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71.
Saha PK Kojima H Martinez-Botas J Sunehag AL Chan L 《The Journal of biological chemistry》2004,279(34):35150-35158
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Sushmita Saha Jennifer Kirkham David Wood Stephen Curran Xuebin B. Yang 《Cell and tissue research》2013,352(3):495-507
A major clinical need exists for cartilage repair and regeneration. Despite many different strategies having been pursued, the identification of an optimised cell type and of pre-treatment conditions remains a challenge. This study compares the cartilage-like tissue generated by human bone marrow stromal cells (HBMSCs) and human neonatal and adult chondrocytes cultured on three-dimensional (3D) scaffolds under various conditions in vitro and in vivo with the aim of informing future cartilage repair strategies based upon tissue-engineering approaches. After 3 weeks in vitro culture, all three cell types showed cartilage-like tissue formation on 3D poly (lactide-co-glycolide) acid scaffolds only when cultured in chondrogenic medium. After 6 weeks of chondro-induction, neonatal chondrocyte constructs revealed the most cartilage-like tissue formation with a prominent superficial zone-like layer, a middle zone-like structure and the thinnest fibrous capsule. HBMSC constructs had the thickest fibrous capsule formation. Under basal culture conditions, neonatal articular chondrocytes failed to form any tissue, whereas HBMSCs and adult chondrocytes showed thick fibrous capsule formation at 6 weeks. After in vivo implantation, all groups generated more compact tissues compared with in vitro constructs. Pre-culturing in chondrogenic media for 1 week before implantation reduced fibrous tissue formation in all cell constructs at week 3. After 6 weeks, only the adult chondrocyte group pre-cultured in chondrogenic media was able to maintain a more chondrogenic/less fibrocartilaginous phenotype. Thus, pre-culture under chondrogenic conditions is required to maintain a long-term chondrogenic phenotype, with adult chondrocytes being a more promising cell source than HBMSCs for articular cartilage tissue engineering. 相似文献
74.
Tan FC Cheng Q Saha K Heinemann IU Jahn M Jahn D Smith AG 《The Biochemical journal》2008,410(2):291-299
UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo. 相似文献
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Adiki Shanta Kumari Perla Chandra Kiran Saha Gargi Katakam Prakash Theendra Vinaykumar 《Biological trace element research》2019,190(1):150-156
Biological Trace Element Research - This research article aims to establish the intake ratio of probiotic Lactobacillus plantarum 299v with iron supplement pearl millet by central composite design... 相似文献
77.
I. Gomes R. K. Saha G. Mohiuddin M. M. Hoq 《World journal of microbiology & biotechnology》1992,8(6):589-592
A thermophilic fungus belonging to the Deuteromyces, having pectinase and xylanase activities, was grown at its optimum temperature of 55°C. It grew over a wide pH range of 4 to 10, being optimal at 6. The fungus grew well on modified Mandels' medium in which cellulose was substituted either with hemicellulose or pectin. With citrus pectin as carbon source, 121 units/ml of pectinase activity were obtained and with larch wood xylan as carbon source, 83 units/ml of xylanase activity were obtained. 相似文献
78.
Removal of cholesterol-containing particles from the circulation is mediated by the low-density lipoprotein (LDL) receptor. Upon ligand binding, the receptor-ligand complex is endocytosed, and the ligand is released. The important biological role of the LDL receptor (LDLR) has been highlighted by the identification of more than 400 LDLR mutations that are associated with familial hypercholesterolemia. The extracellular region of the LDLR is modular in nature and principally comprises multiple copies of ligand binding, epidermal growth factor-like (EGF), and YWTD-type domains. This report describes characterization of the calcium binding properties of the tandem pair of EGF domains. While only the C-terminal EGF module contains the consensus sequence associated with calcium binding, a noncanonical calcium binding site in the N-terminal domain has been revealed using solution NMR spectroscopy. The calcium dissociation constants for the N- and C-terminal sites have been measured under physiologically relevant pH and ionic strength conditions using a combination of solution NMR, intrinsic protein fluorescence, and chromophoric chelator methods to be approximately 50 microM and approximately 10-20 microM, respectively. Identification of the novel calcium binding motif in LDLR sequences from other species suggests that it may confer specificity within the LDLR gene family. Comparison of the K(d) for the C-terminal site with the calcium concentration in late vesicles indicates that the binding properties of this module may be tuned to titrate upon endocytosis of the LDL receptor-ligand complex, and thus calcium binding may play a role in the ligand dissociation process. 相似文献
79.
Removal of the embryonic axis prevents the normal decline of carboxypeptidase (Cpase) I in mung bean seedling cotyledons. Cpase I activity and protein, the latter manifested on western blots, almost completely disappear about 24 h before the cotyledon abscises. Of the 3 proteolytic enzyme patterns, only that of Cpase I can be restored by an exogenous supply of 10 m M CaCl2 in the agar growth medium. The calcium effect is dependent on [CaCl2 ] and is not manifested in the presence of chelators and calcium channel blockers. For detached cotyledons to show the normal low level of Cpase I by the eighth day of growth, calcium had to be supplied during seed imbibition and throughout the entire time from removal of the axis. The difference between detached cotyledons in the absence and presence of calcium was greatest when the cotyledons were detached 4–6 days after seed imbibition. Loss of Cpase I activity and protein can be demonstrated in vitro, with the maximum level of Cpase I-degrading activity measured 4 days after seed imbibition under the same growth conditions used to study the calcium effect. It is sensitive to pepstatin and has a pH optimum of 3, suggesting that this Cpase I-degrading activity is due to an aspartic protease. 相似文献
80.