Cloning of a novel signaling molecule, AMSH-2, that potentiates transforming growth factor β signaling

BACKGROUND: Transforming growth factor-βs (TGF-βs), bone morphogenetic proteins (BMPs) and activins are important regulators of developmental cell growth and differentiation. Signaling by these factors is mediated chiefly by the Smad family of latent transcription factors. RESULTS: There are a large number of uncharacterized cDNA clones that code for novel proteins with homology to known signaling molecules. We have identified a novel molecule from the HUGE database that is related to a previously known molecule, AMSH (associated molecule with the SH3 domain of STAM), an adapter shown to be involved in BMP signaling. Both of these molecules contain a coiled-coil domain located within the amino-terminus region and a JAB (Domain in Jun kinase activation domain binding protein and proteasomal subunits) domain at the carboxy-terminus. We show that this novel molecule, which we have designated AMSH-2, is widely expressed and its overexpression potentiates activation of TGF-β-dependent promoters. Coimmunoprecipitation studies indicated that Smad7 and Smad2, but not Smad3 or 4, interact with AMSH-2. We show that overexpression of AMSH-2 decreases the inhibitory effect of Smad7 on TGF-β signaling. Finally, we demonstrate that knocking down AMSH-2 expression by RNA interference decreases the activation of 3TP-lux reporter in response to TGF-β. CONCLUSIONS: This report implicates AMSH and AMSH-2 as a novel family of molecules that positively regulate the TGF-β signaling pathway. Our results suggest that this effect could be partially explained by AMSH-2 mediated decrease of the action of Smad7 on TGF-β signaling pathway

Evaluation Strategies of Nanomaterials Toxicity

The revolutionary development of nanoscience during the last years has increased the number of studies in the field to evaluate the toxicity and risk levels. The design of different nanomaterials together with biological components has implemented the advances in biomedicine. Specifically, nanoparticles seem to be a promising platform due to their features, including nanoscale dimensions and physical and chemical characteristics than can be modified in function of the final application. Herein, we review the main studies realized with nanoparticles in order to understand and characterize the cellular uptake mechanisms involved in biocompatibility, toxicity, and how they alter the biological processes to avoid disease progression

A systematic approach for peptide characterization of B-cell receptor in chronic lymphocytic leukemia cells

A wide variety of immunoglobulins (Ig) is produced by the immune system thanks to different mechanisms (V(D)J recombination, somatic hypermutation, and antigen selection). The profiling of Ig sequences (at both DNA and peptide levels) are of great relevance to developing targeted vaccines or treatments for specific diseases or infections. Thus, genomics and proteomics techniques (such as Next- Generation Sequencing (NGS) and mass spectrometry (MS)) have notably increased the knowledge in Ig sequencing and serum Ig peptide profiling in a high-throughput manner. However, the peptide characterization of membrane-bound Ig (e.g., B-cell receptors, BCR) is still a challenge mainly due to the poor recovery of mentioned Ig. Herein, we have evaluated three different sample processing methods for peptide sequencing of BCR belonging to chronic lymphocytic leukemia (CLL) B cells identifying up to 426 different peptide sequences (MS/MS data are available via ProteomeXchange with identifier PXD004466). Moreover, as a consequence of the results here obtained, recommended guidelines have been described for BCR-sequencing of B-CLL samples by MS approaches. For this purpose, an in-house algorithm has been designed and developed to compare the MS/MS results with those obtained by molecular biology in order to integrate both proteomics and genomics results and establish the steps to follow when sequencing membrane-bound Ig by MS/MS.We gratefully acknowledge financial support from the Spanish Health Institute Carlos III (ISCIII) for the grants: FIS PI11/02114 and FIS PI114/01538. We also acknowledge Fondos FEDER (EU) and Junta Castilla León (grant BIO/SA07/15). This work has been also sponsored by Fundación Solórzano (FS/23-2015). The Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001, of the PE I+D+I 2013-2016, funded by ISCIII and FEDER. The authors would like to thank all the clinicians and technicians in the Cytometry and Cell Purification Services of the University of Salamanca, the Spanish National DNA Bank (Banco Nacional de DNA Carlos III, University of Salamanca) and the Genomic Unit of Cancer Research Centre (IBMCC, USAL-CSIC) for their support in the data collection for the preparation of this manuscript. P.D. is supported by a JCYL-EDU/346/2013 Ph.D. scholarship.Peer Reviewe

Hypothermia Due to an Ascending Impairment of Shivering in Hyperacute Experimental Allergic Encephalomyelitis in the Lewis Rat

Severe hypothermia and an ascending impairment of shivering are previously undescribed clinical signs in hyperacute experimental allergic encephalomyelitis (EAE) in the Lewis rat. These occurred in hyperacute EAE induced by inoculation with guinea pig spinal cord homogenate and heat-killed Bordetella pertussis. Hypothermia was first detected on day 6-7 post-inoculation, within 12-24 h of the onset of neurological signs, and became more severe as the disease progressed. Rectal temperatures less than or equal to 30 degrees C were common at ambient temperatures of 19-22 degrees C. Shivering was assessed by palpation and by cold tremor electromyography. Shivering was absent in the tail by day 6-7 post-inoculation. The impairment then progressed to affect the hindlimbs, thorax and occasionally the forelimbs. Shivering was absent in hindlimbs with only mild or moderate weakness. Histological studies revealed perivascular inflammation with polymorphonuclear and mononuclear cells, oedema, fibrin deposition, haemorrhage, primary demyelination and axonal degeneration in the spinal cord, dorsal root ganglia and spinal roots. The brainstem was also involved but the cerebral hemispheres, including the hypothalamus, were spared. The close relationship between the severity of hypothermia and the extent of shivering impairment indicates that reduced shivering is an important cause of hypothermia in hyperacute EAE. It is concluded that this impairment of shivering is due not to hypothalamic damage but to lesions elsewhere in the central and peripheral nervous systems

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Human Proteinpedia enables sharing of human protein data [4]

Carta al editor.-- et al.This work was supported by National Institutes of Health grants R21 DK070297 (MRS, PI) and P41 RR02301 (J.L.M.).Peer Reviewe

Regulación de la mielinización por la hormona tiroidea

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 03-07-1996Resumen La hormona tiroidea es fundamental para el desarrollo del sistema nervioso central de los venebrados. Entre los efectos más dramáticos del hipotiroidismo se encuentra la disminución en la cantidad de mielina del cerebro una vez terminado su desarrollo. La mielinización sigue un patrón espacial que comienza en las regiones caudales y termina en las frontales. Este proceso se debe a la diferenciación y maduración de una célula muy especializada, el oligodendrocito. En la presente tesis hemos estudiado las bases mnleculares de la acción de la hormona tiroidea en la mielinización. Hemos demostrado que la expresión de los genes de las proteínas de la mielina, MAG, MBP, PLP y CNP, está alterada en el hipotiroidismo. Este no afecta al patr6n espacial de expresión de dichos genes sino que disminuye los niveles de sus ARNm. Los niveles de expresión de los genes de las proteínas de mielina se recuperaban antes del primer mes postnatal en la mayoría de las regiones excepto en la corteza y el estriado, regiones cuyos niveles se hayan disminuídos más alla de esta edad. Hemos demostrado que el mecanismo común de regulación no es a nivel transcripcional sino que se debe al control que la hormona tiroidea ejerce sobre la diferenciación del oligodendrocito. Los animales hipotiroideos presentan una dramática reducción en el número de dichas células en su nervio óptico. El análisis clonal de la diferenciación de los oligodendrocitos en cultivos puros ha demostrado que la hormona tiroidea promueve de forma directa la diferenciación de los precursores 0-2A a oligodendrocitos. Además el análisis de la acción de la hormona tiroidea en los cultivos de células disociadas de cerebro embrionario nos ha permitido demostrar que la generación inicial de oligodendrocitos en cultivo se produce por diferenciación asimémca.Summary Thyroid hormone plays a fundamental role on the development of vertebnte central nervous system. Amongst the most dramatic effects of hypothyroidism, there is a decrease myelin deposition when brain development is already finished. Myelination follows a spatial and temporal pattem, starting from the caudal regions towards the most rostral ones. This pmess is due to the differentiation and maturation of the oligodendrocyte, a very specialized cell. In this thesis, we have studied the molecular basis of thyroid hormone action on myelination. We have shown that hypothyroidism affects the expression of the myelin protein genes: MAG, MBP, PLP and CNP. Hypothyroidism does not affect the spatial pattem of expression of these genes but decreases their mRNA levels and therefore their protein levels in a transient and coordinated way. In most brain regions myelin protein gene expression levels recovers before the-first postnatal month wiih the exception of conex and smatum in which the impairment remains beyond that age. We have demonstrated that the common mechanism of regulation is not at the transcriptional level, but thyroid hormone controls of oligodendrocyte differentiation. Thus and accordingly, thyroid hormone increases the oligodendrocyte numbers in mixed cultures from optic nerve of P1 and P7 animals. Hypothyroid animals show a smking decrease on oligodendrocyte cell number in the optic nerve. Furthermore, clonal analysis of oligodendrocyte differentiation in pure cultures of 0-2A precursor cells has shown that thyroid hormone promotes directly 0-2A progenitor cell differentiation "in vitro". Finally, our clonal studies on cuihires of embryonic cells have demonstrated that initial generation of oligodendrocytes follow an asymmetric differentiation. ADN

Neonatal hypothyroidism affects the timely expression of myelin-associated glycoprotein in the rat brain

7 pages, 7 figures.Congenital hypothyroidism strongly affects myelination. To assess the role of thyroid hormone on myelin gene expression, we have studied the effect of hypothyroidism on the steady state levels of myelin-associated glycoprotein (MAG) and its mRNA in rat brain during the first postnatal month. As studied by immunoblot analysis of several brain regions, MAG increased from days 10-15 onwards, reaching constant levels by days 20-25. Hypothyroid samples showed a delay in the accumulation of MAG that was more severe in rostral regions, such as cortex and hippocampus. The effect of hypothyroidism on the accumulation of the protein correlated with mRNA levels. MAG mRNA started to accumulate in the cerebrum of normal animals by postnatal day 7, reaching maximal levels by day 20. Hypothyroid rats showed a delay of several days in the onset of mRNA expression, increasing thereafter at the same rate as in normal animals, and eventually reaching similar values. When individual brain regions were analyzed, we found strong regional differences in the effect of hypothyroidism. The cerebral cortex was most affected, with messenger levels lower than in normal animals at all ages. In more caudal regions differences between control and hypothyroid rats were evident only at the earlier stages of myelination, with spontaneous recovery at later ages. By run on analysis, we found no differences in transcriptional activities of the MAG gene in normal, hypothyroid, or T4-treated rats. Therefore, the effects of hypothyroidism on MAG mRNA and protein levels were most likely caused by decreased mRNA stability. We propose that thyroid hormone contributes to enhanced myelin gene expression by affecting the stability of newly transcribed mRNA in the early phases of myelination.This work was supported by grants from Dirección General de Investigación Científica y Técnica (PM88-0006), from Antibióticos-Pharma S.A. (Madrid, Spain), and from the Community of Madrid (C254/90).Peer reviewe

Adult rat brain is sensitive to thyroid hormone. Regulation of RC3/neurogranin mRNA

The mammalian brain is considered to be poorly responsive to thyroid hormone after the so called 'critical periods' of brain development, which occur in the rat before postnatal days 15-20. In a previous work (Munoz, A., A. Rodriguez-Pena, A. Perez-Castillo, B. Ferreiro, J. G. Sutcliffe, and J. Bernal. 1991. Mol. Endocrinol. 5:273-280) we have identified one neuronal gene, RC3, whose expression is influenced by early neonatal hypothyroidism and thyroid hormone treatment. In the present work we show that adult-onset hypothyroidism leads to a reversible decrease of RC3 mRNA. Rats thyroidectomized on postnatal day 40 and killed three months later showed a decreased RC3 mRNA concentration in the cerebral cortex and striatum. The same effect was observed in animals made hypothyroid on postnatal day 32 and killed on postnatal day 52. RC3 expression was normal when hypothyroid animals were treated with T4 five days before being killed. In contrast, the mRNA encoding myelin proteolipid protein showed no changes in either experimental situation. RC3 mRNA levels were not affected by food restriction demonstrating that the effect of hypothyroidism was not related to the lack of weight gain. The control of RC3 mRNA is so far the only molecular event known to be regulated by thyroid hormone once the critical periods of brain development are over and could represent a molecular correlate for the age- independent, reversible alterations induced by hypothyroidism in the adult brain.This work has been supported by grant PM88-0006 from Dirección General de Investigación Científica y Técnica.Peer Reviewe