Acquired and inherited cardiomyopathies: evaluation of cardiotoxic effects in preclinical models

Cardiomyopathies are a heterogeneous group of myocardial disorders defined by structural and functional alterations of the heart. These cardiac diseases can have both non-genetic and genetic origin. Nevertheless, a different etiology can trigger the same phenotype, as in the case of anthracycline-induced cardiotoxicity and desmin-related cardiomyopathy (DRM). Therefore, the aim of this study was to investigate the cellular mechanisms driving the development of these cardiotoxic conditions in in vitro models. Doxorubicin (DOX) is a commonly used antineoplastic drug for the treatment of a wide range of tumors. Besides, its clinical use is restricted because of dose-dependent cardiotoxicity. Our findings provided evidence that phospholipase C Beta 2 (PLCβ2) may have a critical role in DOX-induced cardiotoxicity in undifferentiated and differentiated H9c2 cell line. Interestingly, the results obtained revealed that cardiomyocytes are less sensitive to DOX, following the evaluation of cellular mechanisms such as: oxidative stress, apoptosis and cell proliferation. Nonetheless, the treatment induced a significant upregulation of PLCβ2 associated to morphological changes in both models, demonstrating the implication in a hypertrophic response. On the other hand, a hereditary DRM was associated to a missense mutation of aB crystallin (CRYAB), a chaperone protein involved in the regulation of the intermediate filament network. Since research has only been conducted on transgenic (TG) mice and neonatal rat cardiomyocytes, this study aimed at investigating cellular mechanisms triggered by CRYABR120G mutation in a hiPSC-derived DRM model. Our model confirmed the impairment of the cytoskeletal organization resulting in the formation of desmin and CRYAB aggregates and myofibril misalignment. Moreover, the missense mutation confirmed a hypertrophic cardiomyopathy phenotype, a feature of DRM patients, on cardiac engineered tissues. Lastly, these data obtained suggest that further research on PLCβ2 and CRYAB are needed to comprehend the molecular mechanisms behind the development of these 2 cardiac diseases.

Reorganization of structural proteins in vascular smooth muscle cells grown in collagen gel and basement membrane matrices (Matrigel): A comparison with their in situ counterparts

When smooth muscle cells are enzyme-dispersed from tissues they lose their original filament architecture and extracellular matrix surrounds. They then reorganize their structural proteins to accommodate a 2-D growth environment when seeded onto culture dishes. The aim of the present study was to determine the expression and reorganization of the structural proteins in rabbit aortic smooth muscle cells seeded into 3-D collagen gel and Matrigel (a basement membrane matrix). It was shown that smooth muscle cells seeded in both gels gradually reorganize their structural proteins into an architecture similar to that of their in vivo counterparts. At the same time, a gradual decrease in levels of smooth muscle-specific contractile proteins (mainly smooth muscle myosin heavy chain-2) and an increase in p-nonmuscle actin occur, independent of both cell growth and extracellular matrix components. Thus, smooth muscle cells in 3-D extracellular matrix culture and in vivo have a similar filament architecture in which the contractile proteins such as actin, myosin, and alpha -actinin are organized into longitudinally arranged myofibrils and the vimentin-containing intermediate filaments form a meshed cytoskeletal network, However, the myofibrils reorganized in vitro contain less smooth muscle-specific and more nonmuscle contractile proteins. (C) 2001 Academic Press.

Lesion bypass by the Escherichia coli DNA polymerase V requires assembly of a RecA nucleoprotein filament.

Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOS-regulated proteins UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATPgammaS, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD' along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.

A transgenic mouse model that recapitulates the clinical features of both neonatal and adult forms of the skin disease epidermolytic hyperkeratosis

Keratins are the major structural proteins of keratinocytes, which are the most abundant cell type in the mammalian epidermis. Mutations in epidermal keratin genes have been shown to cause severe blistering skin abnormalities. One such disease, epidermolytic hyperkeratosis (EHK), also known as bullous congenital ichthyosiform erythroderma, occurs as a result of mutations in highly conserved regions of keratins K1 and K10. Patients with EHK first exhibit erythroderma with severe blistering, which later is replaced by thick patches of scaly skin. To assess the effect of a mutated K1 gene on skin biology and to produce an animal model for EHK, we removed 60 residues from the 2B segment of HK1 and observed the effects of its expression in the epidermis of transgenic mice. Phenotypes of the resultant mice closely resembled those observed in the human disease, first with epidermal blisters, then later with hyperkeratotic lesions. In neonatal mice homozygous for the transgene, the skin was thicker, with an increased labeling index, and the spinous cells showed a collapse of the keratin filament network around the nuclei, suggesting that a critical concentration of the mutant HK1, over the endogenous MK1, was required to disrupt the structural integrity of the spinous cells. Additionally, footpad epithelium, which is devoid of hair follicles, showed blistering in the spinous layer, suggesting that hair follicles can stabilize or protect the epidermis from trauma. Blisters were not evident in adult mice, but instead they showed a thick, scaly hyperkeratotic skin with increased mitosis, resulting in an increased number of corneocytes and granular cells. Irregularly shaped keratohyalin granules were also observed. To date, this is the only transgenic model to show the typical morphology found in the adult form of EHK.

Reprint of ""Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury""

Previous studies have suggested that abnormal corneal wound healing in patients after photorefractive keratectomy (PRK) is associated with the appearance of myofibroblasts in the stroma between two and four weeks after surgery. The purpose of this study was to examine potential myofibroblast progenitor cells that might express other filament markers prior to completion of the differentiation pathway that yields alpha-smooth muscle actin (SMA)-expressing myofibroblasts associated with haze localized beneath the epithelial basement membrane after PRK. Twenty-four female rabbits that had -9 diopter PRK were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at -80 degrees C, and analyzed by immunocytochemistry using anti-vimentin, anti-desmin, and anti-SMA antibodies. Double immunostaining was performed for the co-localization of SMA with vimentin or desmin with SMA. An increase in vimentin expression in stromal cells is noted as early as 1 week after PRK in the rabbit cornea. As the healing response continues at two or three weeks after surgery, many stromal cells expressing vimentin also begin to express desmin and SMA. By 4 weeks after the surgery most, if not all, myofibroblasts express vimentin, desmin and SMA. Generalized least squares regression analysis showed that there was strong evidence that each of the marker groups differed in expression over time compared to the other two (p < 0.01). Intermediate filaments - vimentin and desmin co-exist in myofibroblasts along with SMA and may play an important role in corneal remodeling after photorefractive keratectomy. The earliest precursors of myofibroblasts destined to express SMA and desmin are detectible by staining for vimentin at 1 week after surgery. (C) 2009 Elsevier Ltd. All rights reserved.

Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury

Previous studies have suggested that abnormal corneal wound healing in patients after photorefractive keratectomy (PRK) is associated with the appearance of myofibroblasts in the stroma between two and four weeks after surgery. The purpose of this study was to examine potential myofibroblast progenitor cells that might express other filament markers prior to completion of the differentiation pathway that yields a-smooth muscle actin (SMA)-expressing myofibroblasts associated with haze localized beneath the epithelial basement membrane after PRK. Twenty-four female rabbits that had -9 diopter PRK were sacrificed at I week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at -80 degrees C, and analyzed by immunocytochemistry using anti-vimentin, anti-desmin, and anti-SMA antibodies. Double immunostaining was performed for the co-localization of SMA with vimentin or desmin with SMA. An increase in vimentin expression in stromal cells is noted as early as 1 week after PRK in the rabbit cornea. As the healing response continues at two or three weeks after surgery, many stromal cells expressing vimentin also begin to express desmin and SMA. By 4 weeks after the surgery most, if not all, myofibroblasts express vimentin, desmin and SMA. Generalized least squares regression analysis showed that there was strong evidence that each of the marker groups differed in expression over time compared to the other two (p < 0.01). Intermediate filaments - vimentin and desmin co-exist in myofibroblasts along with SMA and may play an important role in corneal remodeling after photorefractive keratectomy. The earliest precursors of myofibroblasts destined to express SMA and desmin are detectible by staining for vimentin at I week after surgery. (C) 2009 Elsevier Ltd. All rights reserved.

Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins

Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. Contractile state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (alpha -SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, beta -NM actin was localised to the cell periphery and basal cortex. The dense body protein alpha -actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In synthetic state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (beta -non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic chan-es in their distribution. The distinct compartmentalisation of structural proteins observed in contractile state SMC was no longer obvious, with proteins more evenly distributed throughout die cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation. (C) 2001 Wiley-Liss, Inc.

Molybdenum-containing proteins from Sulphate Reducing Bacteria: studying proteins with novel cofactors and revealing new features of old enzymes

Dissertação apresentada para a obtenção do Grau de Doutor em Bioquímica, especialidade de Bioquímica-Física pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Untersuchung des humanen Proteins NF110 als Bindungspartner viraler RNAs

In der vorliegenden Masterarbeit wurde das Protein nuclear factor 110 (NF110) in vitro rückgefaltet, chromatographisch gereinigt, durch den Zusatz von L-Arginin stabilisiert und seine Affinität gegenüber RNAs untersucht. Das Ausgangsmaterial wurde mittels Dialyse zurückgefaltet und über drei aufeinander folgende Chromatographieschritte gereinigt: mittels Kationenaustauschchromatographie (capture) zur Entfernung von Fremdproteinen, Größenausschlusschromatographie (intermediate) zur Trennung des monomeren Proteins von höher-oligomeren Spezies und der Affinitätschromatographie (polishing). Diese Proteinlösung wurde unter Zusatz von 500 mM L-Arginin gelagert um eine Proteinaggregation zu unterbinden. Durch Streulichtmessungen und eine analytische Ultrazentrifugation konnte erfolgreich nachgewiesen werden, dass NF110 erst in Puffern mit mindestens 200 mM L-Arginin stabil vorliegt. Im Rahmen der vorliegenden Arbeit konnte erfolgreich bestimmt werden, dass NF110 zwischen doppelsträngiger RNA und DNA diskriminiert, während es gegenüber einzel- bzw. doppelsträngiger RNA ähnlich affin ist. Dies stellt einen großen Unterschied zu NF90 dar. Diese C-terminal verkürzte Variante interagiert mit einzelsträngiger RNA in sehr geringerem Umfang als mit doppelsträngiger RNA (Schmidt, 2015). Daher kann vermutet werden, dass der C-Terminus, insbesondere das dort befindliche GQSY-Motiv, für die Spezifität gegenüber den RNAs verantwortlich ist. Ein weiterer Fokus der Arbeit lag auf dem Einfluss von L-Arginin bei der RNA-Bindung. Mit Hilfe einer linearen freien Enthalpiebeziehung (LFER) konnte so eine Dissoziationskonstante für nicht aggregierendes NF110 ohne L-Arginin ermittelt werden. Außerdem wurde ersichtlich, dass dieser Stabilisator zwar die Aggregation hemmt, aber nur marginal die RNA-Bindung des Proteins beeinflusst

Seasonal variations in the intermediate metabolism of the crayfish Parastacus brasiliensis (Crustacea, Decapoda, Parastacidae) in the natural environment and experimental culture

The goal of this study was to evaluate the effects of seasonal variations on energy metabolism in different tissues of the freshwater crayfish Parastacus brasiliensis (von Martens, 1869). Crayfish were collected monthly from January 2001 to January 2003 in São Francisco de Paula, Rio Grande do Sul, Brazil, in a stream and in a culture tank. Haemolymph samples were collected from each crayfish in the field with a syringe, by puncturing the membrane at the base of the chelipeds. Hepatopancreas, gills, and abdominal muscle were removed for determination of free glucose, glycogen, total lipids, and triglycerides. The haemolymph samples were used for determination of glucose, total proteins, total lipids, and triglycerides. Statistical analysis revealed significant differences in biochemical composition in crayfish collected in the stream compared to the experimental tank during the year, principally in glucose and triglycerides in haemolymph, glycogen and total lipids in all tissues study, and triglycerides only in abdominal muscle. The regular food intake partially modified these seasonal variations of the metabolic pattern. Environmental conditions (e.g., food availability and water temperature) and reproductive period appeared to be the main factors influencing the seasonal patterns of variation in energy metabolism.

Seasonal variation of the effect of high-carbohydrate and high-protein diets on the intermediate metabolism of Parastacus brasiliensis (Crustacea, Decapoda, Parastacidae) maintained in the laboratory

The goal of this study was to evaluate the effect of a high-carbohydrate diet (HC) and a high-protein diet (HP) on the metabolism of the crayfish Parastacus brasiliensis (Von Martens, 1869), collected in different seasons and maintained in the laboratory for 15 days. Crayfish were collected monthly from January 2002 to January 2004 at São Francisco de Paula, Southern Brazil, in Guarapirá stream. In the laboratory, the animals were kept submerged in aquariums under controlled conditions. They were fed ad libitum, for 15 days with either a HC or HP diet. At the end of this period, haemolymph samples were collected, as were hepatopancreas, gills, and abdominal muscle that were removed for determination of glycogen, free glucose, lipids, and triglycerides. The haemolymph samples were used for determination of glucose, proteins, lipids, and triglycerides. Statistical analysis (ANOVA) revealed significant seasonal differences in biochemical composition in crayfish maintained on HC or HP diets. Independent of the diets offered to the animals and the controlled conditions for 15 days, the indications of seasonality were unchanged. The observed changes seemed to be related to the reproductive period. Moreover, the HC diet increased all energy reserves in adult parastacids, which may aid in reproduction.

Isolation and characterization of the full-length cDNA encoding a member of a novel cytochrome p450 family (CYP320A1) from the tropical freshwater snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni

Cytochrome p450s (cyp450s) are a family of structurally related proteins, with diverse functions, including steroid synthesis and breakdown of toxins. This paper reports the full-length sequence of a novel cyp450 gene, the first to be isolated from the tropical freshwater snail Biomphalaria glabrata, an important intermediate host of Schistosoma mansoni. The nucleotide sequence is 2291 bp with a predicted amino acid sequence of 584aa. The sequence demonstrates conserved cyp450 structural motifs, but is sufficiently different from previously reported cyp450 sequences to be given a new classification, CYP320A1. Initially identified as down-regulated in partially resistant snails in response to S. mansoni infection, amplification of this gene using RT-PCR in both totally resistant or susceptible snail lines when exposed to infection, and all tissues examined, suggests ubiquitous expression. Characterization of the first cyp450 from B. glabrata is significant in understanding the evolution of these metabolically important proteins.

Gp74 a membrane glycoprotein of the cis-Golgi network that cycles through the endoplasmic reticulum and intermediate compartment

A monoclonal antibody CC92 (IgM), raised against a fraction of rat liver enriched in Golgi membranes, recognizes a novel Endo H-resistant 74-kD membrane glycoprotein (gp74). The bulk of gp74 is confined to the cis-Golgi network (CGN). Outside the Golgi gp74 is found in tubulovesicular structures and ER foci. In cells incubated at 37 degrees C the majority of gp74 is segregated from the intermediate compartment (IC) marker p58. However, in cells treated with organelle perturbants such as low temperature, BFA, and [AIF4]- the patterns of the two proteins become indistinguishable. Both proteins are retained in the Golgi complex at 20 degrees C and in the IC at 15 degrees C. Incubation of cells with BFA results in relocation of gp74 to p58 positive IC elements. [AIF4]- induces the redistribution of gp74 from the Golgi to p58-positive vesicles and does not retard the translocation of gp74 to IC elements in cells treated with BFA. Disruption of microtubules by nocodazol results in the rapid disappearance of the Golgi elements stained by gp74 and redistribution of the protein into vesicle-like structures. The responses of gp74 to cell perturbants are in sharp contrast with those of cis/middle and trans-Golgi resident proteins whose location is not affected by low temperatures or [AIF4]-, are translocated to the ER upon addition of BFA, and stay in slow disintegrating Golgi elements in cells treated with nocodazol. The results suggest that gp74 is an itinerant protein that resides most of the time in the CGN and cycles through the ER/IC following the pathway used by p58.

Trans-SNARE pairing can precede a hemifusion intermediate in intracellular membrane fusion.

The question concerning whether all membranes fuse according to the same mechanism has yet to be answered satisfactorily. During fusion of model membranes or viruses, membranes dock, the outer membrane leaflets mix (termed hemifusion), and finally the fusion pore opens and the contents mix. Viral fusion proteins consist of a membrane-disturbing 'fusion peptide' and a helical bundle that pin the membranes together. Although SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes form helical bundles with similar topology, it is unknown whether SNARE-dependent fusion events on intracellular membranes proceed through a hemifusion state. Here we identify the first hemifusion state for SNARE-dependent fusion of native membranes, and place it into a sequence of molecular events: formation of helical bundles by SNAREs precedes hemifusion; further progression to pore opening requires additional peptides. Thus, SNARE-dependent fusion may proceed along the same pathway as viral fusion: both use a docking mechanism via helical bundles and additional peptides to destabilize the membrane and efficiently induce lipid mixing. Our results suggest that a common lipidic intermediate may underlie all fusion reactions of lipid bilayers.