In addition, a cluster of aromatic residues was found to be important for guiding and packing of the polysaccharide. The binding cleft of CtCBM11 interacts more strongly with the central glucose units of cellotetraose and cellohexaose, mainly through interactions with the sugar units at positions 2 and 6. This model of binding is supported by saturation transfer difference NMR experiments and linebroadening NMR studies.

Copper complexes with bibracchial lariat ethers: from mono- to binuclear structures

Abstract Copper(II) complexes with a series of bibracchial lariat ethers are described. Independently of the nature of the counterion present (nitrate or perchlorate), the lariat ether N,N′-bis(2-aminobenzyl)-1,10-diaza-15-crown-5 (L1) always forms mononuclear complexes, whereas the lariat ethers N,N′-bis(2-aminobenzyl)-4,13-diaza-18-crown-6 (L2) and N,N′-bis(2-salicylaldiminobenzyl)-4,13-diaza-18-crown-6 (L3) only give binuclear compounds. The X-ray crystal structure of [CuL1](ClO4)2 shows a seven-coordinated copper(II) ion in a distorted (axially compressed) pentagonal-bipyramidal geometry. The X-ray crystal structure of [Cu2(L3-2H)](ClO4)2 confirms the binuclear nature of the compound with both metal ions having identical coordination environments and each one placed out of the crown hole but efficiently encapsulated by the corresponding pendant arm; each copper(II) ion is five-coordinated with an intermediate geometry between trigonal-bipyramidal and square-pyramidal (τ=0.40). The EPR spectra in frozen solution samples are in accordance with a stable coordinate pattern for the metal centre of ligand L1, yielding a rhombic distorted complex with axial compression in solution, in agreement with the X-ray crystal structure of [CuL1](ClO4)2. For the binuclear complexes of L2 and L3, the Cu(II) centres in solution can be distorted from their tetragonally elongated structures via interaction with ethanol and/or the nitrate counterion, leading to more than one species.

Redox properties of Desulfovibrio gigas [Fe3S4] and [Fe4S4] ferredoxins and heterometal cubane-type clusters formed within the [Fe3S4] core. Square wave voltammetric studies

The same polypeptide chain (58 amino acids, 6 cysteines) is used to build up two ferredoxins in Desulfovibrio gigas a sulfate reducing organism. Ferredoxin II (FdII) contains a single [Fe3S4] core and ferredoxin I (FdI) mainly a [Fe4S4] core. The [Fe3S4] core can readily be interconverted into a [Fe4S4] complex (J.J.G. Moura, I. Moura, T.A. Kent, J.D. Lipscomb, B.H. Huynh, J. LeGall, A.V. Xavier, and E. Munck, J. Biol. Chem. 257, 6259 (1982)). This interconversion process suggested that the [Fe3S4] core could be used as a synthetic precursor for the formation of heterometal clusters. Co, Zn, Cd, and Ni derivatives were produced (I. Moura, J.J.G. Moura, E. Munck, V. Papaephthymiou, and J. LeGall, J. Am. Chem. Soc. 108, 349 (1986), K. Sureurs, E. Munck, I. Moura, J.J.G. Moura, and J. LeGall, J. Am. Chem. Soc. 109, 3805 (1986), and A.L. Macedo, I. Moura, J.J.G. Moura, K. Surerus, and E. Munck, unpublished results). The redox properties of a series of heterometal clusters (MFe3S4] are assessed using direct electrochemistry (square wave voltammetry--SWV) promoted by Mg(II) at a glassy carbon electrode (derivatives: Cd (-495 mV), Fe (-420 mV), Ni (-360 mV), and Co (-245 mV) vs normal hydrogen electrode (NHE)). In parallel, the electrochemical behavior (cyclic voltammetry--CV, differential pulse voltammetry--DPV and SWV) of FdI and FdII were investigated as well as the cluster interconversion process. In addition to the +1/0 (3Fe cluster) and +2/+1 (4Fe cluster) redox transitions, a very negative redox step, at -690 mV, was detected for the 3Fe core, reminiscent of a postulated further 2e- reduction step, as proposed for D. africanus ferredoxin III by F.A. Armstrong, S.J. George, R. Cammack, E.C. Hatchikian, and A.J. Thomson, Biochem. J. 264, 265 (1989). The electrochemical redox potential values are compared with those determined by independent methods (namely by electron paramagnetic resonance (EPR) and visible spectroscopy).

The Solution Structure and Heme Binding of the Presequence of Murine 5-Aminolevulinate Synthase

The mitochondrial import of 5‐aminolevulinate synthase (ALAS), the first enzyme of the mammalian heme biosynthetic pathway, requires the N‐terminal presequence. The 49 amino acid presequence transit peptide (psALAS) for murine erythroid ALAS was chemically synthesized, and circular dichroism and 1H nuclear magnetic resonance (NMR) spectroscopies used to determine structural elements in trifluoroethanol/H2O solutions and micellar environments. A well defined amphipathic α‐helix, spanning L22 to F33, was present in psALAS in 50% trifluoroethanol. Further, a short α‐helix, defined by A5–L8, was also apparent in the 26 amino acid N‐terminus peptide, when its structure was determined in sodium dodecyl sulfate. Heme inhibition of ALAS mitochondrial import has been reported to be mediated through cysteine residues in presequence heme regulatory motifs (HRMs). A UV/visible and 1H NMR study of hemin and psALAS indicated that a heme–peptide interaction occurs and demonstrates, for the first time, that heme interacts with the HRMs of psALAS.

The First Structure from the SOUL/HBP Family of Heme-binding Proteins, Murine P22HBP

Murine p22HBP, a 22-kDa monomer originally identified as a cytosolic heme-binding protein ubiquitously expressed in various tissues, has 27% sequence identity to murine SOUL, a heme-binding hexamer specifically expressed in the retina. In contrast to murine SOUL, which binds one heme per subunit via coordination of the Fe(III)-heme to a histidine, murine p22HBP binds one heme molecule per subunit with no specific axial ligand coordination of the Fe(III)-heme. Using intrinsic protein fluorescence quenching, the values for the dissociation constants of p22HBP for hemin and protoporphyrin-IX were determined to be in the low nanomolar range. The three-dimensional structure of murine p22HBP, the first for a protein from the SOUL/HBP family, was determined by NMR methods to consist of a 9-stranded distorted β-barrel flanked by two long α-helices. Although homologous domains have been found in three bacterial proteins, two of which are transcription factors, the fold determined for p22HBP corresponds to a novel α plus β fold in a eukaryotic protein. Chemical shift mapping localized the tetrapyrrole binding site to a hydrophobic cleft formed by residues from helix αA and an extended loop. In an attempt to assess the structural basis for tetrapyrrole binding in the SOUL/HBP family, models for the p22HBP-protoporphyrin-IX complex and the SOUL protein were generated by manual docking and automated methods.

The solution structure of a [3Fe-4S] ferredoxin: oxidised ferredoxin II from Desulfovibrio gigas.

Abstract The use of standard 2D NMR experiments in combination with 1D NOE experiments allowed the assignment of 51 of the 58 spin systems of oxidised [3Fe-4S] ferredoxin isolated from Desulfovibrio gigas. The NMR solution structure was determined using data from 1D NOE and 2D NOESY spectra, as distance constraints, and information from the X-ray structure for the spin systems not detected by NMR in torsion angle dynamics calculations to produce a family of 15 low target function structures. The quality of the NMR family, as judged by the backbone r.m.s.d. values, was good (0.80 Å), with the majority of φ/ψ angles falling within the allowed region of the Ramachandran plot. A comparison with the X-ray structure indicated that the overall global fold is very similar in solution and in the solid state. The determination of the solution structure of ferredoxin II (FdII) in the oxidised state (FdIIox) opens the way for the determination of the solution structure of the redox intermediate state of FdII (FdIIint), for which no X-ray structure is available.

Solution structure of hirsutellin A – new insights into the active site and interacting interfaces of ribotoxins

Hirsutellin (HtA) is intermediate in size between other ribotoxins and less specific microbial RNases, and thus offers a unique chance to determine the minimal structural requirements for activities unique to ribotoxins. Here, we have determined the structure of HtA by NMR methods. The structure consists of one α‐helix, a helical turn and seven β‐strands that form an N‐terminal hairpin and an anti‐parallel β‐sheet, with a characteristic α + β fold and a highly positive charged surface. Compared to its larger homolog α‐sarcin, the N‐terminal hairpin is shorter and less positively charged. The secondary structure elements are connected by large loops with root mean square deviation (rmsd) values > 1 Å, suggesting some degree of intrinsically dynamic behavior. The active site architecture of HtA is unique among ribotoxins. Compared to α‐sarcin, HtA has an aspartate group, D40, replacing a tyrosine, and the aromatic ring of F126, located in the leucine ‘environment’ close to the catalytic H113 in a similar arrangement to that found in RNase T1. This unique active site structure is discussed in terms of its novel electrostatic interactions to understand the efficient cytotoxic activity of HtA. The contributions of the N‐terminal hairpin, loop 2 and loop 5 with regard to protein functionality, protein–protein and protein–lipid interactions, are also discussed. The truncation and reduced charge of the N‐terminal hairpin in HtA may be compensated for by the extension and new orientation of its loop 5. This novel orientation of loop 5 re‐establishes a positive charge on the side of the molecule that has been shown to be important for intermolecular interactions in ribotoxins.

Gla-rich protein function as an anti-inflammatory agent in monocytes/macrophages : Implications for calcification-related chronic inflammatory diseases

Calcification-related chronic inflammatory diseases are multifactorial pathological processes, involving a complex interplay between inflammation and calcification events in a positive feed-back loop driving disease progression. Gla-rich protein (GRP) is a vitamin K dependent protein (VKDP) shown to function as a calcification inhibitor in cardiovascular and articular tissues, and proposed as an anti-inflammatory agent in chondrocytes and synoviocytes, acting as a new crosstalk factor between these two interconnected events in osteoarthritis. However, a possible function of GRP in the immune system has never been studied. Here we focused our investigation in the involvement of GRP in the cell inflammatory response mechanisms, using a combination of freshly isolated human leucocytes and undifferentiated/differentiated THP-1 cell line. Our results demonstrate that VKDPs such as GRP and matrix gla protein (MGP) are synthesized and γ-carboxylated in the majority of human immune system cells either involved in innate or adaptive immune responses. Stimulation of THP-1 monocytes/macrophages with LPS or hydroxyapatite (HA) up-regulated GRP expression, and treatments with GRP or GRP-coated basic calcium phosphate crystals resulted in the down-regulation of mediators of inflammation and inflammatory cytokines, independently of the protein γ-carboxylation status. Moreover, overexpression of GRP in THP-1 cells rescued the inflammation induced by LPS and HA, by down-regulation of the proinflammatory cytokines TNFα, IL-1β and NFkB. Interestingly, GRP was detected at protein and mRNA levels in extracellular vesicles released by macrophages, which may act as vehicles for extracellular trafficking and release. Our data indicate GRP as an endogenous mediator of inflammatory responses acting as an anti-inflammatory agent in monocytes/macrophages. We propose that in a context of chronic inflammation and calcification-related pathologies, GRP might act as a novel molecular mediator linking inflammation and calcification events, with potential therapeutic application.