Zinc transport and metallothionein secretion in the intestinal human cell line Caco-2.

Caco-2, a human cell line, displays several biochemical and morphological characteristics of differentiated enterocytes. Among these is the ability to transport zinc from the apical to the basal compartment. This process was enhanced following exposure by the apical compartment to increasing concentrations of the metal. High pressure liquid chromatography fractionation of the media obtained from cells labeled with radioactive zinc showed that metallothioneins (MTs), small metal-binding, cysteine-rich proteins), were present in the apical and basal media of controls as well as in cells grown in the presence of high concentrations of zinc. Following exposure to the metal, the levels of Zn-MTs in the apical medium increased, while in the basal compartment the greatest part of zinc appeared in a free form with minor changes in the levels of basal MTs. Metabolic labeling experiments with radioactive cysteine confirmed the apical secretion of MTs. A stable transfectant clone of Caco-2 cells (CL11) was selected for its ability to express constitutively high levels of the mouse metallothionein I protein. This cell line showed an enhanced transport of the metal following exposure to high concentrations of zinc and a constitutive secretion of the mouse metallothionein I protein in the apical compartment. Together, these findings strongly support the hypothesis of a functional role between the biosynthesis and secretion of MTs and the transport of zinc in intestinal cells

Spectroscopic and metal binding properties of a de novo metalloprotein binding a tetrazinc cluster

De novo design provides an attractive approach, which allows one to test and refine the principles guiding metalloproteins in defining the geometry and reactivity of their metal ion cofactors. Although impressive progress has been made in designing proteins that bind transition metal ions including iron–sulfur clusters, the design of tetranuclear clusters with oxygen‐rich environments remains in its infancy. In previous work, we described the design of homotetrameric four‐helix bundles that bind tetra‐Zn2+ clusters. The crystal structures of the helical proteins were in good agreement with the overall design, and the metal‐binding and conformational properties of the helical bundles in solution were consistent with the crystal structures. However, the corresponding apo‐proteins were not fully folded in solution. In this work, we design three peptides, based on the crystal structure of the original bundles. One of the peptides forms tetramers in aqueous solution in the absence of metal ions as assessed by CD and NMR. It also binds Zn2+ in the intended stoichiometry. These studies strongly suggest that the desired structure has been achieved in the apo state, providing evidence that the peptide is able to actively impart the designed geometry to the metal cluster

The complex interplay among atherosclerosis, inflammation, and degeneration in ascending thoracic aortic aneurysms

OBJECTIVE: To assess the histopathological findings of a large series of ascending thoracic aortic aneurysm (TAA) surgical specimens applying the updated classification on noninflammatory degenerative and inflammatory aortic diseases proposed by the Association for European Cardiovascular Pathology and the Society for Cardiovascular Pathology clinicopathological correlations. METHODS: A total of 255 patients surgically treated for ascending TAA were enrolled. Surgical ascending aorta specimens were examined. RESULTS: The histopathological substrate of ascending TAAs was mainly degenerative (67.5%), but with a remarkable prevalence of atherosclerotic lesions (18.8%) and aortitis (13.7%). Degenerative patients more frequently had bicuspid aortic valve (37.2%; P = .002). Patients in the atherosclerotic group were older (median age, 69 years; P < .001), more often with a history of hypertension (87.5%; P = .059), hypercholesterolemia (75%; P = .019), diabetes (16.6%; P = .054), current smoking (22.9%; P = .066), and a history of coronary artery disease (18.7%; P = .063). Patients with aortitis represented the older group (median age, 75 years, P < .001), were mostly females (68.6%; P < .001), and had a larger ascending aorta diameter (median, 56 mm; P < .001). Both patients with atherosclerosis and aortitis presented a higher incidence of concomitant abdominal aortic aneurysm (20.8% and 22.8%, respectively; P < .001). CONCLUSIONS: Although degenerative histopathology is the most frequent substrate in ascending TAA, atherosclerosis and inflammation significantly contribute to the development of chronic aortic thoracic disease

Amyloidosis: What does pathology offer? The evolving field of tissue biopsy

Since the mid-nineteenth century pathology has followed the convoluted story of amyloidosis, recognized its morphology in tissues and made identification possible using specific staining. Since then, pathology studies have made a significant contribution and advanced knowledge of the disease, so providing valuable information on the pathophysiology of amyloid aggregation and opening the way to clinical studies and non-invasive diagnostic techniques. As amyloidosis is a heterogeneous disease with various organ and tissue deposition patterns, histology evaluation, far from offering a simple yes/no indication of amyloid presence, can provide a wide spectrum of qualitative and quantitative information related to and changing with the etiology of the disease, the comorbidities and the clinical characteristics of patients. With the exception of cardiac transthyretin related amyloidosis cases, which today can be diagnosed using non-biopsy algorithms when stringent clinical criteria are met, tissue biopsy is still an essential tool for a definitive diagnosis in doubtful cases and also to define etiology by typing amyloid fibrils. This review describes the histologic approach to amyloidosis today and the current role of tissue screening biopsy or targeted organ biopsy protocols in the light of present diagnostic algorithms and various clinical situations, with particular focus on endomyocardial and renal biopsies. Special attention is given to techniques for typing amyloid fibril proteins, necessary for the new therapies available today for cardiac transthyretin related amyloidosis and to avoid patients receiving inappropriate chemotherapy in presence of plasma cell dyscrasia unrelated to amyloidosis. As the disease is still burdened with high mortality, the role of tissue biopsy in early diagnosis to assure prompt treatment is also mentioned

A biomimetic metalloporphyrin catalyzes indole oxidation with high selectivity

Indole is one of the most common heterocyclic scaffolds available in nature. It occurs in several natural compounds, including alkaloids, plant hormones, flower scents and dyes.1 Despite the structural simplicity of this molecule, indole oxidation commonly results in the formation of a large number of products, including the 2- or 3-oxygenated compounds, di-oxygenated and more complex molecules. For this reason, indole oxidation has become a widespread model reaction to test the efficacy of both biological catalysts2,3 and their synthetic analogues.4,5 Most of the catalysts examined so far gave poor selectivity toward any of the oxidation products.2-5 Here we present the results concerning oxidation of indole and its derivatives catalyzed by Mn-Mimochrome VI*a (Mn-MC6*a). Mn-MC6*a is a synthetic peptide-porphyrin conjugate conceived to act as a miniaturized heme-protein model.6 Mn-MC6*a is able to oxidize indole under unprecedented site-selective conditions, yielding to 3-oxindolenine as single product. Additionally, the reaction selectivity is dramatically altered when 1- or 3-methyl-substituted indoles are used as substrates. The formation and isolation of the reactive 3-oxindolenine is highly important, since it is believed to represent a useful synthon in organic synthesis. Accordingly, the exploitation of its reactivity with nucleophiles, in order to provide one pot transformations, is currently ongoing, with the aim to further increase the synthetic potential of our catalyst. 1. Burton, T.C. in Heterocyclic scaffolds II: Reactions and applications of indoles; Gribble, G.W., Ed.; Springer-Verlag Berlin Heidelberg, 2011. 2. Kuo, H. H. and Mauk, A. G.; Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 13966–13971. 3. Barrios, D. A. et al. J. Am. Chem. Soc. 2014, 136, 7914-7925. 4. Linhares, M. et al. Appl. Catal. A. 2014, 470, 427–433. 5. Poon L. C.-H. et al. J. Am. Chem. Soc. 2011, 133, 1877–1884. 6. Caserta, G. et al. ChemBioChem 2018 (doi: 10.1002/cbic.201800200

Occurrence of Fatal and Nonfatal Adverse Outcomes after Heart Transplantation in Patients with Pretransplant Noncytotoxic HLA Antibodies

HLA antibodies (HLA ab) in transplant candidates have been associated with poor outcome. However, clinical relevance of noncytotoxic antibodies after heart transplant (HT) is controversial. By using a Luminex-based HLA screening, we retested pretransplant sera from HT recipients testing negative for cytotoxic HLA ab and for prospective crossmatch. Out of the 173 consecutive patients assayed (52±13y; 16% females; 47% ischemic etiology), 32 (18%) showed pretransplant HLA ab, and 12 (7%) tested positive against both class I and class II HLA. Recipients with any HLA ab had poorer survival than those without (65±9 versus 82±3%; P=0.02), accounting for a doubled independent mortality risk (P=0.04). In addition, HLA-ab detection was associated with increased prevalence of early graft failure (35 versus 15%; P=0.05) and late cellular rejection (29 versus 11%; P=0.03). Of the subgroup of 37 patients suspected for antibody mediated rejection (AMR), the 9 with pretransplant HLA ab were more likely to display pathological AMR grade 2 (P=0.04). By an inexpensive, luminex-based, HLA-screening assay, we were able to detect non-cytotoxic HLA ab predicting fatal and nonfatal adverse outcomes after heart transplant. Allocation strategies and desensitization protocols need to be developed and prospectively tested in these patients

Site-selective indole oxidation catalyzed by a Mn-containing artificial metalloenzyme

Metalloenzymes have become attractive tools for application in oxidation catalysis, since a complex protein environment exerts a highly specific control on the reactivity of the metal center.1 Compared to synthetic catalysts, enzymes cover only a limited repertoire of reactions and substrates. The development of hybrid catalysts, obtained by anchoring catalytic metal complexes to native or artificial biomolecular scaffolds, is aimed at merging the advantages of both systems while overcoming the drawbacks.2,3 In this area, our research is devoted to the development of peptide-porphyrin conjugates resembling natural heme-proteins, called “Mimochromes”.3,4 Among them, Mimochrome VIa (MC6a) is the most promising catalyst, thanks to its robust but flexible scaffold. MC6a, in its MnIII complex, (Mn-MC6a) is an efficient catalyst with enzyme-like properties, because fast and chemoselective reactions with a peroxygenase-like mechanism were found in the oxidation of thioethers. Even more remarkably, Mn-MC6a selectively exhibits either peroxygenase- or catalase-like activity depending on the reaction conditions. Here we present the oxidation of indole and its derivatives catalyzed by Mn-MC6a, with the aim of exploiting the catalytic properties of this artificial enzyme in reactions with potential synthetic applications. Indole is one of the most common heterocyclic scaffolds available in nature. It occurs in several natural compounds (such as alkaloids and plant hormones) and is part of many pharmaceuticals.5-8 Despite the structural simplicity of this molecule, indole oxidation leads to a large number of products, including mono- and di-oxygenated compounds. Indole oxidation has been studied with both biological5,6 and synthetic7,8 catalysts. In all the approaches described so far, no or weak selectivity toward any of the oxidation products has been reported.5-8 Conversely, Mn-MC6a is able to oxidize indole under unprecedented site-selective conditions, yielding to 3-oxindolenine as single product. Additionally, the reaction selectivity is dramatically altered when 1- or 3-methyl-substituted indoles are used as substrates. A detailed mechanistic analysis will help to rationalize the outstanding selectivity of the catalyst. References: 1. Sheldon, R. A. and Woodley, J. M. Chem. Rev. 2018, 118, 801–838. 2. Schwizer, F. et al. Chem. Rev. 2018, 118, 142-231. 3. Chino, M. et al. Biopolymers 2018 (doi: 10.1002/bip.23107). 4. Nastri, F. et al. Chem. Soc. Rev., 2016, 45, 5020-5054. 5. Kuo, H. H. and Mauk, A. G.; Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 13966–13971. 6. Barrios, D. A. et al. J. Am. Chem. Soc. 2014, 136, 7914-7925. 7. Linhares, M. et al. Appl. Catal. A. 2014, 470, 427–433. 8. Poon L. C.-H. et al. J. Am. Chem. Soc. 2011, 133, 1877–1884

Histopathological comparison of intramural coronary artery remodeling and myocardial fibrosis in obstructive versus end-stage hypertrophic cardiomyopathy.

Abstract Background Although imaging techniques have demonstrated the existence of microvascular abnormalities in hypertrophic cardiomyopathy (HCM), a detailed histopathological assessment is lacking as well as a comparison between different phases of the disease. We aimed to compare microvasculopathy and myocardial fibrosis in hypertrophic obstructive cardiomyopathy (HOCM) versus end-stage (ES) HCM. Methods 27 myectomy specimens of HOCM patients and 30 ES-HCM explanted hearts were analyzed. Myocardial fibrosis was quantitatively determined with dedicated software and qualitatively classified as scar-like or interstitial. Intramural coronary arteries were evaluated separately according to lumen diameter: 100–500 μ versus Results Median value of fibrosis in the anterobasal septum of explanted hearts was 34.6% as opposed to 10.3% of myectomy specimens (p  Conclusions Microvasculopathy is an intrinsic feature of HCM with similar characteristics across the natural phases of the disease. Conversely, myocardial fibrosis changes over time with ES hearts showing a three-fold greater amount, mainly scar-like. ES showed a closer association between microvasculopathy and replacement fibrosis