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161.
Hickery MS Bayliss MT Dudhia J Lewthwaite JC Edwards JC Pitsillides AA 《The Journal of biological chemistry》2003,278(52):53063-53071
Cartilage glycosaminoglycan (GAG) synthesis and composition, upon which its structural integrity depends, varies with age, is modified by anabolic and catabolic stimuli, and is regulated by UDP-glucuronate availability. However, how such stimuli, prototypically represented by transforming growth factor-beta1 (TGF-beta1) and IL-1alpha, modify GAG synthesis during aging of normal human articular cartilage is not known. Using explants, we show that chondroitin sulfate (CS):total GAG ratios decrease, whereas C6S:C4S ratios increase with cartilage maturation, and that chondrocytes in the cartilage mid-zone, but not the superficial or deep zones, exhibit uridine 5'-diphosphoglucose dehydrogenase (UDPGD) activity, which is also increased in mature cartilage. We also show that IL-1alpha treatment reduces both total GAG and CS synthesis, decreases C6S:C4S ratios (less C6S), but fails to modify chondrocyte UDPGD activity at all ages. On the other hand, TGF-beta1 increases total GAG synthesis in immature, but not mature, cartilage (stimulates CS but not non-CS), age-independently decreases C6S:C4S (more C4S), and increases chondrocyte UDPGD activity in a manner inversely correlated with age. Our findings show that TGF-beta1, but not IL-1alpha, modifies matrix synthesis such that its composition more closely resembles "less mature" articular cartilage. These effects of TGF-beta1, which appear to be restricted to periods of skeletal immaturity, are closely associated although not necessarily mechanistically linked with increases in chondrocyte UDPGD activity. The antianabolic effects of IL-1alpha are, on the other hand, likely to be independent of any direct modification in UDPGD activity and manifest equally in human cartilage of all ages. 相似文献
162.
Ko YH Delannoy M Hullihen J Chiu W Pedersen PL 《The Journal of biological chemistry》2003,278(14):12305-12309
The terminal step of ATP synthesis in intact mitochondria is catalyzed by the ATP synthase (F(0)F(1)) that works in close synchrony with the P(i) and ADP/ATP carriers. Each carrier consists of only a single polypeptide chain in dimeric form, while the ATP synthase is highly complex consisting in animals of 17 known subunit types and more than 30 total subunits. Although structures at high resolution have been obtained for the water-soluble F(1) part of the ATP synthase consisting of only five subunit types, such structures have not been obtained for either the complete ATP synthase or the P(i) and ADP/ATP carriers. Here, we report that all three proteins are localized in highly purified cristae-like vesicles obtained by extensive subfractionation of the mitochondrial inner membrane. Moreover, using a multiwell detergent screening assay, 4 nonionic detergents out of 80 tested were found to disperse these cristae-like vesicles into single soluble complexes or "ATP synthasomes" that contain the ATP synthase in association with the P(i) and ADP/ATP carriers. These studies offer new mechanistic insights into the terminal steps of oxidative phosphorylation in mitochondria and set the stage for future structural efforts designed to visualize in atomic detail the entire complex involved. They also provide evidence that the cristae are a subcompartment of the inner membrane. 相似文献
163.
M phase phosphoprotein 1 is a human plus-end-directed kinesin-related protein required for cytokinesis 总被引:4,自引:0,他引:4
Abaza A Soleilhac JM Westendorf J Piel M Crevel I Roux A Pirollet F 《The Journal of biological chemistry》2003,278(30):27844-27852
The human M phase phosphoprotein 1 (MPP1), previously identified through a screening of a subset of proteins specifically phosphorylated at the G2/M transition (Matsumoto-Taniura, N., Pirollet, F., Monroe, R., Gerace, L., and Westendorf, J. M. (1996) Mol. Biol. Cell 7, 1455-1469), is characterized as a plus-end-directed kinesin-related protein. Recombinant MPP1 exhibits in vitro microtubule-binding and microtubule-bundling properties as well as microtubule-stimulated ATPase activity. In gliding experiments using polarity-marked microtubules, MPP1 is a slow molecular motor that moves toward the microtubule plus-end at a 0.07 microm/s speed. In cycling cells, MPP1 localizes mainly to the nuclei in interphase. During mitosis, MPP1 is diffuse throughout the cytoplasm in metaphase and subsequently localizes to the midzone to further concentrate on the midbody. MPP1 suppression by RNA interference induces failure of cell division late in cytokinesis. We conclude that MPP1 is a new mitotic molecular motor required for completion of cytokinesis. 相似文献
164.
Although the expression of the metastases-associated gene MTA1 correlates with tumor metastases, its role in regulating type IV collagenase expression is unknown. Enforced MTA1 expression in HT1080 cells reduced basal and 12-myristate 13-acetate-induced 92-kDa type IV collagenase (MMP-9) protein/mRNA levels. DNase I hypersensitivity and PstI accessibility assays revealed multiple regions of the MMP-9 promoter (-650/-450 and -120/+1), showing reduced hypersensitivity in the MTA1-expressing cells. Chromatin immunoprecipitation assays demonstrated MTA1 binding to the distal region, which spans several regulatory cis elements. Co-immunoprecipitation and chromatin immunoprecipitation assay experiments revealed histone deacetylase 2 (HDAC2)-MTA1 protein-protein interactions and the MTA1-dependent recruitment of HDAC2 to the distal MMP-9 promoter region, yielding diminished histone H3/H4 acetylation. However, HDAC2 binding and H3/H4 acetylation at the proximal MMP-9 region were unaffected by MTA1 expression. Furthermore, trichostatin treatment only partially relieved MTA1-repressed MMP-9 expression, indicating a HDAC-insensitive component possibly involv ing the nucleosome-remodeling Mi2 activity, which was recruited to the promoter by MTA1. In summary, (a) MMP-9 adds to a short list of MTA1-regulated genes, which so far only includes c-myc and pS2, and (b) MTA1 binds to the MMP-9 promoter, thereby repressing expression of this type IV collagenase via histone-dependent and independent mechanisms. 相似文献
165.
Stanniocalcin 1 is an autocrine modulator of endothelial angiogenic responses to hepatocyte growth factor 总被引:4,自引:0,他引:4
Zlot C Ingle G Hongo J Yang S Sheng Z Schwall R Paoni N Wang F Peale FV Gerritsen ME 《The Journal of biological chemistry》2003,278(48):47654-47659
Stanniocalcin 1 (STC1) is a secreted glycoprotein originally described as a hormone involved in calcium and phosphate homeostasis in bony fishes. We recently identified the mammalian homolog of this molecule to be highly up-regulated in an in vitro model of angiogenesis, as well as focally and intensely expressed at sites of pathological angiogenesis (e.g. tumor vasculature). In the present study, we report that STC1 is a selective modulator of hepatocyte growth factor (HGF)-induced endothelial migration and morphogenesis, but not proliferation. STC1 did not inhibit proliferative or migratory responses to vascular endothelial growth factor or basic fibroblast growth factor. The mechanism of STC1 inhibitory effects on HGF-induced endothelial migration seem to occur secondary to receptor activation because STC1 did not inhibit HGF-induced c-met receptor phosphorylation, but did block HGF-induced focal adhesion kinase activation. In the mouse femoral artery ligation model of angiogenesis, STC1 expression closely paralleled that of the endothelial marker CD31, and the peak level of STC1 expression occurred after an increase in HGF expression. We propose that STC1 may play a selective modulatory role in angiogenesis, possibly serving as a "stop signal" or stabilizing factor contributing to the maturation of newly formed blood vessels. HGF is a mesenchyme-derived pleiotropic factor with mitogenic, motogenic, and morphogenic activities on a number of different cell types. HGF effects are mediated through a specific tyrosine kinase, c-met, and aberrant HGF and c-met expression are frequently observed in a variety of tumors. Recent studies have shown HGF to be a potent growth factor implicated in wound healing, tissue regeneration, and angiogenesis. 相似文献
166.
Castronuevo P Thornton MA McCarthy LE Klimas J Schick BP 《The Journal of biological chemistry》2003,278(49):48704-48712
167.
Berson JF Theos AC Harper DC Tenza D Raposo G Marks MS 《The Journal of cell biology》2003,161(3):521-533
Lysosome-related organelles are cell type-specific intracellular compartments with distinct morphologies and functions. The molecular mechanisms governing the formation of their unique structural features are not known. Melanosomes and their precursors are lysosome-related organelles that are characterized morphologically by intralumenal fibrous striations upon which melanins are polymerized. The integral membrane protein Pmel17 is a component of the fibrils and can nucleate their formation in the absence of other pigment cell-specific proteins. Here, we show that formation of intralumenal fibrils requires cleavage of Pmel17 by a furin-like proprotein convertase (PC). As in the generation of amyloid, proper cleavage of Pmel17 liberates a lumenal domain fragment that becomes incorporated into the fibrils; longer Pmel17 fragments generated in the absence of PC activity are unable to form organized fibrils. Our results demonstrate that PC-dependent cleavage regulates melanosome biogenesis by controlling the fibrillogenic activity of a resident protein. Like the pathologic process of amyloidogenesis, the formation of other tissue-specific organelle structures may be similarly dependent on proteolytic activation of physiological fibrillogenic substrates. 相似文献
168.
OBJECTIVE: To evaluate and compare the quality and cost of urine cytology using the Cytospin method (Shandon, ThermoElectron Corporation, Waltham, Massachusetts, U.S.A.) and the AutoCyte PREP (TriPath Imaging, Burlington, North Carolina, U.S.A.) in a general laboratory. STUDY DESIGN: A study of differences between the Cytospin method and AutoCyte PREP in the areas of specimen preparation, staining, number and quality of diagnostic cells, background, screener preference, and cost was undertaken over a 3-month period in 2000. Sixty fresh voided urine samples from 25 patients with known transitional cell carcinoma were prepared by the Cytospin method and the AutoCyte PREP according to the manufacturers' instructions. RESULTS: The Cytospin method had longer preparation time but shorter screening time than the AutoCyte PREP. The number of diagnostic cells was higher in the Cytospin method. Fixation quality and staining clarity were better in the Cytospin method. Qualitative assessment of cell arrangements, cell and nuclear size and shape, nuclear/cytoplasmic ratio and nuclear membrane irregularity showed no significant differences between the 2 methods. Cellular details and nuclear chromatin patterns were clearer and better preserved in the Cytospin method, but the AutoCyte PREP showed less blood and inflammatory cells and debris. CONCLUSION: In the majority of cases the screeners preferred the Cytospin method due to its better overall cytologic quality. However, the amount of blood, inflammation and debris was much lower in the AutoCyte PREP. This reduced the need to make a second, diluted specimen and made turnaround time faster. The AutoCyte PREP was 7 times more expensive than the Cytospin method. 相似文献
169.
11F8 is a murine anti-ssDNA monoclonal autoantibody isolated from a lupus prone autoimmune mouse. This mAb binds sequence specifically, and prior studies have defined the thermodynamic and kinetic basis for sequence-specific recognition of ssDNA (Ackroyd, P. C., et al. (2001) Biochemistry 40, 2911-2922; Beckingham, J. A. and Glick, G. D. (2001) Bioorg. Med. Chem. 9, 2243-2252). Here we present experiments designed to identify the residues on 11F8 that mediate sequence-specific, noncognate, and nonspecific recognition of ssDNA and their contribution to the overall binding thermodynamics. Site-directed mutagenesis of an 11F8 single-chain construct reveals that six residues within the complementarity determining regions of 11F8 account for ca. 80% of the binding free energy and that there is little cooperativity between these residues. Germline-encoded aromatic and hydrophobic side chains provides the basis for nonspecific recognition of single-stranded thymine nucleobases. Sequence-specific recognition is controlled by a tyrosine in the heavy chain along with a somatically mutated arginine residue. Our data show that the manner in which 11F8 achieves sequence-specific recognition more closely resembles RNA-binding proteins such as U1A than other types of nucleic acid binding proteins. In addition, comparing the primary sequence of 11F8 with clonally related antibodies that differ by less than five amino acids suggests that somatic mutations which confer sequence specificity may be a feature that distinguishes glomerulotrophic pathogenic anti-DNA from those that are benign. 相似文献
170.
CTP:phosphocholine cytidylyltranferase (CCT) regulates phosphatidylcholine (PC) biosynthesis. Its activity is controlled by reversible interactions with membrane lipids, mediated by an internal segment referred to as domain M. Although domain M peptides adopt an amphipathic alpha-helical structure when membrane bound, the structure of this domain in the context of the whole enzyme in the lipid-free and lipid-bound state is unknown. Here we derive lipid-induced secondary structural changes in CCTalpha using circular dichroism and three deconvolution programs. The analysis of two fragments, CCT236 (CCT1-236, housing the catalytic domain) and a synthetic domain M peptide (CCT237-293) aided the assignment of structural change to specific domains. To carry out this study, we developed a micellar lipid activating system that would avoid generation of CCT-induced lipid vesicle aggregates that interfere with the CD analysis. Lysophosphatidylcholine/phosphatidylglycerol (LPC/PG) mixed micelles supported full activation of CCT and caused an increase in the alpha-helix content of full-length CCT from 25 to 41%, at the expense of all other conformations. LPC/PG also induced an increase in alpha-helix content of the domain M peptide from 7 to 85% at the expense of all other conformers. This lipid system did not significantly affect the secondary structure of CCT236, nor did it affect the proteolytic fragmentation pattern of this region within full-length CCT, suggesting that the region containing the catalytic domain changes very little upon membrane activation of CCT. Our data suggest that lipids trigger a conformational switch in domain M from a mixed structure to an alpha-helix, thus creating a hydrophobic face for membrane insertion. Our results negate the idea that domain M is entirely helical in both the soluble and membrane-bound forms of CCT. 相似文献