Uremic CPPs and EVs are important players in the mechanisms of widespread calcification in CKD. We propose a major role for cGRP as inhibitory factor to prevent calcification at systemic and tissue levels.

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 γ-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, γ-carboxylated GRP but not undercarboxylated GRP was shown to inhibit calcification and osteochondrogenic differentiation through α-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 γ-carboxylation status, with potential clinical relevance.

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.

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Iron−sulfur clusters with [3Fe−4S] cores are widely distributed in biological systems. In the oxidized state, designated [3Fe−4S]+, these electron-transfer agents have an electronic ground state with S = 1/2, and they exhibit EPR signals centered at g = 2.01. It has been established by Mössbauer spectroscopy that the three iron sites of the cluster are high-spin Fe3+, and the general properties of the S = 1/2 ground state have been described with the exchange Hamiltonian H exch = J 12 S 1·S 2 + J 23 S 2·S 3 + J 13 S 1·S 3. Some [3Fe−4S]+ clusters (type 1) have their g-values confined to the range between g = 2.03 and 2.00 while others (type 2) exhibit a continuous distribution of g-values down to g ≈ 1.85. Despite considerable efforts in various laboratories no model has emerged that explains the g-values of type 2 clusters. The 4.2 K spectra of all [3Fe−4S]+ clusters have broad features which have been simulated in the past by using 57Fe magnetic hyperfine tensors with anisotropies that are unusually large for high-spin ferric sites. It is proposed here that antisymmetric exchange, H AS = d·(S 1 × S 2 + S 2 × S 3 + S 3 × S 1), is the cause of the g-value shifts in type 2 clusters. We have been able to fit the EPR and Mössbauer spectra of the 3Fe clusters of beef heart aconitase and Desulfovibrio gigas ferredoxin II by using antisymmetric exchange in combination with distributed exchange coupling constants J 12, J 13, and J 23 (J-strain). While antisymmetric exchange is negligible for aconitase (which has a type 1 cluster), fits of the ferredoxin II spectra require |d| ≈ 0.4 cm-1. Our studies show that the data of both proteins can be fit using the same isotropic 57Fe magnetic hyperfine coupling constant for the three cluster sites, namely a = −18.0 MHz for aconitase and a = −18.5 MHz for the D. gigas ferredoxin. The effects of antisymmetric exchange and J-strain on the Mössbauer and EPR spectra are discussed.

Dissimilatory nitrite reduction, carried out by hexaheme proteins, gives ammonia as the final product. Representatives of this enzyme group from 3 bacterial species can also reduce NO to either ammonia or NzO. The redox regulation of the nitrite/nitric oxide activities is discussed in the context of the denitrifying pathway, [25]. Two pairs of magneti~~ly interacting hemes (low-spin/low-spin; low-spinlhighspin) operate in this complex, The high-spin heme binds NO and appears to be the enzyme-active site. Reaction of nitrite with fuIly reduced enzyme reoxidizes the lowspin hemes, but the EPR spectrum reveals persistence of the high-spin heme in the NO-bound form. Since the six-electron reduction of nitrite yields no NO as a free intermediate [25,26], NO appears to exist as an enzymebound and not as a gaseous product during the nitriteto-ammonia transit.Ammonia-generating nitrite reductases have typically been purified from spinach and display a complex active site comprised of a siroheme coupled to a single {4Fe,4S] center fl9]. Nitrite reduction by hexaheme enzymes from strictly and facultatively anaerobic bacteria, such as Desuvovibrio desulfuricans (ATTC 27774) [20], Woiinellu succinogenes [21j, Escherichia co& [223, and V. jkcfzeri [24], also give rise directly to ammonia. In concert, EPR andIn view of such involvement of NO with the hexaheme nitrite reductases, we have studied the capacity of representatives of this group to reduce free NO. Their influence in the pathway, NOz-to Nz is discussed as is the redox regulation of the nitrite/nitric oxide reduction activities in these multi-heme systems. MATERIALS AND METHODS

With the implementation of a computerized maintenance management system for a corporate perspective in the Organization ANA, SA. Aeroportos de Portugal, combined with several parameterizations related to core business processes in place, naturally there was a set of opportunities to improve business processes through research and metrics development allowing more analyses. These were previously dealt without the same depth and without access to the same database and data model. On this basis, the idealization of the maintenance management procedure, based on the framework for maintenance management in force in the organization, moved quickly to the reality. The development of the procedure starts by identifying problems, setting goals and objectives to be achieved, research literature in maintenance area and best practice in service management with a focus on ITIL, study existing maintenance standards with respect to concepts and KPI calculation methods, presentation of business processes in production at the Organization, development of the procedure for maintenance management with appropriated methods and outcome analysis, and finally the main conclusions of the work. A major contribution of this work has been considered a series of actions to improve and/or correct existing business processes that may prove technical, organizational and economic benefits for the maintenance management in that Organization.

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 hemebinding 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 ␤-barrel flanked by two long ␣-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 ␣ plus ␤ fold in a eukaryotic protein. Chemical shift mapping localized the tetrapyrrole binding site to a hydrophobic cleft formed by residues from helix ␣ A 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.Heme synthesis occurs mainly in erythroid cells (ϳ85%) and hepatocytes, although heme is synthesized in virtually all tissues. In hepatocytes, heme is required for incorporation into cytochromes, in particular, the P450 class of cytochromes that are important for detoxification. Numerous other cytochromes of the oxidative-phosphorylation pathway also contain heme. 5-Aminolevulinic acid synthase (ALAS) 2 catalyzes the first, and rate-limiting, step in hepatic heme biosynthesis. Hemin, the Fe(III) oxidation product of heme, acts as a feedback inhibitor of ALAS as well as an inhibitor of mitochondrial transport of ALAS; via an interaction with the ALAS presequence (1), hemin prevents ALAS from reaching its mitochondrial, mature, active form (2).Because porphyrins and metallated porphyrins (e.g. heme) are extremely reactive and poorly soluble in aqueous solution under physiological conditions, it was hypothesized that one or more intracellular heme-binding proteins act as a buffer during induced heme synthesis (3). p22HBP, a 22-kDa protein, was first purified from mouse liver cell extracts and characterized as a cytosolic, heme-binding protein by Taketani et al. in 1998 (4). Blackmon et al. (3) subsequently determined that p22HBP binds other tetrapyrroles in addition to hemin, although its functional role in the cell remains unknown. However, a recent proteomic study, involving metabolic labeling with 59 Fe-hemin of murine erythroleukemia cells induced to undergo differentiation, demonstrated that p22HBP is a component in one of the four identified multiprotein complexes related to hemoglobin biosynthesis (5). The investigat...