ABSTRACT
"Laminopathies" refers to a wide spectrum of myopathies caused by mutations in the LMNA gene. These myopathies include limb girdle muscular dystrophy type 1B (LGMD1B) and dilated cardiomyopathy 1 A (DCM1A), which are both autosomal dominant neurogenetic diseases. There have been few studies on mosaicism in laminopathies. Herein, a Han Chinese family with laminopathies was enrolled in our study. Genetic analysis revealed that the proband carried a novel splice site mutation, c. 1158-3 C > T, in the LMNA gene due to her mother having de novo somatic and gonadal mosaicism. Reverse-transcription polymerase chain reaction (RT-PCR) analysis revealed reduced levels of LMNA mRNA in the proband, which were probably due to nonsense-mediated mRNA decay (NMD). Western blotting revealed reduced lamin A/C protein levels in the skeletal muscle tissue of the proband. In this family, the clinical phenotypes of the proband's mother were normal, and the c. 1158-3 C > T splicing mutation was identified in the blood sample of the proband's mother. Thus, the mutation could be easily considered to be nonpathogenic. Our study emphasizes the importance of mosaicism in the identification of pathogenic variants and genetic counseling.
Subject(s)
Lamin Type A , Laminopathies , Mosaicism , Muscular Diseases , Female , Humans , East Asian People , Lamin Type A/genetics , Laminopathies/blood , Laminopathies/genetics , Muscle, Skeletal/chemistry , Muscle, Skeletal/metabolism , Muscular Diseases/blood , Muscular Diseases/genetics , Muscular Dystrophies, Limb-Girdle/genetics , Lamins/analysis , Lamins/bloodABSTRACT
O tomateiro é uma das hortaliças de maior importância econômica a nível mundial. No entanto, sua produção pode ser limitada por diversos fatores, sendo o manejo da água o principal fator limitante. Dessa forma, o uso de tecnologias que melhorem a eficiência no uso da água é de extrema importância, destacando-se entre estas o uso de hidrogel. Nesse sentido, objetivou-se nesse trabalho avaliar as taxas de crescimento e produção do tomateiro sob lâminas de irrigação e volumes de hidrogel. O experimento foi conduzido em esquema fatorial 3x4, em blocos ao acaso com quatro repetições, sendo os fatores: três volumes de hidrogel previamente hidratado (0, 50 e 100 ml por planta); e 4 lâminas de irrigação (40, 60, 80 e 100% da evapotranspiração da cultura). Foram avaliadas as taxas de crescimento absoluto e relativo da altura de planta e diâmetro do caule, massa média dos frutos e a produtividade por planta. Os resultados evidenciaram que a redução das lâminas de irrigação levou a redução linear das taxas de crescimento absolutas e relativas de altura e diâmetro. Perante essas mesmas condições, também houve redução da massa média dos frutos e da produtividade por planta. O uso de hidrogel não afetou nenhuma das características avaliadas, dessa forma, recomenda-se a sua não utilização nas condições desse estudo. Indica-se a utilização da lâmina de reposição de 100% da ETc.(AU)
Tomato is one of the most economically relevant vegetables worldwide. However, its production can be limited by several factors, with water management being the main limiting factor. Thus, the use of technologies that improve efficiency in the use of water are extremely important, with emphasis on the use of hydrogel. In this sense, the objective of this study was to evaluate the growth and production rates of tomato under irrigation depths and hydrogel volumes. The experiment was carried out in a 3x4 factorial scheme, in randomized blocks with four replications, with the following factors: three volumes of previously hydrated hydrogel (0, 50 and 100 ml per plant); and 4 irrigation depths (40, 60, 80 and 100% evapotranspiration of the crop). The absolute and relative growth rates of plant height and stem diameter, average fruit mass, and productivity per plant were evaluated. The results showed that the reduction of irrigation depths led to a linear reduction in absolute and relative growth rates in both height and diameter. Under these same conditions, there was also a reduction in the average fruit mass and productivity per plant. The use of hydrogel did not affect any of the evaluated characteristics; therefore, it is recommended not to use it under the conditions of this study. It is recommended to use the 100% ETc replacement blade.(AU)
El tomate es una de las hortalizas de mayor importancia económica a nivel mundial. Sin embargo, su producción puede verse limitada por varios factores, siendo la gestión del agua el principal factor limitante. Por ello, el uso de tecnologías que mejoren la eficiencia en el uso del agua es de suma importancia, con énfasis en el uso de hidrogel. En ese sentido, el objetivo de este estudio fue evaluar las tasas de crecimiento y producción de tomate en láminas de riego y volúmenes de hidrogel. El experimento se realizó en un esquema factorial 3x4, en bloques al azar con cuatro repeticiones, siendo los factores: tres volúmenes de hidrogel previamente hidratado (0, 50 y 100 ml por planta); y 4 láminas de riego (40, 60, 80 y 100% evapotranspiración del cultivo). Se evaluaron las tasas de crecimiento absoluto y relativo de la altura de la planta y el diámetro del tallo, la masa promedio de frutos y la productividad por planta. Los resultados mostraron que la reducción de las láminas de riego condujo a una reducción lineal en las tasas de crecimiento absoluto y relativo en altura y diámetro. En estas mismas condiciones, también se redujo la masa media de frutos y de la productividad por planta. El uso de hidrogel no afectó ninguna de las características evaluadas, por lo que se recomienda no utilizarlo en las condiciones de ese estudio. Se recomienda utilizar la lámina de repuesto del 100% del ETc.(AU)
Subject(s)
Solanum lycopersicum/growth & development , Hydrogels , Lamins/analysis , Agricultural Irrigation , TechnologyABSTRACT
Cells perceive and relay external mechanical forces into the nucleus through the nuclear envelope. Here we examined the effect of lowering substrate stiffness as a paradigm to address the impact of altered mechanical forces on nuclear structure-function relationships. RNA sequencing of cells on softer matrices revealed significant transcriptional imbalances, predominantly in chromatin associated processes and transcriptional deregulation of human Chromosome 1. Furthermore, 3-Dimensional fluorescence in situ hybridization (3D-FISH) analyses showed a significant mislocalization of Chromosome 1 and 19 Territories (CT) into the nuclear interior, consistent with their transcriptional deregulation. However, CT18 with relatively lower transcriptional dysregulation, also mislocalized into the nuclear interior. Furthermore, nuclear Lamins that regulate chromosome positioning, were mislocalized into the nuclear interior in response to lowered matrix stiffness. Notably, Lamin B2 overexpression retained CT18 near the nuclear periphery in cells on softer matrices. While, cells on softer matrices also activated emerin phosphorylation at a novel Tyr99 residue, the inhibition of which in a phospho-deficient mutant (emerinY99F), selectively retained chromosome 18 and 19 but not chromosome 1 territories at their conserved nuclear locations. Taken together, emerin functions as a key mechanosensor, that modulates the spatial organization of chromosome territories in the interphase nucleus.
Subject(s)
Chromosome Positioning , Membrane Proteins/metabolism , Nuclear Proteins/metabolism , Biomechanical Phenomena , Cell Line, Tumor , Cell Nucleus/chemistry , Cell Nucleus/genetics , Cell Nucleus/metabolism , Chromosomes, Human , Chromosomes, Human, Pair 18 , Gene Expression Regulation , Histone Code , Humans , Lamin Type B/metabolism , Lamins/analysis , Membrane Proteins/chemistry , Nuclear Proteins/chemistry , Phosphorylation , Transcription, Genetic , Tyrosine/metabolismABSTRACT
Although many protein labeling probes have been developed to elucidate the trafficking and turnover processes of cell surface proteins, real-time tracking of intracellular proteins remains a challenging task. Herein, we describe a new design to construct a cell-permeable, photostable, and far-red fluorescent turn-on probe to enable no-wash, organelle-specific, and long-term visualization of intracellular SNAP-tagged proteins in living cells. When the probe was used in dual-color pulse chase labeling experiments to differentiate between preLamin and mature Lamin, our results reveal that the shape of mature Lamin can be altered by the newly synthesized preLamin and that this alteration is progressive, cumulative, and due to a concentration-dependent dominant-negative effect of preLamin. We believe that this probe can also be applied to other intracellular proteins whose cellular localization and synthesis changes dynamically in response to external stimuli.
Subject(s)
Fluorescent Dyes/chemistry , Lamins/analysis , Fluorescent Dyes/metabolism , Humans , Lamins/metabolism , MCF-7 Cells , Nuclear Envelope/chemistry , Nuclear Envelope/metabolism , Optical Imaging/methods , Protein Processing, Post-TranslationalABSTRACT
The inner nuclear membrane (INM) protein Nemp1/TMEM194A has previously been suggested to be involved in eye development in Xenopus, and contains two evolutionarily conserved sequences in the transmembrane domains (TMs) and the C-terminal region, named region A and region B, respectively. To elucidate the molecular nature of Nemp1, we analyzed its interacting proteins through those conserved regions. First, we found that Nemp1 interacts with itself and lamin through the TMs and region A, respectively. Colocalization of Nemp1 and lamin at the INM suggests that the interaction with lamin participates in the INM localization of Nemp1. Secondly, through yeast two-hybrid screening using region B as bait, we identified the small GTPase Ran as a probable Nemp1-binding partner. GST pulldown and co-immunoprecipitation assays using region B and Ran mutants revealed that region B binds directly to the GTP-bound Ran through its effector domain. Immunostaining experiments using transfected COS-7 cells revealed that full-length Nemp1 recruits Ran near the nuclear envelope, suggesting a role for Nemp1 in the accumulation of RanGTP at the nuclear periphery. At the neurula-to-tailbud stages of Xenopus embryos, nemp1 expression overlapped with ran in several regions including the eye vesicles. Co-knockdown using antisense morpholino oligos for nemp1 and ran caused reduction of cell densities and severe eye defects more strongly than either single knockdown alone, suggesting their functional interaction. Finally we show that Arabidopsis thaliana Nemp1-orthologous proteins interact with A. thaliana Ran, suggesting their evolutionally conserved physical and functional interactions possibly in basic cellular functions including nuclear transportation. Taken together, we conclude that Nemp1 represents a new type of RanGTP-binding protein.
Subject(s)
Carrier Proteins/metabolism , Nuclear Proteins/metabolism , Xenopus Proteins/metabolism , Xenopus laevis/metabolism , ran GTP-Binding Protein/metabolism , Animals , Arabidopsis/chemistry , Arabidopsis/metabolism , Arabidopsis Proteins/analysis , Arabidopsis Proteins/metabolism , COS Cells , Carrier Proteins/analysis , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Chlorocebus aethiops , Lamins/analysis , Lamins/metabolism , Membrane Proteins , Mice , Nuclear Proteins/analysis , Phosphorylation , Protein Interaction Maps , Xenopus Proteins/analysis , Xenopus laevis/embryology , ran GTP-Binding Protein/analysisABSTRACT
This study investigated the profiling of polycyclic aromatic hydrocarbon- (PAH-) induced genotoxicity in cell lines and zebrafish. Each type of cells displayed different proportionality of apoptosis. Mitochondrial DNA (mtDNA) copy number was dramatically elevated after 5-day treatment of fluoranthene and pyrene. The notable deregulated proteins for PAHs exposure were displayed as follows: lamin-A/C isoform 3 and annexin A1 for benzopyrene; lamin-A/C isoform 3 and DNA topoisomerase 2-alpha for pentacene; poly[ADP-ribose] polymerase 1 (PARP-1) for fluoranthene; and talin-1 and DNA topoisomerase 2-alpha for pyrene. Among them, lamin-A/C isoform 3 and PARP-1 were further confirmed using mRNA and protein expression study. Obvious morphological abnormalities including curved backbone and cardiomegaly in zebrafish were observed in the 54 hpf with more than 400 nM of benzopyrene. In conclusion, the change of mitochondrial genome (increased mtDNA copy number) was closely associated with PAH exposure in cell lines and mesenchymal stem cells. Lamin-A/C isoform 3, talin-1, and annexin A1 were identified as universal biomarkers for PAHs exposure. Zebrafish, specifically at embryo stage, showed suitable in vivo model for monitoring PAHs exposure to hematopoietic tissue and other organs.
Subject(s)
Biomarkers/analysis , DNA Copy Number Variations/drug effects , DNA, Mitochondrial/drug effects , Lamins/analysis , Poly(ADP-ribose) Polymerases/analysis , Polycyclic Aromatic Hydrocarbons/toxicity , Animals , Apoptosis/drug effects , Biomarkers/metabolism , Cell Line , Cell Shape/drug effects , Cell Survival/drug effects , DNA Damage/drug effects , Embryo, Nonmammalian , Humans , Lamins/metabolism , Poly(ADP-ribose) Polymerases/metabolism , ZebrafishABSTRACT
Differences in the microscopic morphology of the hoof in forelimbs and hindlimbs of horses have been scarcely reported in the literature, especially concerning the distribution of primary and secondary epidermal laminae in the different regions. This study aimed to determine the density of primary and secondary epidermal laminae in the hoof of horses. For this, it was used fore and hindlimbs of 16 adult mixed breed horses. With a cross section 0.5 cm above the sole, it was quantified the primary epidermal laminae in the regions of the toe, and of lateral and medial quarters. Fragments with about 1cm ³ were taken from the proximal, middle and distal thirds of the hooves, in the different regions, subjected to conventional histological techniques and examined with an optical microscope. Data were statistically analyzed in relation to the fore and hindlimbs and between their various regions. The density of primary epidermal laminae varied around the hoof circumference, with greater values in the hoof toe, which gradually decreased towards the bulb of the hoof, without difference between thoracic and pelvic limbs. The average density of the secondary epidermal laminae per primary epidermal lamina does not change around the circumference of the hoof. Our findings indicated that the density of epidermal laminae is not different between fore and hindlimbs. The variation in the density of primary epidermal laminae around the hoof seems to be part of an adaptive response to different stresses in each region. A better understanding of the structural morphology contributes to a better understanding of the diagnosis, pathophysiology, and treatment of disorders that affect the hoof.
Diferenças na morfologia microscópica dos cascos dos membros pélvicos e torácicos dos equinos têm sido pouco relatadas na literatura, principalmente no tocante a distribuição de lâminas epidérmicas primárias e secundárias nas diversas regiões. O propósito deste estudo foi quantificar a densidade de lâminas epidérmicas primárias e secundárias no casco de equinos. Foram utilizados membros torácicos e pélvicos de oito equinos adultos e sem raça definida. Em uma secção transversal de aproximadamente 0,5cm de altura da sola dos cascos foi quantificada a densidade das lâminas epidérmicas primárias tanto na região da pinça quanto dos quartos lateral e medial. Fragmentos com aproximadamente 1cm³ foram retirados dos terços proximal, médio e distal do casco, nas diferentes regiões e submetidos a técnica histológica convencional, a densidade de lâminas epidérmicas secundárias foi quantificada com auxilio de microscópio óptico. Os dados foram analisados estatisticamente em relação aos membros torácicos e pélvicos e entre suas diversas regiões. A densidade de lâminas epidérmicas primárias varia ao redor da circunferência do casco, sendo maior na região da pinça do casco e diminui gradualmente em direção ao bulbo do casco, não existindo diferença entre membros pélvicos e torácicos. A densidade média de lâminas epidérmicas secundárias por lâmina epidérmica primária não varia em torno da circunferência dos cascos, assim como, quando comparada entre os membros torácicos e pélvicos. A variação da densidade das lâminas epidérmicas primárias em torno do casco parece fazer parte de uma resposta adaptativa às diferentes tensões existentes em cada região. O melhor entendimento da morfologia das estruturas do casco contribui na melhor compreensão do diagnóstico, fisiopatologia e tratamento das afecções que as acometem.
Subject(s)
Animals , Hoof and Claw/anatomy & histology , Horses/anatomy & histology , Lamins/analysis , Hindlimb , Upper ExtremityABSTRACT
The cellular cytoskeleton is composed of three fibrillar systems, namely actin microfilaments, microtubules and intermediate filaments (IFs). It not only is a structural system, which mediates functional compartmentalization, but also contributes to many cellular processes such as transport, mitosis, secretion, formation of cell extensions, intercellular communication and apoptosis. In this study, we have examined the distribution of four groups of IFs [cytokeratins (CKs), vimentin, desmin and lamins] in the somatic and germinal cells of the bovine ovary using RT-PCR and immunohistochemical techniques. Using RT-PCR, specific transcripts for all intermediate proteins studied (CK8, CK18, desmin, vimentin, lamin A/C and lamin B1) were detected. A characteristic immunohistochemical staining pattern was observed for the different IFs within the ovary. In this study, we used antibodies against type I CK (acidic CKs: CK14, CK18 and CK19) and type II CK (basic CKs: CK5 and CK8). Among these, only antibodies against CK18 gave a characteristic pattern of immunostaining in the ovary, which included the surface epithelium, the follicle cells, the endothelium of blood vessels and rete ovarii. Antibodies against all other CKs resulted in a weak staining of a limited number of cellular structures (CK5 and CK19) or were completely negative (CK8 and CK14, apart from the surface epithelium). Vimentin antibodies resulted occasionally in a weak staining of the granulosa cells of primary and secondary follicles. In late secondary follicles, the basal and the most apical follicle cells contacting the zona pellucida usually showed a marked immunostaining for vimentin. In antral follicles, three different immunostaining patterns for vimentin were observed. Desmin immunostaining was confined to the smooth muscle cells of blood vessels. Although mRNA for lamin A/C and lamin B1 could be demonstrated using RT-PCR, no immunostaining was found for lamins, neither in the follicle cells nor in the oocytes.
Subject(s)
Cattle/physiology , Intermediate Filaments/genetics , Intermediate Filaments/metabolism , Ovarian Follicle/physiology , Animals , Antibodies/immunology , Desmin/analysis , Desmin/immunology , Female , Granulosa Cells , Immunohistochemistry , Keratins/analysis , Keratins/immunology , Lamins/analysis , Lamins/immunology , Oocytes , Vimentin/analysis , Vimentin/immunologyABSTRACT
A thorough understanding of fat cell biology is necessary to counter the epidemic of obesity. Although molecular pathways governing adipogenesis are well delineated, the structure of the nuclear lamina and nuclear-cytoskeleton junction in this process are not. The identification of the 'linker of nucleus and cytoskeleton' (LINC) complex made us consider a role for the nuclear lamina in adipose conversion. We herein focused on the structure of the nuclear lamina and its coupling to the vimentin network, which forms a cage-like structure surrounding individual lipid droplets in mature adipocytes. Analysis of a mouse and human model system for fat cell differentiation showed fragmentation of the nuclear lamina and subsequent loss of lamins A, C, B1 and emerin at the nuclear rim, which coincides with reorganization of the nesprin-3/plectin/vimentin complex into a network lining lipid droplets. Upon 18 days of fat cell differentiation, the fraction of adipocytes expressing lamins A, C and B1 at the nuclear rim increased, though overall lamin A/C protein levels were low. Lamin B2 remained at the nuclear rim throughout fat cell differentiation. Light and electron microscopy of a subcutaneous adipose tissue specimen showed striking indentations of the nucleus by lipid droplets, suggestive for an increased plasticity of the nucleus due to profound reorganization of the cellular infrastructure. This dynamic reorganization of the nuclear lamina in adipogenesis is an important finding that may open up new venues for research in and treatment of obesity and nuclear lamina-associated lipodystrophy.
Subject(s)
Adipocytes/cytology , Adipogenesis , Cytoskeleton/metabolism , Nuclear Lamina/metabolism , 3T3-L1 Cells , Adipocytes/metabolism , Adipocytes/ultrastructure , Animals , Cells, Cultured , Cytoskeleton/ultrastructure , Humans , Immunohistochemistry , Lamins/analysis , Lamins/biosynthesis , Mice , Nuclear Lamina/ultrastructureABSTRACT
Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentially expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.
Subject(s)
Liver Neoplasms/metabolism , Matrix Attachment Region Binding Proteins/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Nuclear Matrix/metabolism , Proteomics/methods , Animals , Blotting, Western , Cell Cycle Proteins , Electrophoresis, Gel, Two-Dimensional , Heat-Shock Proteins/analysis , Heat-Shock Proteins/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/analysis , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Keratins, Type II/analysis , Keratins, Type II/metabolism , Lamins/analysis , Lamins/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/ultrastructure , Male , Matrix Attachment Region Binding Proteins/analysis , Microscopy, Electron , Nuclear Matrix/chemistry , Nuclear Matrix/ultrastructure , Nuclear Matrix-Associated Proteins/analysis , Nuclear Proteins/analysis , Nuclear Proteins/metabolism , Protein Binding , RNA, Nuclear/metabolism , RNA-Binding Proteins/analysis , RNA-Binding Proteins/metabolism , Rats , Rats, Inbred F344 , Ribonucleosides/chemistry , Ribonucleosides/metabolism , Tandem Mass Spectrometry/methods , Time Factors , Vanadates/chemistry , Vanadates/metabolismABSTRACT
Nuclear margin irregularity is an important diagnostic feature of malignant cells. The exact cause of nuclear margin irregularity is not fully understood. The distortion of the nuclear envelope is probably the major factor in nuclear margin irregularity. Multiple proteins on the nuclear envelope, particularly nuclear lamin, are responsible for the distortion of the nuclear envelope. The extracellular matrix may also indirectly affect the nuclear position and shape by the closely connected network of actin-nespirin-SUN-lamin links. The alteration of nuclear matrix protein and RET-oncogene expression may play a role in nuclear envelope distortion and in margin irregularity. In this review, the probable causes and impact of nuclear margin irregularities are discussed.
Subject(s)
Neoplasms/pathology , Nuclear Envelope/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Lamins/analysis , Lamins/genetics , Neoplasms/genetics , Nuclear Envelope/chemistry , Nuclear Envelope/geneticsABSTRACT
En este trabajo se analizaron los cambios en los parámetros de color (L, a, b, Æ, IB) de láminas de sardina durante la deshidratación osmótica a diferentes condiciones de concentración y temperatura de la solución osmótica. Las sardinas (Sardinella aurita) se cortaron en láminas (20,1×15,0×6,4 mm3), se les midió el color y se formaron 175 grupos experimentales de 4 láminas cada uno. Se introdujeron simultáneamente siete grupos en una solución osmótica de concentración y temperatura dadas para someterlos a deshidratación osmótica, posteriormente se removió un grupo a los 20; 40; 60; 90; 120; 180 y 240 min de transcurrido el proceso osmótico y se midió el color en las láminas. Este procedimiento se efectuó para cada condición de acuerdo a un diseño factorial 5x5 donde la temperatura y concentración eran 30; 32; 34; 36 y 38°C, 0,15; 0,18; 0,21; 0,24 y 0,27 g NaCl/g, respectivamente. Se obtuvieron valores iniciales de L (36,48 ± 0,77), a (6,47 ± 0,64), b (8,74 ± 0,49), Æ (0) y IB (37,42 ± 0,69). Los valores finales variaron para L entre 40 y 47, a entre 4,2 y 2,6, b entre 7,5 y 5,7, IB entre 39,5 y 45,0 y Æ entre 5,3 y 10,4 dependiendo de las condiciones de deshidratación. Los valores de a y b disminuyeron (P<0,05) al incrementar el tiempo de deshidratación y la concentración, mientras que los de L, Æ y IB aumentaron (P < 0,05). Las disminuciones en a y b fueron menores al incrementarse la temperatura mientras que los aumentos en L, Æ y IB fueron mayores. Se obtuvieron modelos de predicción de los cambios en el color en función de las condiciones de la deshidratación osmótica.
The changes on the color parameters (L, a, b, Æ, whiteness index) of sardine sheets during osmotic dehydration were analyzed at different temperatures and brine concentrations. Sardines (Sardinella aurita) were cut into sheets (20.1x15.0x6.4 mm3), color was measured and 175 groups with 4 sheets in each were formed. Seven groups were introduced simultaneously in an osmotic solution of a desired concentration and temperature for carry out osmotic dehydration, one group was removed at 20; 40; 60; 90; 120; 180 and 240 min, and color of sheets was determined. This procedure was performed for each test condition according to a 5x5 factorial design where the temperature and concentration were 30; 32; 34; 36 and 38°C, 0.15, 0.18, 0.21, 0.24, and 0.27 g NaCl/g brine, respectively. Initial values for L (36.48 ± 0.77), a (6.47 ± 0.64), b (8.74 ± 0.49), Æ (0) and WI (37.42 ± 0.69) were obtained. Final values ranged for L from 40 to 47, for a from 4.2 to 2.6, for b from 7.5 to 5.7, for WI from 39.5 to 45.0 and for Æ from 5.3 to 10.4 according to dehydration conditions. Values for a and b decreased (P<0.05) with increasing both dehydration time and temperature while those for L, Æ and WI index increased (P < 0.05). The decreases in a and b values were lesser with increasing temperature while increases in L, Æ and WI values were higher. Models for prediction of color changes as functions of the conditions of osmotic dehydration were obtained.
Subject(s)
Food Coloring Agents/radiation effects , Food Preservation/methods , Fishes , Lamins/analysis , Osmotic PressureABSTRACT
The peripheral nuclear lamina, which is largely but not entirely associated with inactive chromatin, is considered to be an important determinant of nuclear structure and gene expression. We present here an inducible system to target a genetic locus to the nuclear lamina in living mammalian cells. Using three-dimensional time-lapse microscopy, we determined that targeting of the locus requires passage through mitosis. Once targeted, the locus remains anchored to the nuclear periphery in interphase as well as in daughter cells after passage through a subsequent mitosis. Upon transcriptional induction, components of the gene expression machinery are recruited to the targeted locus, and we visualized nascent transcripts at the nuclear periphery. The kinetics of transcriptional induction at the nuclear lamina is similar to that observed at an internal nuclear region. This new cell system provides a powerful approach to study the dynamics of gene function at the nuclear periphery in living cells.
Subject(s)
Gene Expression Regulation/physiology , Nuclear Lamina/genetics , Transcription, Genetic/physiology , Cell Line , Chromosomes/physiology , Humans , Interphase , Kinetics , Lamins/analysis , Lamins/metabolism , Luminescent Proteins/analysis , Mitosis/physiology , Models, Genetic , Nuclear Lamina/metabolism , Nuclear Lamina/ultrastructureABSTRACT
We report stimulated emission depletion (STED) fluorescence microscopy with continuous wave (CW) laser beams. Lateral fluorescence confinement from the scanning focal spot delivered a resolution of 29-60 nm in the focal plane, corresponding to a 5-8-fold improvement over the diffraction barrier. Axial spot confinement increased the axial resolution by 3.5-fold. We observed three-dimensional (3D) subdiffraction resolution in 3D image stacks. Viable for fluorophores with low triplet yield, the use of CW light sources greatly simplifies the implementation of this concept of far-field fluorescence nanoscopy.
Subject(s)
Lasers , Microscopy, Fluorescence/methods , Optics and Photonics , Animals , Cell Line, Tumor , Fluorescent Dyes/chemistry , Humans , Imaging, Three-Dimensional , Immunohistochemistry , Lamins/analysis , Microscopy, Confocal , Microscopy, Fluorescence/instrumentation , Microspheres , Neurofilament Proteins/analysis , Nuclear Lamina/chemistry , Nuclear Lamina/metabolism , PC12 Cells , Qa-SNARE Proteins/analysis , Rats , Spectrometry, FluorescenceABSTRACT
The process of carcinogenesis is characterized by definite changes in the protein composition of the nuclear matrix. We have recently found that lamins form, in addition to the nuclear lamina, an intranuclear web of thin fibrils. This finding prompted us to address the question of whether changes in the expression of lamins occur in the course of tumor development. In prostate cancer, lamin B undergoes a significant increase; interestingly, its nuclear content strongly correlates with tumor differentiation. Moreover, all the lamins show reproducible alterations in the distribution of the isoelectric variants, suggesting that dephosphorylation events could trigger changes in the pattern of gene expression by inducing structural rearrangements of the nuclear scaffold.
Subject(s)
Cell Nucleus/chemistry , Lamins/analysis , Prostatic Neoplasms/metabolism , Aged , Blotting, Western , Electrophoresis, Gel, Two-Dimensional , Humans , Intermediate Filaments/chemistry , Lamin Type A/analysis , Lamin Type B/analysis , Male , Middle Aged , Nuclear Matrix/chemistry , Prostate/chemistry , Prostate/pathology , Prostatic Neoplasms/pathologyABSTRACT
The him-8 gene is essential for proper meiotic segregation of the X chromosomes in C. elegans. Here we show that loss of him-8 function causes profound X chromosome-specific defects in homolog pairing and synapsis. him-8 encodes a C2H2 zinc-finger protein that is expressed during meiosis and concentrates at a site on the X chromosome known as the meiotic pairing center (PC). A role for HIM-8 in PC function is supported by genetic interactions between PC lesions and him-8 mutations. HIM-8 bound chromosome sites associate with the nuclear envelope (NE) throughout meiotic prophase. Surprisingly, a point mutation in him-8 that retains both chromosome binding and NE localization fails to stabilize pairing or promote synapsis. These observations indicate that stabilization of homolog pairing is an active process in which the tethering of chromosome sites to the NE may be necessary but is not sufficient.
Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/genetics , Cell Cycle Proteins/physiology , Chromosome Pairing/physiology , Meiosis/physiology , X Chromosome/metabolism , Animals , Caenorhabditis elegans/physiology , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosome Pairing/genetics , Disorders of Sex Development/genetics , Female , In Situ Hybridization, Fluorescence , Lamins/analysis , Male , Meiosis/genetics , Meiotic Prophase I/genetics , Microscopy, Fluorescence , Mutation/genetics , Nuclear Envelope/chemistry , Nuclear Envelope/physiology , Pachytene Stage/genetics , Phenotype , Point Mutation/genetics , Synaptonemal Complex/genetics , Synaptonemal Complex/metabolism , X Chromosome/genetics , Zinc Fingers/geneticsABSTRACT
BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare sporadic disorder with an incidence of approximately 1 per 8 million live births. The phenotypic appearance consists of short stature, sculptured nose, alopecia, prominent scalp veins, small face, loss of subcutaneous fat, faint mid-facial cyanosis, and dystrophic nails. HGPS is caused by mutations in LMNA, the gene that encodes nuclear lamins A and C. The most common mutation in subjects with HGPS is a de novo single-base pair substitution, G608G (GGC>GGT), within exon 11 of LMNA. This creates an abnormal splice donor site, leading to expression of a truncated protein. RESULTS: We studied a new case of a 5 year-old girl with HGPS and found a heterozygous point mutation, G608G, in LMNA. Complementary DNA sequencing of RNA showed that this mutation resulted in the deletion of 50 amino acids in the carboxyl-terminal tail domain of prelamin A. We characterized a primary dermal fibroblast cell line derived from the subject's skin. These cells expressed the mutant protein and exhibited a normal growth rate at early passage in primary culture but showed alterations in nuclear morphology. Expression levels and overall distributions of nuclear lamins and emerin, an integral protein of the inner nuclear membrane, were not dramatically altered. Ultrastructural analysis of the nuclear envelope using electron microscopy showed that chromatin is in close association to the nuclear lamina, even in areas with abnormal nuclear envelope morphology. The fibroblasts were hypersensitive to heat shock, and demonstrated a delayed response to heat stress. CONCLUSION: Dermal fibroblasts from a subject with HGPS expressing a mutant truncated lamin A have dysmorphic nuclei, hypersensitivity to heat shock, and delayed response to heat stress. This suggests that the mutant protein, even when expressed at low levels, causes defective cell stability, which may be responsible for phenotypic abnormalities in the disease.