scholarly journals Chondrons from articular cartilage. (IV). Immunolocalization of proteoglycan epitopes in isolated canine tibial chondrons.

1991 ◽  
Vol 39 (9) ◽  
pp. 1175-1187 ◽  
Author(s):  
C A Poole ◽  
T T Glant ◽  
J R Schofield
Keyword(s):  
Core Protein ◽  
Polyclonal Antibodies ◽  
Keratan Sulfate ◽  
Thin Layers ◽  
Pericellular Matrix ◽  
Binding Region ◽  
The Core ◽  
Pre Treatment ◽  
Testicular Hyaluronidase

Chondrons have recently been extracted from adult articular cartilages and techniques developed to study their structure and composition in isolation. This study introduces methods to immobilize isolated canine chondrons in thin layers of agarose gel for immunohistochemistry and future in vitro studies. An antibody to Type VI collagen which stained the chondron in suspension was used to successfully validate the system and its feasibility for immunoelectron microscopy. Monoclonal and polyclonal antibodies to a variety of epitopes on the proteoglycan molecule were tested on fresh and fixed plugs cored from chondron-agarose gels. Plugs were immunolabeled with peroxidase-diaminobenzidine before or after digestion with testicular hyaluronidase or chondroitinase ABC. Trypsin/chymotrypsin were used to challenge epitopes of the core protein. The results indicate that epitopes to keratan sulfate, chondroitin sulfate, hyaluronate binding region, and core protein are localized in the chondron. Consistent staining was found in the tail and interconnecting segments between chondrons, whereas staining of the pericellular matrix and capsule adjacent to the chondrocyte varied according to the enzyme pre-treatment employed. We conclude that isolated chondrons are rich in proteoglycan monomer, which is particularly concentrated in the tail and interconnecting segments of the chondron where it could function to protect and stabilize the chondrocyte.

scholarly journals Immunohistochemical localization of glycosaminoglycans and proteoglycans in predentin and dentin of rat incisors.

1990 ◽  
Vol 38 (3) ◽  
pp. 319-324 ◽  
Author(s):  
M Takagi ◽  
H Hishikawa ◽  
Y Hosokawa ◽  
A Kagami ◽  
F Rahemtulla
Keyword(s):  
Dermatan Sulfate ◽  
Core Protein ◽  
Polyclonal Antibodies ◽  
Keratan Sulfate ◽  
Immunohistochemical Localization ◽  
Immunoperoxidase Staining ◽  
Weak Staining ◽  
Dentin Matrix ◽  
Pre Treatment ◽  
Definite Difference

We examined immunocytochemically the type and distribution of glycosaminoglycans and proteoglycans (PG) in predentin and dentin demineralized with EDTA after aldehyde fixation of rat incisors using (a) four monoclonal antibodies (1-B-5,9-A-2,3-B-3, and 5-D-4) which recognize epitopes in unsulfated chondroitin (C0-S), chondroitin 4-sulfate (C4-S), chondroitin 6-sulfate (C6-S), and keratan sulfate (KS) associated with the PG, and (b) monoclonal (5-D-5) and polyclonal antibodies specific for the core protein of large and small dermatan sulfate (DS) PG. Light microscope immunoperoxidase staining after pre-treatment of tissue sections with chondroitinase ABC localized the majority of stainable PG (C4-S, KS, DSPG, C0-S, and C6-S) in predentin and, to a lesser extent (C4-S and small DSPG), in the dentin matrix. The former site demonstrated relatively homogeneous PG distribution, whereas the latter site revealed that strong staining of C4-S and small DSPG was confined mostly to dentinal tubules surrounding odontoblastic processes, with only weak staining in the rest of the dentin matrix. These results indicate that there is not only a definite difference between PG of predentin and dentin but also a selective decrease in the concentration or alteration of these macromolecules during dentinogenesis and mineralization.

scholarly journals Chondrocyte-mediated depletion of articular cartilage proteoglycans in vitro

1985 ◽  
Vol 225 (2) ◽  
pp. 493-507 ◽  
Author(s):  
J A Tyler
Keyword(s):  
Articular Cartilage ◽  
Core Protein ◽  
Fold Increase ◽  
Acid Proteinase ◽  
Binding Region ◽  
Guanidinium Chloride ◽  
The Core ◽  
The Matrix ◽  
Average Size

The degradation of proteoglycan was examined in cultured slices of pig articular cartilage. Pig leucocyte catabolin (10 ng/ml) was used to stimulate the chondrocytes and induce a 4-fold increase in the rate of proteoglycan loss from the matrix for 4 days. Material in the medium of both control and depleted cultures was mostly a degradation product of the aggregating proteoglycan. It was recovered as a very large molecule slightly smaller than the monomers extracted with 4M-guanidinium chloride and lacked a functional hyaluronate binding region. The size and charge were consistent with a very limited cleavage or conformational change of the core protein near the hyaluronate binding region releasing the C-terminal portion of the molecule intact from the aggregate. The ‘clipped’ monomer diffuses very rapidly through the matrix into the medium. The amount of proteoglycan extracted with 4M-guanidinium chloride decreased during culture from both the controls and depleted cartilage, and the average size of the molecules initially remained the same. However, the proportion of molecules with a smaller average size increased with time and was predominant in explants that had lost more than 70% of their proteoglycan. All of this material was able to form aggregates when mixed with hyaluronate, and glycosaminoglycans were the same size and charge as normal, indicating either that the core protein had been cleaved in many places or that larger molecules were preferentially released. A large proportion of the easily extracted and non-extractable proteoglycan remained in the partially depleted cartilage and the molecules were the same size and charge as those found in the controls. There was no evidence of detectable glycosidase activity and only very limited sulphatase activity. A similar rate of breakdown and final distribution pattern was found for newly synthesized proteoglycan. Increased amounts of latent neutral metalloproteinases and acid proteinase activities were present in the medium of depleted cartilage. These were not thought to be involved in the breakdown of proteoglycan. Increased release of proteoglycan ceased within 24h of removal of the catabolin, indicating that the effect was reversible and persisted only while the stimulus was present.

Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP

1991 ◽  
Vol 11 (11) ◽  
pp. 5516-5526 ◽  
Author(s):  
M Cross ◽  
A Günzl ◽  
Z Palfi ◽  
A Bindereif
Keyword(s):  
Trypanosoma Brucei ◽  
Core Protein ◽  
Rnase H ◽  
Spliced Leader ◽  
The Core ◽  
In Vitro Reconstitution ◽  
U2 Snrnp ◽  
Protein Components ◽  
Small Nuclear Ribonucleoproteins

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.

scholarly journals The N-terminal half of the core protein of hepatitis C virus is sufficient for nucleocapsid formation

2004 ◽  
Vol 85 (4) ◽  
pp. 971-981 ◽  
Author(s):  
Nathalie Majeau ◽  
Valérie Gagné ◽  
Annie Boivin ◽  
Marilène Bolduc ◽  
Josée-Anne Majeau ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Host Cells ◽  
Yeast Cells ◽  
Main Function ◽  
C Protein ◽  
Multifunctional Protein ◽  
The Core

The core (C) protein of hepatitis C virus (HCV) appears to be a multifunctional protein that is involved in many viral and cellular processes. Although its effects on host cells have been extensively discussed in the literature, little is known about its main function, the assembly and packaging of the viral genome. We have studied the in vitro assembly of several deleted versions of recombinant HCV C protein expressed in E. coli. We demonstrated that the 75 N-terminal residues of the C protein were sufficient to assemble and generate nucleocapsid-like particles (NLPs) in vitro. However, homogeneous particles of regular size and shape were observed only when NLPs were produced from at least the first 79 N-terminal amino acids of the C protein. This small protein unit fused to the endoplasmic reticulum-anchoring domain also generated NLPs in yeast cells. These data suggest that the N-terminal half of the C protein is important for formation of NLPs. Similarities between the HCV C protein and C proteins of other members of the Flaviviridae are discussed.

scholarly journals E6AP Ubiquitin Ligase Mediates Ubiquitylation and Degradation of Hepatitis C Virus Core Protein

2006 ◽  
Vol 81 (3) ◽  
pp. 1174-1185 ◽  
Author(s):  
Masayuki Shirakura ◽  
Kyoko Murakami ◽  
Tohru Ichimura ◽  
Ryosuke Suzuki ◽  
Tetsu Shimoji ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Core Protein ◽  
Protein Levels ◽  
The Core ◽  
Hcv Core Protein ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Viral Nucleocapsid

ABSTRACT Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.

scholarly journals Nuclear Localization of Japanese Encephalitis Virus Core Protein Enhances Viral Replication

2005 ◽  
Vol 79 (6) ◽  
pp. 3448-3458 ◽  
Author(s):  
Yoshio Mori ◽  
Tamaki Okabayashi ◽  
Tetsuo Yamashita ◽  
Zijiang Zhao ◽  
Takaji Wakita ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Cell Lines ◽  
Japanese Encephalitis ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Core Protein ◽  
Encephalitis Virus ◽  
Wild Type ◽  
The Core

ABSTRACT Japanese encephalitis virus (JEV) core protein was detected in both the nucleoli and cytoplasm of mammalian and insect cell lines infected with JEV or transfected with the expression plasmid of the core protein. Mutation analysis revealed that Gly42 and Pro43 in the core protein are essential for the nuclear and nucleolar localization. A mutant M4243 virus in which both Gly42 and Pro43 were replaced by Ala was recovered by plasmid-based reverse genetics. In C6/36 mosquito cells, the M4243 virus exhibited RNA replication and protein synthesis comparable to wild-type JEV, whereas propagation in Vero cells was impaired. The mutant core protein was detected in the cytoplasm but not in the nucleus of either C6/36 or Vero cell lines infected with the M4243 virus. The impaired propagation of M4243 in mammalian cells was recovered by the expression of wild-type core protein in trans but not by that of the mutant core protein. Although M4243 mutant virus exhibited a high level of neurovirulence comparable to wild-type JEV in spite of the approximately 100-fold-lower viral propagation after intracerebral inoculation to 3-week-old mice of strain Jcl:ICR, no virus was recovered from the brain after intraperitoneal inoculation of the mutant. These results indicate that nuclear localization of JEV core protein plays crucial roles not only in the replication in mammalian cells in vitro but also in the pathogenesis of encephalitis induced by JEV in vivo.

scholarly journals Cartilage proteoglycans. Assembly with hyaluronate and link protein as studied by electron microscopy

1988 ◽  
Vol 253 (1) ◽  
pp. 175-185 ◽  
Author(s):  
M Mörgelin ◽  
M Paulsson ◽  
T E Hardingham ◽  
D Heinegård ◽  
J Engel
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Core Protein ◽  
Protein Domains ◽  
Globular Domain ◽  
Binding Region ◽  
Link Protein ◽  
The Core ◽  
Cartilage Proteoglycan ◽  
Cartilage Proteoglycans

Aggregates formed by the interaction of cartilage proteoglycan monomers and fragments thereof with hyaluronate were studied by electron microscopy by use of rotary shadowing [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333]. The differences in shape and packing of the proteins bound along the hyaluronate strand in aggregates formed in the presence and in the absence of link protein were examined in detail. The high resolution of the method allowed examination of the involvement in hyaluronate binding of the globular core-protein domains G1, G2 and G3 [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333; Paulsson, Mörgelin, Wiedemann, Beardmore-Gray, Dunham, Hardingham, Heinegård, Timpl & Engel (1987) Biochem. J. 245, 763-772]. Fragments comprising the globular hyaluronate-binding region G1 form complexes with hyaluronate with an appearance of necklace-like structures, statistically interspaced by free hyaluronate strands. The closest centre-to-centre distance found between adjacent G1 domains was 12 nm. Another fragment comprising the binding region G1 and the adjacent second globular domain G2 attaches to hyaluronate only by one globule. Also, the core protein obtained by chondroitinase digestion of proteoglycan monomer binds only by domain G1, with domain G3 furthest removed from the hyaluronate. Globule G1 shows a statistical distribution along the hyaluronate strands. In contrast, when link protein is added, binding is no longer random, but instead uninterrupted densely packed aggregates are formed.

scholarly journals The Core Protein of Classical Swine Fever Virus Is Dispensable for Virus Propagation In Vitro

2012 ◽  
Vol 8 (3) ◽  
pp. e1002598 ◽  
Author(s):  
Christiane Riedel ◽  
Benjamin Lamp ◽  
Manuela Heimann ◽  
Matthias König ◽  
Sandra Blome ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Core Protein ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Virus Propagation ◽  
The Core

scholarly journals A Dynamic Scaffold of Pre-snoRNP Factors Facilitates Human Box C/D snoRNP Assembly

2007 ◽  
Vol 27 (19) ◽  
pp. 6782-6793 ◽  
Author(s):  
Kenneth Scott McKeegan ◽  
Charles Maurice Debieux ◽  
Séverine Boulon ◽  
Edouard Bertrand ◽  
Nicholas James Watkins
Keyword(s):  
Protein Interactions ◽  
Core Protein ◽  
The Novel ◽  
Protein Protein Interactions ◽  
Core Complex ◽  
The Core ◽  
Snornp Biogenesis ◽  
The Individual ◽  
Vitro Protein

ABSTRACT The box C/D small nucleolar RNPs (snoRNPs) are essential for the processing and modification of rRNA. The core box C/D proteins are restructured during human U3 box C/D snoRNP biogenesis; however, the molecular basis of this is unclear. Here we show that the U8 snoRNP is also restructured, suggesting that this may occur with all box C/D snoRNPs. We have characterized four novel human biogenesis factors (BCD1, NOP17, NUFIP, and TAF9) which, along with the ATPases TIP48 and TIP49, are likely to be involved in the formation of the pre-snoRNP. We have analyzed the in vitro protein-protein interactions between the assembly factors and core box C/D proteins. Surprisingly, this revealed few interactions between the individual core box C/D proteins. However, the novel biogenesis factors and TIP48 and TIP49 interacted with one or more of the core box C/D proteins, implying that they mediate the assembly of the pre-snoRNP. Consistent with this, we show that NUFIP bridges interactions between the core box C/D proteins in a partially reconstituted pre-snoRNP. Restructuring of the core complex probably reflects the conversion of the pre-snoRNP, where core protein-protein interactions are maintained by the bridging biogenesis factors, to the mature snoRNP.

scholarly journals Keratan sulfate-based glycomimetics using Langerin as a target for COPD: lessons from studies on Fut8 and core fucose

2021 ◽  
Author(s):  
Yuki Ohkawa ◽  
Yoichiro Harada ◽  
Naoyuki Taniguchi
Keyword(s):  
Posttranslational Modification ◽  
Therapeutic Strategy ◽  
Keratan Sulfate ◽  
Therapeutic Strategies ◽  
Chronic Obstructive ◽  
Obstructive Pulmonary Disease ◽  
Cooperative Action ◽  
The Core ◽  
Area Of Interest

Glycosylation represents one of the most abundant posttranslational modification of proteins. Glycosylation products are diverse and are regulated by the cooperative action of various glycosyltransferases, glycosidases, substrates thereof: nucleoside sugars and their transporters, and chaperons. In this article, we focus on a glycosyltransferase, α1,6-fucosyltransferase (Fut8) and its product, the core fucose structure on N-glycans, and summarize the potential protective functions of this structure against emphysema and chronic obstructive pulmonary disease (COPD). Studies of FUT8 and its enzymatic product, core fucose, are becoming an emerging area of interest in various fields of research including inflammation, cancer and therapeutics. This article discusses what we can learn from studies of Fut8 and core fucose by using knockout mice or in vitro studies that were conducted by our group as well as other groups. We also include a discussion of the potential protective functions of the keratan sulfate (KS) disaccharide, namely L4, against emphysema and COPD as a glycomimetic. Glycomimetics using glycan analogs is one of the more promising therapeutics that compensate for the usual therapeutic strategy that involves targeting the genome and the proteome. These typical glycans using KS derivatives as glycomimetics, will likely become a clue to the development of novel and effective therapeutic strategies.

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