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1.
Sci Rep ; 10(1): 8340, 2020 05 20.
Article in English | MEDLINE | ID: mdl-32433527

ABSTRACT

A previous study identified kartogenin (KGN) as a potent modulator of bone marrow mesenchymal stem/stromal cell (BMSC) chondrogenesis. This initial report did not contrast KGN directly against transforming growth factor-beta 1 (TGF-ß1), the most common growth factor used in chondrogenic induction medium. Herein, we directly compared the in vitro chondrogenic potency of TGF-ß1 and KGN using a high resolution micropellet model system. Micropellets were cultured for 7-14 days in medium supplemented with TGF-ß1, KGN, or both TGF-ß1 + KGN. Following 14 days of induction, micropellets exposed to TGF-ß1 alone or TGF-ß1 + KGN in combination were larger and produced more glycosominoglycan (GAG) than KGN-only cultures. When TGF-ß1 + KGN was used, GAG quantities were similar or slightly greater than the TGF-ß1-only cultures, depending on the BMSC donor. BMSC micropellet cultures supplemented with KGN alone contracted in size over the culture period and produced minimal GAG. Indicators of hypertrophy were not mitigated in TGF-ß1 + KGN cultures, suggesting that KGN does not obstruct BMSC hypertrophy. KGN appears to have weak chondrogenic potency in human BMSC cultures relative to TGF-ß1, does not obstruct hypertrophy, and may not be a viable alternative to growth factors in cartilage tissue engineering.


Subject(s)
Anilides/pharmacology , Chondrogenesis/drug effects , Mesenchymal Stem Cells/drug effects , Phthalic Acids/pharmacology , Tissue Engineering/methods , Transforming Growth Factor beta1/pharmacology , Cartilage/growth & development , Cell Differentiation/drug effects , Cells, Cultured , Culture Media/pharmacology , Humans , Mesenchymal Stem Cells/physiology , Primary Cell Culture/methods , Recombinant Proteins/pharmacology
2.
Cell Tissue Res ; 374(3): 541-553, 2018 Dec.
Article in English | MEDLINE | ID: mdl-30136155

ABSTRACT

Bone marrow-derived mesenchymal stem/stromal cells (BMSC) may facilitate bone repair through secretion of factors that stimulate endogenous repair processes or through direct contribution to new bone through differentiation into osteoblast-like cells. BMSC microtissue culture and differentiation has been widely explored recently, with high-throughput platforms making large-scale manufacture of microtissues increasingly feasible. Bone-like BMSC microtissues could offer an elegant method to enhance bone repair, especially in small-volume non-union defects, where small diameter microtissues could be delivered orthoscopically. Using a high-throughput microwell platform, our data demonstrate that (1) BMSC in 3D microtissue culture result in tissue compaction, rather than growth, (2) not all mineralised bone-like matrix is incorporated in the bulk microtissue mass and (3) a significant amount of lipid vacuole formation is observed in BMSC microtissues exposed to BMP-2. These factors should be considered when optimising BMSC osteogenesis in microtissues or developing BMSC microtissue-based therapeutic delivery processes.


Subject(s)
Adipogenesis/drug effects , Bone Morphogenetic Protein 2/pharmacology , Culture Media/pharmacology , Mesenchymal Stem Cells/cytology , Osteogenesis/drug effects , Tissue Culture Techniques , Tissue Engineering , Calcium/metabolism , Female , Humans , Male , Middle Aged , Young Adult
3.
Acta Virol ; 56(3): 217-26, 2012.
Article in English | MEDLINE | ID: mdl-23043601

ABSTRACT

UNLABELLED: Hepatitis C virus (HCV) core (C) protein is thought to bind to viral RNA before it undergoes oligomerization leading to RNA encapsidation. Details of these events are so far unknown. The 5ʹ-terminal C protein coding sequence that includes an adenine (A)-rich tract is a part of an internal ribosome entry site (IRES). This nucleotide sequence but not the corresponding protein sequence is needed for proper initiation of translation of viral RNA by an IRES-dependent mechanism. In this study, we examined the importance of this sequence for the ability of the C protein to bind to viral RNA. Serially truncated C proteins with deletions from 10 up to 45 N-terminal amino acids were expressed in Escherichia coli, purified and tested for binding to viral RNA by a gel shift assay. The results showed that truncation of the C protein from its N-terminus by more than 10 amino acids abolished almost completely its expression in E. coli. The latter could be restored by adding a tag to the N-terminus of the protein. The tagged proteins truncated by 15 or more amino acids showed an anomalous migration in SDS-PAGE. Truncation by more than 20 amino acids resulted in a complete loss of ability of tagged C protein to bind to viral RNA. These results provide clues to the early events in the C protein - RNA interactions leading to C protein oligomerization, RNA encapsidation and virion assembly. KEYWORDS: hepatitis C virus; core protein; RNA binding.


Subject(s)
Hepacivirus/metabolism , Open Reading Frames , Peptides/metabolism , RNA, Viral/metabolism , Viral Core Proteins/chemistry , Viral Core Proteins/metabolism , 5' Untranslated Regions , Adenine/chemistry , Adenine/metabolism , Amino Acid Sequence , Animals , Base Sequence , Hepacivirus/chemistry , Hepacivirus/genetics , Molecular Sequence Data , Nucleic Acid Conformation , Peptides/genetics , Protein Binding , RNA, Viral/chemistry , RNA, Viral/genetics , Viral Core Proteins/genetics
4.
Proc Natl Acad Sci U S A ; 98(9): 4916-21, 2001 Apr 24.
Article in English | MEDLINE | ID: mdl-11296247

ABSTRACT

Cobalamins are stored in high concentrations in the human liver and thus are available to participate in the regulation of hepatotropic virus functions. We show that cyanocobalamin (vitamin B12) inhibited the HCV internal ribosome entry site (IRES)-dependent translation of a reporter gene in vitro in a dose-dependent manner without significantly affecting the cap-dependent mechanism. Vitamin B12 failed to inhibit translation by IRES elements from encephalomyocarditis virus (EMCV) or classical swine fever virus (CSFV). We also demonstrate a relationship between the total cobalamin concentration in human sera and HCV viral load (a measure of viral replication in the host). The mean viral load was two orders of magnitude greater when the serum cobalamin concentration was above 200 pM (P < 0.003), suggesting that the total cobalamin concentration in an HCV-infected liver is biologically significant in HCV replication.


Subject(s)
Hepacivirus/drug effects , Hepacivirus/physiology , Hepatitis C/virology , Vitamin B 12/pharmacology , Antiviral Agents/blood , Antiviral Agents/pharmacology , Base Sequence , Classical Swine Fever Virus/drug effects , Classical Swine Fever Virus/genetics , Dose-Response Relationship, Drug , Encephalomyocarditis virus/drug effects , Encephalomyocarditis virus/genetics , Hepacivirus/genetics , Hepacivirus/pathogenicity , Hepatitis C/blood , Humans , Liver/virology , Molecular Sequence Data , Protein Binding/drug effects , Protein Biosynthesis/drug effects , Regulatory Sequences, Nucleic Acid/genetics , Ribosomes/metabolism , Substrate Specificity , Thermodynamics , Viral Load , Virus Replication/drug effects , Vitamin B 12/blood
5.
J Virol ; 75(7): 3095-104, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11238836

ABSTRACT

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(-)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (-)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.


Subject(s)
Anti-HIV Agents/pharmacology , HIV Reverse Transcriptase/antagonists & inhibitors , HIV-1/drug effects , Reverse Transcriptase Inhibitors/pharmacology , Transcription, Genetic , DNA, Single-Stranded/biosynthesis , DNA, Viral/biosynthesis , Gene Products, tat/antagonists & inhibitors , HIV-1/genetics , Humans , Integrase Inhibitors/pharmacology , Jurkat Cells , tat Gene Products, Human Immunodeficiency Virus
6.
Arch Virol ; 145(10): 2163-71, 2000.
Article in English | MEDLINE | ID: mdl-11087098

ABSTRACT

An assay for the bovine viral diarrhoea virus (BVDV) replicase was developed using extracts from BVDV-infected cells. The replicase activity was maximal approximately 8 h post-infection as measured by the generation of a genomic length radiolabelled RNA. Using a semi-denaturing gel system, three virus-specific in vitro radiolabelled nascent RNA species were identified. A fast-migrating RNA was demonstrated to be the double-stranded replicative form (RF). A second form was shown to be a partially single-stranded/partially double-stranded RNA, characteristic of the replicative intermediate (RI). A third form, which was often undetectable, migrated between the RF and RI and was probably genomic viral RNA. The optimal replicase activity was dependent on 5-10 mM Mg2+ and although it was also active in 1-2 mM Mn2+ it was inhibited at higher concentrations. The optimum KCl concentration for labelling of the RI and RF were different, suggestive of at least two distinct replicase activities. These results are supportive of a semi-conservative model of BVDV RNA replication.


Subject(s)
Diarrhea Viruses, Bovine Viral/enzymology , RNA-Dependent RNA Polymerase/metabolism , Animals , Cattle , Cell Line , Diarrhea Viruses, Bovine Viral/physiology , Electrophoresis, Polyacrylamide Gel , Kidney , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/chemistry
7.
Proc Natl Acad Sci U S A ; 95(2): 542-7, 1998 Jan 20.
Article in English | MEDLINE | ID: mdl-9435228

ABSTRACT

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 microM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and > 50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and > 37.5 microM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2'-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5'-oxygen in the transition state. We suggest structural reasons why the Mg(2+)-La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction.


Subject(s)
Metals/chemistry , RNA, Catalytic/metabolism , RNA/metabolism , Binding Sites , Metals/metabolism , RNA/chemistry , RNA, Catalytic/chemistry , Substrate Specificity
8.
Proc Natl Acad Sci U S A ; 94(6): 2290-4, 1997 Mar 18.
Article in English | MEDLINE | ID: mdl-9122187

ABSTRACT

Significant cleavage by hammerhead ribozymes requires activation by divalent metal ions. Several models have been proposed to account for the influence of metal ions on hammerhead activity. A number of recent papers have presented data that have been interpreted as supporting a one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium isotope effect has been taken as evidence against a proton transfer in the rate-limiting step of the cleavage reaction. We propose that these data are more easily explained by a two-metal-ion mechanism that does not involve a metal hydroxide, but does involve a proton transfer in the rate-limiting step.


Subject(s)
Cations, Divalent , Nucleic Acid Conformation , RNA, Catalytic/chemistry , RNA, Catalytic/metabolism , Base Sequence , Binding Sites , Calcium , Catalysis , Cobalt , Crystallography, X-Ray , Kinetics , Lead , Magnesium , Manganese , Molecular Sequence Data , Thionucleotides
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