Alreration of nuclear matrix-intermediate filament system and differential expression of nuclear matrix proteins during human hepatocarcinoma cell differentiation

AIM:To investigate the association between the configurational and compositional changes of nuclear matrix and the differentiation of carcinoma cells. METHODS: Cells cultured with or without 5 x 10"3 mmol/L of hexamethylene bisacetamide (HMBA) on Nickel grids were treated by selective extraction and prepared for whole mount observation under electron microscopy. The samples were examined under transmission electron microscope. Nuclear matrix proteins were selectively extracted and subjected to subcellular proteomics study. The protein expression patterns were analyzed by PDQuest software. Spots of differentially expressed nuclear matrix proteins were excised and subjected to in situ digestion with trypsin. The peptides were analyzed by matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Data were submitted for database searching using Mascot tool (www.matrixscience.com). RESULTS: The nuclear matrix (NM) and intermediate filament (IF) in SMMC-7721 hepatocarcinoma cells were found relatively sparse and arranged irregularly. The nuclear lamina was non-uniform,and two kinds of filaments were not tightly connected. After induction for differentiation by HMBA,the NM-IF filaments were concentrated and distributed uniformly. The heterogeneous population of filaments,including highly branched utrathin filaments could also be seen in the regular meshwork. The connection between the two kinds of filaments and the relatively thin,condensed and sharply demarcated lamina composed of intermediate-sized filaments was relatively fastened. Meanwhile,21 NM proteins changed remarkably during SMMC-7721 cell differentiation. Four proteins,i.e. mutant Pyst1,hypothetical protein,nucleophosminl,and LBP were downregulated,whereas four other proteins,eIF6,p44 subunit,(3-tubulin,and SIN3B were upregulated with the last one,SR2/ASF found only in the differentiated SMMC-7721 cells. CONCLUSION: The induced differentiation of SMMC-7721 cells by HMBA is accompanied by the configurational changes of nuclear matrix-intermediate filament (NM-IF) system and the compositional changes of nuclear matrix protein expression. These changes may be important morphological or functional indications of the cancer cell reversion.

THE RELATIONSHIPS BETWEEN ERYTHROBLAST DENUCLEATION AND THE NUCLEAR MATRIX－INTERMEDIATE FILAMENTS

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Mechanical properties of crosslinks controls failure mechanism of hierarchical intermediate filament networks

Intermediate filaments are one of the key components of the cytoskeleton in eukaryotic cells, and their mechanical properties are found to be equally important for physiological function and disease. While the mechanical properties of single full length filaments have been studied, how the mechanical properties of crosslinks affect the mechanical property of the intermediate filament network is not well understood. This paper applies a mesoscopic model of the intermediate network with varied crosslink strengths to investigate its failure mechanism under the extreme mechanical loading. It finds that relatively weaker crosslinks lead to a more flaw tolerant intermediate filament network that is also 23% stronger than the one with strong crosslinks. These findings suggest that the mechanical properties of interfacial components are critical for bioinspired designs which provide intriguing mechanical properties.

Computational and theoretical modeling of intermediate filament networks:Structure,mechanics and disease

Intermediate filaments, in addition to microtubules and actin microfilaments, are one of the three major components of the cytoskeleton in eukaryotic cells. It was discovered during the recent decades that in most cells, intermediate filament proteins play key roles to reinforce cells subjected to large-deformation, and that they participate in signal transduction, and it was proposed that their nanome- chanical properties are critical to perform those functions. However, it is still poorly understood how the nanoscopic structure, as well as the combination of chemical composition, molecular structure and interfacial properties of these protein molecules contribute to the biomechanical properties of filaments and filament networks. Here we review recent progress in computational and theoretical studies of the intermediate filaments network at various levels in the protein＇s structure. A multiple scale method is discussed, used to couple molecular modeling with atomistic detail to larger-scale material properties of the networked material. It is shown that a finer-trains-coarser method- ology as discussed here provides a useful tool in understanding the biomechanical property and disease mechanism of intermediate filaments, coupling experiment and simulation. It further allows us to improve the understanding of associated disease mechanisms and lays the foundation for engineering the mechanical properties of biomaterials.

The keratin intermediate filament-like system in maize protoplasts

The application of Penman's method of cell fractionation to plant protoplasts leads to our finding of keratin intermediate filament(IF)-like system in maize protoplasts,which was identified by using immunogold labelling with monoclonal antibody of cytokeratin from animal cells.Many gold particles were found to be bound on filaments,linked by 3 nm filaments.After further digestion and extraction with DNase I and ammonium sulphate.IF-like framework-lamina-nuclear matrix system was shown under electron microscope.That IF system exists in plant protoplasts just like in animal cells,and their main component is keratin-like protein.

广州管圆线虫抗原IF的体外表达系统的建立和分析

【目的】建立广州管圆线虫特异性抗原中间纤维(IF)基因的体外表达系统,研究IF蛋白的结构和功能关系。【方法】将IF基因亚克隆入原核表达载体pET-30a-c(+)并在原核中诱导表达,Westernblot鉴定融合表达产物的免疫特性,用组氨酸(His)亲合层析柱分离纯化表达产物,鉴定表达体系效率。生物信息学分析目的蛋白结构和功能关系。【结果】抗原基因IF在原核中与6个组氨酸进行了融合表达,以可溶性表达为主,并能在该系统中大量、稳定表达,其相对分子质量Mr=48×103,该表达产物具有较好的抗原性,可被大鼠感染血清识别,抗原IF具有典型的中间纤维家族特征,该蛋白具有多个抗原表位存在。【结论】建立了IF的有效原核表达系统,IF抗原蛋白的结构决定了它的属性和功能。

广州管圆线虫抗原IF的中间纤维蛋白组织来源分析和定位

目的对广州管圆线虫抗原IF进行中间纤维蛋白家族分类和细胞组织定位。方法IPTG诱导重组基因pET-IF在大肠杆菌中表达广州管圆线虫抗原IF,用组氨酸亲和层析纯化表达产物,用Western blot方法鉴定抗原IF的中间纤维蛋白类型;小鼠皮下多点注射法制备抗IF抗体血清,用免疫组织化学方法检测抗原IF在虫体组织和细胞上的定位。结果广州管圆线虫抗原IF在体外成功表达并纯化,其属于中间纤维蛋白家族的角蛋白,定位于广州管圆线虫成虫肠管壁,存在于细胞质中。结论广州管圆线虫抗原IF分布于虫体肠管壁,同时从组织学角度证明广州管圆线虫存在中间纤维结构。

HMBA诱导胃癌细胞MGc80-3分化过程中NM-LA-IF体系的变化

目的研究环六亚甲基双乙酰胺（ＨＭＢＡ）对胃癌细胞ＭＧｃ８０－３核骨架－核周层状体－中间纤维（ＮＭ－ＬＡ－ＩＦ）体系的影响。方法使用整装电镜及双向电泳技术观察诱导分化前后ＭＧｃ８０－３细胞ＮＭ－ＬＡ－ＩＦ体系结构及蛋白组成的变化。结果（１）ＨＭＢＡ诱导处理后细胞ＮＭ－ＬＡ－ＩＦ系统结构清晰有序，形成相互作用的骨架网络系统。（２）多肽组成变化显著，除了ＬａｍｉｎａＡ、Ｂ、Ｃ以及两种波形纤维蛋白没有变化外，其余蛋白的含量和种类均有减少。结论该系统结构及多肽组成的差异与胃癌细胞恶性表型逆转有关，且诱导后的细胞分化程度提高。

Intermediate Filaments in Higher Plant Cells and Their Reconstitution in vitro

Delicate networks of 10-nm filaments were found to exist widely in suspension cells of cultured carrot and root tip cells of pea by selective extraction combined with whole mount and diethylene glycol distearate plastic embedment-free section for electron microscopy. These filament components are polypeptides of 52, 58, 62—64 and 50kD using Western blot analysis. A relatively uniform 10-nm filaments, as well as bundle of such filaments could be reconstituted in vitro. The reconstituted filaments were indistinguishable from native plant intermediate filaments in morphology and protein components. Thus the intermediate filaments are actually present in cytoplasm of higher plants. We also found no obvious specificity in tissues and species by comparison of the proteins among various types of plant cells.

Assembly characteristics of plant keratin intermediate filaments in vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembled in vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filaments in vitro. In higher magnification images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24—25 nm periodic structural repeat alone the axis of beth the 10 nm filaments and protofilaments. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments.

Joining actions: crosstalk between intermediate filaments and actin orchestrates cellular physical dynamics and signaling

Many key cellular functions are regulated by the interplay of three distinct cytoskeletal networks, made of actin filaments,microtubules, and intermediate filaments(IFs), which is a hitherto poorly investigated area of research. However, there are growing evidence in the last few years showing that the IFs cooperate with actin filaments to exhibit strongly coupled functions.This review recapitulates our current knowledge on how the crosstalk between IFs and actin filaments modulates the migration properties, mechano-responsiveness and signaling transduction of cells, from both biophysical and biochemical point of view.

Bone morphogenetic protein-7 induced bone marrow stromal cells differentiate into neuron-like cells

Bone morphogenetic protein-7 is widely accepted as an inducer for bone marrow stem cells differentiating into osteoblasts and chondrocytes. Whether bone marrow stromal cells differentiate into neuron-like cells remains unclear. The current study examined the presence of positive cells for intermediate filament protein and microtubule associated protein-2 in the cytoplasm of bone marrow stromal cells induced by bone morphogenetic protein-7 under an inverted microscope, while no expression of glial fibrillary acidic protein was found. Reverse transcription PCR electrophoresis also revealed a positive target band for intermediate filament protein and microtubule-associated protein 2 mRNA. These results confirmed that bone morphogenetic protein-7 induces rat bone marrow stromal cells differentiating into neuron-like cells.

Immunocytochemical Study of Cells in the Vitreous of Proliferative Vitreoretretinopathy

Purpose: To identify the cellular components of vitreous samples obtained during vit-rectomy for proliferative vitreoretinopathy(PVR).Methods: With the use of three intermediate filament (IF) proteins, vimentin, glialfibrillary acidic protein (GFAP), and cytokeratin (CK), cytocentrifuge slides of 14fresh vitreous aspirates were detected with immunohistochemical technique.Results: All the specimens contained epithelial-like proliferative cells with or withoutpigment and some membrane-like pieces. Immunocytochemical staining showed that76.0-90.0% cells stained for CK, 17.4-29.6% cells expressed GFAP, and 80.1-91.0% cells were positive for vimentin.Conclusions : Majority of cells in the vitreous samples originated from retinal pigmentepithelial cells (RPE) and glial cells in PVR. Expression of IF proteins may be determinedby tissue of origin and local microenvironment. Eye Science 1999 ; 15; 13 - 16.

Time course of expression of intermediate filament protein vimentin,nestin and desmin in rat renal glomerular injury

Podocytes in renal glomemlus express unusual intermediate filament proteins （IFs） for visceral epithelial cells. IFs cytoskeleton is mainly composed of vimentin, nestin and desmin. Tissue injury is often accompanied by changes in gene expression of IFs. Enhanced desmin staining in variety of rat experimental including 2 4 puromycin nephrosis. It has not podocytes are observed in a models of podocyte injury aminonucleoside （PAN） been elucidated whether expression of vimentin and nestin is up-regulated in podocyte injury. To further gain insight into expression of IFs in podocytes, we investigated the time course of vimentin, nestin and desmin in PAN nephrosis.

THE KERATIN INTERMEDIATE FILAMENTLIKE SYSTEM IN THE PLANT MESOPHYLL CELLS

A delicate intermediate filament-like network of mesophyll cells was observed both in maize and tobacco, using selective extraction together with whole-mount cell preparation for electron microscopy. The filament of the network is about 10 nm in diameter. Further test using immuno-gold labeling with anti-keratin antibodies indicated that the component of the intermediate filament-like system was keratin-like protein. Such a keratin-like intermediate filament system existing in plant cells was demonstrated for the first time. Meanwhile, 3-nm size filaments and their connection with 10-nm filaments were also shown in maize and tobacco protoplasts.

Sequence analysis of keratin-like proteins and cloning of intermediate filament-like cDNA from higher plant cells

Two keratin-like proteins of 64 and 55 ku were purified from suspension cells of Caucus carota L, and their partial amino acid sequences were determined. The homological analysis showed that the sequence from the 64 ku protein was highly homological to p-glucosidase, and that from the 55 ku protein had no significant homologue in GenBank. Using conservative sequence of animal IF proteins as primer, we cloned a cDNA fragment from Daucus carota L. Southern blot and Northern blot results indicated that this cDNA fragment was a single copy gene and expressed both in suspension cells and leaves. Homological analysis revealed that it had moderate homology to a variety of a-helical proteins. Our results might shed more light on molecular characterization of IF existence in higher plant.

Existence of Cage-like Structure Consisting of Intermediate Filament-like Fibrils in Basal Bodies of Chlamydomonas sp

Chlamydomonas is a primitive unicellar eukaryote with two flagella of equal length at its anterior end. At the root of flagella is the organella called the basal body which consists of nine triple sets of microtubules. The basal bodies are homologous to the centriole, and are the center of organizing microtubule in flagellum

THE KERATIN-LIKE INTERMEDIATE FILAMENT IN PLANT ROOT TIP CELLS

Ⅰ. INTRODUCTION Intermediate filaments with a diameter of 10 nm, known as a cytoskeleton system, are extensively present in animal cells. Whether the intermediate filaments are present in plant cells remains to be a question. Some investigators attempt to seek answers from various aspects, but it is still unsolved. Powell observed 7-nm filament in carrot suspension cells after treatment with high salt and Triton X-100. Dawson and Miller showed

一个广州管圆线虫特异性抗原候选基因的克隆与表达

目的用免疫探针交叉筛选前期实验获得的广州管圆线虫候选抗原基因,将筛选出的特异抗原候选基因进行原核表达、鉴定。方法制备抗血吸虫和抗旋毛虫多克隆抗体血清,交叉筛选广州管圆线虫抗原候选基因,从中获得特异性抗原候选基因,将其从重组噬菌体状态转化为质粒状态,再亚克隆入原核表达载体pET-30a-c(+)进行融合表达,SDS-PAGE鉴定表达产物,同时对该基因进行生物信息学分析。结果获得了一个广州管圆线虫特异性抗原候选基因,经生物信息学分析该抗原基因属中间纤维家族基因(intermediate filament,IF),原核表达产物为48KDa。结论获得了一个广州管圆线虫特异性抗原候选基因及其原核表达产物。