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31.
Extracellular cell matrices deposited by cells stimulate cell proliferation. However, their generation is cumbersome and time consuming. We show here that controlled fixation of fibronectin layers after coating culture vessels significantly enhances expansion of murine and human mesenchymal stem cells (MSCs) and, to a lesser extent, primary fibroblasts. In contrast, fibronection fixation did not stimulate proliferation of established cancer cell lines. Fixed vitronectin or collagen IV layers also enhanced proliferation of murine MSCs. Thus, controlled formaldehyde fixation of layers formed by fibronectin or some other extracellular matrix components represents a simple and reproducible way to enhance proliferation of primary cells. 相似文献
32.
Low A Chandrashekaran IR Adda CG Yao S Sabo JK Zhang X Soetopo A Anders RF Norton RS 《Biopolymers》2007,87(1):12-22
Merozoite surface protein 2 (MSP2) is a GPI-anchored protein on the surface of the merozoite stage of the malaria parasite Plasmodium falciparum. It is largely disordered in solution, but has a propensity to form amyloid-like fibrils under physiological conditions. The N-terminal conserved region (MSP2(1-25)) is part of the protease-resistant core of these fibrils. To investigate the structure and dynamics of this region, its ability to form fibrils, and the role of individual residues in these properties, we have developed a bacterial expression system that yields > or =10 mg of unlabeled or (15)N-labeled peptide per litre of culture. Two recombinant versions of MSP2(1-25), wild-type and a Y7A/Y16A mutant, have been produced. Detailed conformational analysis of the wild-type peptide and backbone (15)N relaxation data indicated that it contains beta-turn and nascent helical structures in the central and C-terminal regions. Residues 6-21 represent the most ordered region of the structure, although there is some flexibility around residues 8 and 9. The 10-residue sequence (MSP2(7-16)) (with two Tyr residues) was predicted to have a higher propensity for beta-aggregation than the 8-mer sequence (MSP2(8-15)), but there was no significant difference in conformation between MSP2(1-25) and [Y7A,Y16A]MSP2(1-25) and the rate of fibril formation was only slightly slower in the mutant. The peptide expression system described here will facilitate further mutational analyses to define the roles of individual residues in transient structural elements and fibril formation, and thus contribute to the further development of MSP2 as a malaria vaccine candidate. 相似文献
33.
Han F Gilbert JR Harrison G Adams CS Freeman T Tao Z Zaka R Liang H Williams C Tuan RS Norton PA Hickok NJ 《Experimental cell research》2007,313(8):1518-1532
Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that includes the type II repeat extra domain B (EDB) but excludes extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for the progression of chondrogenesis. To test this, we treated the pre-chondrogenic cell line ATDC5 with transforming growth factor-beta1, which has been shown to modulate expression of the EDA and EDB exons, as well as the late markers of chondrocyte maturation; it also slightly accelerates the early acquisition of a sulfated proteoglycan matrix without affecting cell proliferation. When chondrocytes are treated with TGF-beta1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with the regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. To determine if overexpression of the SRp40 isoforms altered FN and FN EDA organization, cDNAs encoding these isoforms were overexpressed in ATDC5 cells. Overexpression of the long-form of SRp40 yielded an FN organization similar to TGF-beta1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-beta1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression. 相似文献
34.
Fibronectin alternative exon EIIIA is largely included in undifferentiated mesenchymal cells of the developing limb bud, whereas the exon is excluded in differentiated chondrocytes. Inclusion of exon EIIIA in chondrocytic cells is increased by overexpression of SRp40, and, to a lesser extent, SRp75, but not SRp55. RT-PCR analysis using real-time PCR revealed that the levels of the mRNAs for these three proteins did not vary significantly in chick chondrocytes versus mesenchymal cells of the developing limb bud. However, a variant spliced form of SRp40, termed, SRp40LF, is detected preferentially in chondrocytes and in chondrifying mesenchymal cells. Forced overexpression of SRp40 or SRp75, but not SRp55, enhanced chondrogenic differentiation of chick limb mesenchymal cells in a high-density micromass assay. Overexpression of SRp40LF, which produces a truncated form of SRp40, also was strongly pro-chondrogenic. In a HeLa cell-based assay, SRp40LF fails to substitute for SRp40 in mediating an increase in exon EIIIA inclusion, suggesting that the latter event is not essential for the pro-chondrogenic effect. These results demonstrate the ability of these highly conserved splicing factors to modulate chondrogenesis and are consistent with earlier results that implicated exon EIIIA-containing isoforms of fibronectin in formation of chondrogenic condensations. 相似文献
35.
Morita Y Araki H Sugimoto T Takeuchi K Yamane T Maeda T Yamamoto Y Nishi K Asano M Shirahama-Noda K Nishimura M Uzu T Hara-Nishimura I Koya D Kashiwagi A Ohkubo I 《FEBS letters》2007,581(7):1417-1424
Legumain/asparaginyl endopeptidase (EC 3.4.22.34) is a novel cysteine protease that is abundantly expressed in the late endosomes and lysosomes of renal proximal tubular cells. Recently, emerging evidence has indicated that legumain might play an important role in control of extracellular matrix turnover in various pathological conditions such as tumor growth/metastasis and progression of atherosclerosis. We initially found that purified legumain can directly degrade fibronectin, one of the main components of the extracellular matrix, in vitro. Therefore, we examined the effect of legumain on fibronectin degradation in cultured mouse renal proximal tubular cells. Fibronectin processing can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and can be enhanced by the overexpression of legumain, indicating that fibronectin degradation occurs in the presence of legumain in lysosomes from renal proximal tubular cells. Furthermore, in legumain-deficient mice, unilateral ureteral obstruction (UUO)-induced renal interstitial protein accumulation of fibronectin and renal interstitial fibrosis were markedly enhanced. These findings indicate that legumain might have an important role in extracellular matrix remodeling via the degradation of fibronectin in renal proximal tubular cells. 相似文献
36.
Debasish Haldar Arindam Banerjee 《International journal of peptide research and therapeutics》2007,13(3):439-446
The terminally protected peptide Boc-Leu-Val-Phe-Phe-Ala-OMe bearing sequence similarity with the central hydrophobic cluster
(CHC) of Alzheimer’s Aβ17–21 peptide self-assembles to produce amyloid-like straight unbranched fibrils from organic solvents. The fibrils readily bind
with a physiological dye Congo red (CR) and exhibits green gold birefringence under polarized light, a characteristic feature
of amyloid plaque obtained from many neurodegenerative diseases. FTIR spectroscopy and in silico energy minimization study shed some light on the antiparallel supramolecular β-sheet aggregation of the peptide. 相似文献
37.
Goki Tanaka Tomoyuki Yamanaka Yoshiaki Furukawa Naoko Kajimura Kaoru Mitsuoka Nobuyuki Nukina 《生物化学与生物物理学报:疾病的分子基础》2019,1865(6):1410-1420
Synucleinopathies comprise a diverse group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. These share a common pathological feature, the deposition of alpha-synuclein (a-syn) in neurons or oligodendroglia. A-syn is highly conserved in vertebrates, but the primary sequence of mouse a-syn differs from that of human at seven positions. However, structural differences of their aggregates remain to be fully characterized. In this study, we found that human and mouse a-syn aggregated in vitro formed morphologically distinct amyloid fibrils exhibiting twisted and straight structures, respectively. Furthermore, we identified different protease-resistant core regions, long and short, in human and mouse a-syn aggregates. Interestingly, among the seven unconserved amino acids, only A53T substitution, one of the familial PD mutations, was responsible for structural conversion to the straight-type. Finally, we checked whether the structural differences are transmissible by seeding and found that human a-syn seeded with A53T aggregates formed straight-type fibrils with short protease-resistant cores. These results suggest that a-syn aggregates form sequence-dependent polymorphic fibrils upon spontaneous aggregation but become seed structure-dependent upon seeding. 相似文献
38.
Calero M Pawlik M Soto C Castaño EM Sigurdsson EM Kumar A Gallo G Frangione B Levy E 《Journal of neurochemistry》2001,77(2):628-637
Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild-type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild-type protein forms amorphous aggregates. We also show by circular dichroism, steady-state fluorescence and Fourier-transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing alpha-helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural differences between variant and wild-type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis. 相似文献
39.
Extracellular matrix components that flank the fissura prima, a primary surface infolding of the cerebellum in birds and mammals, were examined in the embryonic chick using light and transmission electron microscopy. Cerebella dissected from Day 10 embryos were perfused with a paraformaldehyde-glutaraldehyde-tannic acid primary fixative and sectioned in the sagittal plane through the mid-vermis. Ultrastructural analysis revealed a distinct, continuous basal lamina separating the organ parenchyma (epithelia) from pia mater (mesenchyme) at the fissure surface (arbitrarily labeled; fissure floor, folia wall, and folia apex). The basal lamina was significantly thicker (P < 0.001) at the fissure floor compared to that found at the folia wall, which was significantly thicker (P < 0.001) than that observed at the folia apex. Folds in the basal lamina were observed exclusively at the fissure floor. Surface-associated collagen fibrils were distributed in an aligned, relatively dense manner at the fissure floor, compared with fibrils observed in various orientations and widely separated or absent at the folia wall and folia apex. Metachromasia was more pronounced in the fissure floor than in either the folia wall or folia apex in methylene blue-stained tissue sections. Together, the thicker, folded basal lamina and densely aligned collagen fibrils at the fissure floor provide a chemical rationale for this color change. These findings suggest that the differential accumulation of extracellular matrix at the fissura prima is positioned to play a structural and/or biochemical role in the maintenance of this fold. 相似文献
40.
Matrix loading: assembly of extracellular matrix collagen fibrils during embryogenesis 总被引:1,自引:0,他引:1
Kadler K 《Birth defects research. Part C, Embryo today : reviews》2004,72(1):1-11
Nothing in biology stimulates the imagination like the development of a single fertilized egg into a newborn child. Consequently, a major focus of biomedical research is aimed at understanding cell differentiation, proliferation, and specialization during child health and human development. However, the fact that the increase in size and shape of the growing embryo has as much to do with the extracellular matrix (ECM) as with the cells themselves, is largely overlooked. Cells in developing tissues are surrounded by a fiber-composite ECM that transmits mechanical stimuli, maintains the shape of developing tissues, and functions as a scaffold for cell migration and attachment. The major structural element of the ECM is the collagen fibril. The fibrils, which are indeterminate in length, are arranged in different tissues in exquisite supramolecular architectures, including parallel bundles, orthogonal lamellae, and concentric weaves. This article reviews our current understanding of the synthesis and assembly of collagen fibrils, and discusses challenging questions about how cells assemble an organized ECM during embryogenesis. 相似文献