RESUMEN

OBJECTIVES: Workplace health promotion activities have a positive effect on emotions. Zentangle art relaxes the body and mind through the process of concentrating while painting, achieving a healing effect. This study aimed to promote the physical and mental health of rural healthcare workers through Zentangle art-based intervention. STUDY DESIGN: This was a quasi-experimental pilot study. METHODS: A Zentangle art workshop was held from November 2019 to July 2020. A total of 40 healthcare workers were recruited. The participants were asked to provide baseline data, and the Brief Symptom Rating Scale (BSRS-5), work stress management effectiveness self-rating scale, General Self-Efficacy Scale (GSES), and Workplace Spirituality Scale (WSS) were administered before and after the workshop. SPSS 22.0 statistical package software was used to conduct the data analysis. RESULTS: The median age (interquartile range [IQR]) was 32.00 years (23.00-41.75 years). The Wilcoxon signed-rank test revealed that the median (IQR) BSRS-5 postintervention score was 4.0 (1.25-5.0), which was lower than the preintervention score (P = 0.004). The postintervention score for the work stress management effectiveness self-rating scale was 36.5 (31.0-40.0), which was also lower than the preintervention score (P = 0.009). A higher score for the GSES or WSS indicated improvements in stress management and self-efficacy. The GSES postintervention score 25.00 (21.0-30.75) was significantly higher than the preintervention score (P = 0.010), and the WSS postintervention score 104.0 (88.0-111.75) was significantly higher than the preintervention score (P = 0.005). CONCLUSIONS: The study provides evidence that painting therapy can effectively relieve stress, reduce workplace stress and frustration, enhance self-efficacy, and increase commitment to work among healthcare workers, thus improving their physical, mental, and spiritual well-being. Zentangle art provides employees with multiple channels for expressing their emotions and can improve the physical and mental health of healthcare workers in the workplace. It is beneficial and cost-effective and can serve as a benchmark for peer learning.

Asunto(s)

Personal de Salud , Lugar de Trabajo , Adulto , Promoción de la Salud , Humanos , Proyectos Piloto , Encuestas y Cuestionarios

RESUMEN

OBJECTIVE: To illustrate the role of interleukin 6 (IL-6) in the progression of non-small cell lung cancer (NSCLC) via activating STAT1. PATIENTS AND METHODS: The level of IL-6 mRNA in 48 paired NSCLC tissues and matched normal ones was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Kaplan-Meier curves were depicted for assessing the overall survival of NSCLC patients with high or low level of IL-6 mRNA. Subsequently, the ZEB2-AS1 level in A549 cells treated with different doses of IL-6 for different time points was determined. After A549 cells were treated with different doses of IL-6, wound closure assays were performed to assess the migration of cells. Protein levels of pSTAT1 and STAT1 in IL-6-treated A549 cells were detected by Western blot. The regulatory effect of STAT1 on IL-6-induced migration of A549 cells was also evaluated. The interaction between ZEB2-AS1 and STAT1 was explored through Chromatin Immunoprecipitation (ChIP) assay. Finally, the role of ZEB2-AS1/STAT1 axis in regulating NSCLC cells was investigated through rescue experiments. RESULTS: Our results indicated that IL-6 was upregulated in NSCLC tissues and cancer cell lines. NSCLC patients with T3-T4 or accompanied with lymphatic metastasis had a higher IL-6 abundance than those with T1-T2 or without metastatic foci. The worse prognosis was identified in NSCLC patients with high expression of IL-6 compared to those with low expression. ZEB2-AS1 showed dose-dependent and time-dependent increase in IL-6-treated A549 cells. IL-6 treatment gradually enhanced the migration ability of A549 cells in a dose-dependent manner. In IL-6-treated A549 cells, protein level of pSTAT1 was remarkably upregulated, and knockdown of STAT1 significantly reversed the promotive effect of IL-6 on migration ability of A549 cells. The results of ChIP assay verified the interaction between ZEB2-AS1 and STAT1. In addition, ZEB2-AS1 could reverse the regulatory effect of STAT1 on the migration ability of A549 cells. CONCLUSIONS: IL-6 was upregulated in NSCLC and accelerated the progression of NSCLC via activating STAT1/ ZEB2-AS1.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Interleucina-6/metabolismo , Neoplasias Pulmonares/metabolismo , ARN Largo no Codificante/metabolismo , Factor de Transcripción STAT1/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/metabolismo , Progresión de la Enfermedad , Humanos , Interleucina-6/genética , Neoplasias Pulmonares/patología , ARN Largo no Codificante/genética , Células Tumorales Cultivadas , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genética

RESUMEN

Animal size is a highly variable trait regulated by complex interactions between biological and environmental processes. Despite the importance of understanding the mechanistic bases of growth, predicting size variation in early stages of development remains challenging. Pedigreed lines of the Pacific oyster (Crassostrea gigas) were crossed to produce contrasting growth phenotypes to analyze the metabolic bases of growth variation in larval stages. Under controlled environmental conditions, substantial growth variation of up to 430% in shell length occurred among 12 larval families. Protein was the major biochemical constituent in larvae, with an average protein-to-lipid content ratio of 2.8. On average, 86% of protein synthesized was turned over (i.e. only 14% retained as protein accreted), with a regulatory shift in depositional efficiency resulting in increased protein accretion during later larval growth. Variation in protein depositional efficiency among families did not explain the range in larval growth rates. Instead, changes in protein synthesis rates predicted 72% of growth variation. High rates of protein synthesis to support faster growth, in turn, necessitated greater allocation of the total ATP pool to protein synthesis. An ATP allocation model is presented for larvae of C. gigas that includes the major components (82%) of energy demand: protein synthesis (45%), ion pump activity (20%), shell formation (14%) and protein degradation (3%). The metabolic trade-offs between faster growth and the need for higher ATP allocation to protein synthesis could be a major determinant of fitness for larvae of different genotypes responding to the stress of environmental change.

Asunto(s)

Crassostrea/crecimiento & desarrollo , Crassostrea/metabolismo , Biosíntesis de Proteínas , Adenosina Trifosfato/metabolismo , Exoesqueleto/crecimiento & desarrollo , Animales , Crassostrea/química , Crassostrea/genética , Genotipo , Larva/química , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Fenotipo

RESUMEN

Exogenous environmental factors alter growth rates, yet information remains scant on the biochemical mechanisms and energy trade-offs that underlie variability in the growth of marine invertebrates. Here we study the biochemical bases for differential growth and energy utilization (as adenosine triphosphate [ATP] equivalents) during larval growth of the bivalve Crassostrea gigas exposed to increasing levels of experimental ocean acidification (control, middle, and high pCO2, corresponding to ∼400, ∼800, and ∼1100 µatm, respectively). Elevated pCO2 hindered larval ability to accrete both shell and whole-body protein content. This negative impact was not due to an inability to synthesize protein per se, because size-specific rates of protein synthesis were upregulated at both middle and high pCO2 treatments by as much as 45% relative to control pCO2. Rather, protein degradation rates increased with increasing pCO2. At control pCO2, 89% of cellular energy (ATP equivalents) utilization was accounted for by just 2 processes in larvae, with protein synthesis accounting for 66% and sodium-potassium transport accounting for 23%. The energetic demand necessitated by elevated protein synthesis rates could be accommodated either by reallocating available energy from within the existing ATP pool or by increasing the production of total ATP. The former strategy was observed at middle pCO2, while the latter strategy was observed at high pCO2. Increased pCO2 also altered sodium-potassium transport, but with minimal impact on rates of ATP utilization relative to the impact observed for protein synthesis. Quantifying the actual energy costs and trade-offs for maintaining physiological homeostasis in response to stress will help to reveal the mechanisms of resilience thresholds to environmental change.

Asunto(s)

Dióxido de Carbono/farmacología , Crassostrea/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Estrés Fisiológico/fisiología , Animales , Crassostrea/crecimiento & desarrollo , Metabolismo Energético/efectos de los fármacos , Concentración de Iones de Hidrógeno , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Agua de Mar/química

RESUMEN

The stress response of Oncorhynchus mykiss in high-altitude farms in central Mexico was investigated over two seasons: the cool (9·1-13·7° C) dry winter season, and the warmer (14·7-15·9° C), wetter summer season. Fish were subjected to an acute stress test followed by sampling of six physiological variables: blood cortisol, glucose, lactate, total antioxidant capacity, haemoglobin concentration and per cent packed cell volume (VPC %). Multivariate analyses revealed that lactate and total antioxidant capacity were significantly higher in the summer, when water temperatures were warmer and moderate hypoxia (4·9-5·3 mg l(-1) ) prevailed. In contrast, plasma cortisol was significantly higher in the winter (mean ± s.e.: 76·7 ± 4·0 ng ml(-1) ) when temperatures were cooler and dissolved oxygen levels higher (6·05-7·9 mg l(-1) ), than in the summer (22·7 ± 3·8 ng ml(-1) ). Haemoglobin concentrations (mg dl(-1) ) were not significantly different between seasons, but VPC % was significantly higher in the summer (50%) than in the winter (35%). These results suggest that in summer, effects of high altitude on farmed fish are exacerbated by stresses of high temperatures and hypoxia, resulting in higher blood lactate, increased total antioxidant capacity and elevated VPC % levels.

Asunto(s)

Altitud , Oncorhynchus mykiss/fisiología , Estrés Fisiológico , Animales , Antioxidantes/química , Glucemia/química , Hemoglobinas/química , Hidrocortisona/sangre , Hipoxia , Ácido Láctico/sangre , México , Estaciones del Año , Temperatura

RESUMEN

Energy is required to maintain physiological homeostasis in response to environmental change. Although responses to environmental stressors frequently are assumed to involve high metabolic costs, the biochemical bases of actual energy demands are rarely quantified. We studied the impact of a near-future scenario of ocean acidification [800 µatm partial pressure of CO2 (pCO2)] during the development and growth of an important model organism in developmental and environmental biology, the sea urchin Strongylocentrotus purpuratus. Size, metabolic rate, biochemical content, and gene expression were not different in larvae growing under control and seawater acidification treatments. Measurements limited to those levels of biological analysis did not reveal the biochemical mechanisms of response to ocean acidification that occurred at the cellular level. In vivo rates of protein synthesis and ion transport increased ∼50% under acidification. Importantly, the in vivo physiological increases in ion transport were not predicted from total enzyme activity or gene expression. Under acidification, the increased rates of protein synthesis and ion transport that were sustained in growing larvae collectively accounted for the majority of available ATP (84%). In contrast, embryos and prefeeding and unfed larvae in control treatments allocated on average only 40% of ATP to these same two processes. Understanding the biochemical strategies for accommodating increases in metabolic energy demand and their biological limitations can serve as a quantitative basis for assessing sublethal effects of global change. Variation in the ability to allocate ATP differentially among essential functions may be a key basis of resilience to ocean acidification and other compounding environmental stressors.

Asunto(s)

Metabolismo Energético , Expresión Génica , Strongylocentrotus purpuratus/genética , Strongylocentrotus purpuratus/metabolismo , Ácidos/química , Factores de Edad , Análisis de Varianza , Animales , Dióxido de Carbono/metabolismo , Femenino , Concentración de Iones de Hidrógeno , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Masculino , Océanos y Mares , Proteínas/genética , Proteínas/metabolismo , Agua de Mar/química , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Strongylocentrotus purpuratus/crecimiento & desarrollo , Factores de Tiempo

RESUMEN

Understanding the complex interactions that regulate growth and form is a central question in developmental physiology. We used experimental crosses of pedigreed lines of the Pacific oyster, Crassostrea gigas, to investigate genetically determined variations in larval growth and nutrient transport. We show that (i) transport rates at 10 and 100 µM glycine scale differentially with size; (ii) size-specific maximum transport capacity (Jmax) is genetically determined; and (iii) Jmax serves as an early predictive index of subsequent growth rate. This relationship between genetically determined Jmax and growth suggests the potential use of transporter genes as biomarkers of growth potential. Analysis of the genome of C. gigas revealed 23 putative amino acid transporter genes. The complexity of gene families that underpin physiological traits has additional precedents in this species and others and warrants caution in the use of gene expression as a biomarker for physiological state. Direct in vivo measurements of physiological processes using species with defined genotypes are required to understand genetically determined variance of nutrient flux and other processes that regulate development and growth.

Asunto(s)

Crassostrea/genética , Animales , Crassostrea/crecimiento & desarrollo , Crassostrea/metabolismo , Cruzamientos Genéticos , Perfilación de la Expresión Génica , Variación Genética , Genoma , Glicina/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Transporte de Proteínas

RESUMEN

Understanding and predicting biological stability and change in the face of rapid anthropogenic modifications of ecosystems and geosystems are grand challenges facing environmental and life scientists. Physiologically, organisms withstand environmental stress through changes in biochemical regulation that maintain homeostasis, which necessarily demands tradeoffs in the use of metabolic energy. Evolutionarily, in response to environmentally forced energetic tradeoffs, populations adapt based on standing genetic variation in the ability of individual organisms to reallocate metabolic energy. Combined study of physiology and genetics, separating "Nature and Nurture," is, thus, the key to understanding the potential for evolutionary adaptation to future global change. To understand biological responses to global change, we need experimentally tractable model species that have the well-developed physiological, genetic, and genomic resources necessary for partitioning variance in the allocation of metabolic energy into its causal components. Model species allow for discovery and for experimental manipulation of relevant phenotypic contrasts and enable a systems-biology approach that integrates multiple levels of analyses to map genotypic-to-phenotypic variation. Here, we illustrate how combined physiological and genetic studies that focus on energy metabolism in developmental stages of a model marine organism contribute to an understanding of the potential to adapt to environmental change. This integrative research program provides insights that can be readily incorporated into individual-based ecological models of population persistence under global change.

Asunto(s)

Organismos Acuáticos/fisiología , Cambio Climático , Metabolismo Energético , Modelos Animales , Adaptación Biológica , Animales , Organismos Acuáticos/genética , Organismos Acuáticos/crecimiento & desarrollo , Organismos Acuáticos/metabolismo , Modelos Biológicos , Biología de Sistemas

RESUMEN

The ontogeny of cardiac hypoxic responses, and how such responses may be modified by rearing environment, are poorly understood in amphibians. In this study, cardiac performance was investigated in Xenopus laevis from 2 to 25 days post-fertilization (dpf). Larvae were reared under either normoxia or moderate hypoxia (PO2 = 110 mmHg), and each population was assessed in both normoxia and acute hypoxia. Heart rate (f(H)) of normoxic-reared larvae exhibited an early increase from 77 ± 1 beats min⁻¹ at 2 dpf to 153 ± 1 beats min⁻¹ at 4 dpf, followed by gradual decreases to 123 ± 3 beats min⁻¹ at 25 dpf. Stroke volume (SV), 6 ± 1 nl, and cardiac output (CO), 0.8 ± 0.1 µl min⁻¹, at 5 dpf both increased by more than 40-fold to 25 dpf with rapid larval growth (~30-fold increase in body mass). When exposed to acute hypoxia, normoxic-reared larvae increased f(H) and CO between 5 and 25 dpf. Increased SV in acute hypoxia, produced by increased end-diastolic volume (EDV), only occurred before 10 dpf. Hypoxic-reared larvae showed decreased acute hypoxic responses of EDV, SV and CO at 7 and 10 dpf. Over the period of 2-25 dpf, cardiac scaling with mass showed scaling coefficients of -0.04 (f(H)), 1.23 (SV) and 1.19 (CO), contrary to the cardiac scaling relationships described in birds and mammals. In addition, f(H) scaling in hypoxic-reared larvae was altered to a shallower slope of -0.01. Collectively, these results indicate that acute cardiac hypoxic responses develop before 5 dpf. Chronic hypoxia at a moderate level can not only modulate this cardiac reflex, but also changes cardiac scaling relationship with mass.

Asunto(s)

Corazón/fisiología , Hipoxia/fisiopatología , Xenopus laevis/fisiología , Animales , Cámaras de Exposición Atmosférica , Tamaño Corporal , Corazón/crecimiento & desarrollo , Corazón/fisiopatología , Frecuencia Cardíaca , Larva/crecimiento & desarrollo , Larva/fisiología , Miocardio/patología , Tamaño de los Órganos , Volumen Sistólico , Xenopus laevis/crecimiento & desarrollo

RESUMEN

High-resolution heat-capacity and optical-reflectivity measurements have been conducted near the smectic-A to hexatic-B transition in thin free-standing films of the liquid-crystal compound 64COOBC. We find an unexpected dependence on film thickness of the integrated magnitude of the heat-capacity anomalies as the films undergo layer-by-layer transitions. We measure the penetration depths of the ordering from the surface and next-to-surface layers which are pertinent to the highly correlated thermal behavior.

RESUMEN

Electron diffraction and optical reflectivity have provided the direct confirmation of the existence of layer-by-layer surface transitions from the smectic-A immediately to the crystal-B phase in a liquid-crystal material, without going through an intermediate hexatic phase. The molecular interactions are found to be through retarded van der Waals forces. Our results suggest that a smectic-A film can transform into a crystal-B through three possible scenarios.

RESUMEN

This paper describes the framework for the development of a ground motion analysis software based on MapInfo, a geographical information system (GIS). The GIS-based system maintains and manipulates the soil stratum information of Singapore, interfaces with WAVES, a site response analysis software, to compute the peak ground accelerations (PGAs) at selected sites, and integrates with MATLAB a numeric processor for the 2-D spatial interpolation of PGAs. The interpolated PGA values are shown as a fringe plot overlaid on the Singapore map

Asunto(s)

Redes de Monitoreo de la Calidad de la Agua , Programas Informáticos , Sistemas de Información , Bases de Datos como Asunto

RESUMEN

Fibulin-1 is a 90 kDa calcium-binding protein present in the extracellular matrix and in the blood. Two major variants, C and D, differ in their C-termini as well as the ability to bind the basement membrane protein nidogen. Here we characterized genomic clones encoding the mouse fibulin-1 gene, which contains 18 exons spanning at least 75 kb of DNA. The two variants are generated by alternative splicing of exons in the 3' end. By searching the database we identified most of the exons encoding the human fibulin-1 gene and showed that its exon-intron organization is similar to that of the mouse gene.

Asunto(s)

Proteínas de Unión al Calcio/genética , Clonación Molecular , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Unión al Calcio/química , Cromosomas Humanos Par 22/genética , Cósmidos/genética , ADN Complementario/genética , Bases de Datos Factuales , Exones/genética , Biblioteca Genómica , Humanos , Intrones/genética , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Homología de Secuencia de Ácido Nucleico

RESUMEN

A CA dinucleotide repeat has been identified in an intron of the human alpha3(VI) collagen gene (COL6A3) located on chromosome 2q37. Analysis of 100 chromosomes in unrelated Caucasians has demonstrated the existence of eight alleles, and the allelic frequencies have been determined.

Asunto(s)

Cromosomas Humanos Par 2 , Colágeno/genética , Polimorfismo Genético , Alelos , Enfermedades del Colágeno/genética , Repeticiones de Dinucleótido/genética , Frecuencia de los Genes , Humanos , Población Blanca/genética

RESUMEN

To study the mechanism of basement membrane formation, we determined by immunochemistry temporal and spatial expression of laminin-5 (Ln-5), laminin-1 (Ln-1) and their integrin receptors during early skin morphogenesis. A 3-dimensional skin culture was used that allows the study of the sequential molecular events of basement membrane formation at the epidermodermal interface. During early anchorage of keratinocytes to the extracellular matrix there is expression of Ln-5, BP-230 antigen and alpha3, beta1 integrin subunits. During epidermal stratification and prior to the formation of the lamina densa there is assembly of Ln-5, Ln-1, collagen IV and nidogen accompanied by keratinocyte basal clustering of alpha2, alpha3, alpha6, beta1, and beta4+ integrin subunits. The assembly pattern of Ln-1 and Ln-5 can be disturbed with functional antibodies against the beta1 (AIIB2) and alpha6 (GoH3) integrin subunits. Ln-1 assembly can also be disturbed with antibodies against its E8 domain and by competitive inhibition with a synthetic peptide (AG-73) derived from its G-4 domain. Quantitative RT-PCR showed that the dermis contributes about 80% of the laminin gamma)1 chain mRNA while 20% is produced by the epidermis which emphasizes its dual tissue origin and the major contribution of the mesenchyma in laminin production. The laminin gamma2 chain mRNA, present in Ln-5, was mostly of epidermal origin. This study presents evidence that during the initiation of basement membrane formation, laminins bind to keratinocyte plasma membrane receptors and thus may serve as nucleation sites for further polymerization of these compounds by a self-assembly process.

Asunto(s)

Membrana Basal/fisiología , Epidermis/fisiología , Queratinocitos/fisiología , Laminina/biosíntesis , Piel/citología , Citoesqueleto de Actina/fisiología , Citoesqueleto de Actina/ultraestructura , Secuencia de Aminoácidos , Membrana Basal/citología , Comunicación Celular , Membrana Celular/fisiología , Células Cultivadas , Desmosomas/fisiología , Desmosomas/ultraestructura , Células Epidérmicas , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Inmunohistoquímica , Integrina beta1/biosíntesis , Queratinocitos/citología , Laminina/síntesis química , Laminina/metabolismo , Masculino , Morfogénesis , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Fenómenos Fisiológicos de la Piel

RESUMEN

The Bethlem myopathy is a rare autosomal dominant proximal myopathy characterized by early childhood onset and joint contractures. Evidence for linkage and genetic heterogeneity has been established, with the majority of families linked to 21q22.3 and one large family linked to 2q37, implicating the three type VI collagen subunit genes, COL6A1 (chromosome 21), COL6A2 (chromosome 21) and COL6A3 (chromosome 2) as candidate genes. Mutations of the invariant glycine residues in the triple-helical domain-coding region of COL6A1 and COL6A2 have been reported previously in the chromosome 21-linked families. We report here the identification of a G-->A mutation in the N-terminal globular domain-coding region of COL6A3 in a large American pedigree (19 affected, 12 unaffected), leading to the substitution of glycine by glutamic acid in the N2 motif, which is homologous to the type A domains of the von Willebrand factor. This mutation segregated to all affected family members, to no unaffected family members, and was not identified in 338 unrelated Caucasian control chromosomes. Thus mutations in either the triple-helical domain or the globular domain of type VI collagen appear to cause Bethlem myopathy.

Asunto(s)

Colágeno/genética , Distrofias Musculares/genética , Mutación Puntual , Factor de von Willebrand/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Cromosomas Humanos Par 2/genética , Contractura/genética , Femenino , Fibroblastos , Ligamiento Genético , Ácido Glutámico/genética , Glicina/genética , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Distrofias Musculares/etiología , Linaje , Estructura Terciaria de Proteína

RESUMEN

Collagen VI is a microfibrillar protein found in the extracellular matrix of virtually all connective tissues. Three genetically distinct subunits, the alpha1(VI), alpha2(VI), and alpha3(VI) chains, associate intracellularly to form triple-helical monomers, which then assemble into disulfide-bonded dimers and tetramers before secretion. Although sequence considerations suggest that collagen VI monomers composed of all three chains are the most stable isoform, the precise chain composition of collagen VI remains controversial and alternative assemblies containing only alpha1(VI) and alpha2(VI) chains have also been proposed. To address this question directly and study the role of the alpha3(VI) chain in assembly, we have characterized collagen VI biosynthesis and in vitro matrix formation by a human osteosarcoma cell line (SaOS-2) that is deficient in alpha3(VI) production. Northern analysis showed an abundance of alpha1(VI) and alpha2(VI) mRNAs, but no detectable alpha3(VI) mRNA was apparent in SaOS-2 cells. By day 30 of culture, however, small amounts of alpha3(VI) mRNA were detected, although the level of expression was still much less than alpha1(VI) and alpha2(VI). Collagen VI protein was not detected in SaOS-2 medium or cell layer samples until day 30 of culture, demonstrating that despite the abundant synthesis of alpha1(VI) and alpha2(VI), no stable collagen VI protein was produced without expression of alpha3(VI). The alpha1(VI) and alpha2(VI) chains produced in the absence of alpha3(VI) were non-helical and were largely retained intracellularly and degraded. The critical role of the alpha3(VI) chain in collagen VI assembly was directly demonstrated after stable transfection of SaOS-2 cells with an alpha3(VI) cDNA expression construct that lacked 4 of the 10 N-terminal type A subdomains. The transfected alpha3(VI) N6-C5 chains associated with endogenous alpha1(VI) and alpha2(VI) and formed collagen VI dimers and tetramers, which were secreted and deposited into an extensive network in the extracellular matrix. These data demonstrated that alpha3(VI) is essential for the formation of stable collagen VI molecules and subdomains N10-N7 are not required for molecular assembly.

Asunto(s)

Colágeno/química , Ácido Ascórbico/farmacología , Línea Celular , Colágeno/biosíntesis , Colágeno/genética , Disulfuros/química , Electroforesis en Gel de Poliacrilamida , Humanos , ARN Mensajero/metabolismo , Relación Estructura-Actividad

RESUMEN

Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.

Asunto(s)

Membrana Basal/metabolismo , Colágeno/metabolismo , Integrina beta1/metabolismo , Piel/crecimiento & desarrollo , Piel/metabolismo , Anticuerpos Monoclonales/farmacología , Membrana Basal/citología , Membrana Basal/crecimiento & desarrollo , Técnicas de Cocultivo , Colágeno/química , Fibroblastos/citología , Humanos , Queratinocitos/citología , Microscopía Electrónica , Fragmentos de Péptidos/farmacología , Piel/citología , Factores de Tiempo , Distribución Tisular

RESUMEN

The purpose of this study was to investigate ultratrace levels of metals in serum of patients with Blackfoot disease (BFD). BFD is an endemic peripheral vascular disorder confined to a limited area along the southwest coast of Taiwan. In this study, graphite furnace atomic absorption spectrophotometry with stabilized temperature platform furnace conditions was used for the determination of selenium, manganese, cobalt, chromium and zinc. This technique includes a dilution of serum with 12 mM ultrapure nitric acid and 1% Triton X-100. The results showed that total manganese, cobalt, chromium and zinc levels in the BFD patients were significantly different from those in normal controls (P < 0.05). The total selenium level in the BFD patients was not different from the normal controls (P > 0.05). The possible connection of these elements with the etiology of the disease is discussed.

Asunto(s)

Metales/sangre , Enfermedades Vasculares/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Cobre/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Selenio/sangre , Zinc/sangre

RESUMEN

Collagen VI is a microfibrillar component of the extracellular matrix that is predicted to have an important structural role in matrix organization and a biological function in mediating cell-matrix interactions. Secreted collagen VI molecules are composed of three distinct subunits, the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains. To determine when, and in which tissues, collagen VI is likely to have a role in embryonic processes, we have analyzed the expression patterns of the three subunit chains during postimplantation mouse development by reverse transcriptase-PCR (RT-PCR), in situ hybridization, and immunofluorescence. No collagen VI protein could be detected in the mouse embryo until Day 11.5 of gestation, when low levels were localized within the mesoderm layer of the visceral yolk sac, the subepidermal matrix of branchial arches, and the vessel wall of the dorsal aorta. Levels of collagen VI mRNA and protein increased during the period from Days 12.5 to 14.5 in the visceral yolk sac, subepidermal mesenchyme, lung, gut, meninges, muscle, perichondrium, and vertebral column. The cartilage matrix of ribs and developing long bones was not stained with collagen VI antisera, but pericellular staining of chondrocytes was seen in both tissues. Low levels of collagen VI mRNA and protein were seen in the fetal liver except for the connective tissue of the liver capsule, which was highly stained. Collagen VI was first detected at significant levels in the developing heart on Day 14.5. These data demonstrate a tissue-specific onset of collagen VI synthesis and deposition in the extracellular matrix of developing mouse embryos at a much later stage of development than that reported for fibronectin or collagen I. Sensitive RT-PCR assays showed that alpha 1(VI) and alpha 2(VI) mRNAs were amplified from extracts of embryonic tissues as early as Day 7.5, while alpha 3(VI) mRNA was not detected until Day 10.5. Expression of the alpha 3(VI) gene immediately preceded the appearance of collagen VI protein in embryonic tissues. This correlation is consistent with the proposal that expression of alpha 3(VI) chains regulates the formation and secretion of collagen VI trimers and collagen VI matrix deposition during development.

Asunto(s)

Colágeno/análisis , Colágeno/genética , Desarrollo Embrionario y Fetal , Matriz Extracelular/química , Regulación del Desarrollo de la Expresión Génica/fisiología , Animales , Secuencia de Bases , Colágeno/biosíntesis , Embrión de Mamíferos/química , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/análisis