Search

Beta-tricalcium phosphate (β-TCP) serves as a bone substitute in clinical practice because it is resorbable, biocompatible, osteointegrative, and osteoconductive. Particles of β-TCP are also inflammatory mediators although the mechanism of this function has not been fully elucidated. Regardless, the ability of β-TCP to stimulate the immune system might be useful for immunomodulation. The present study aimed to determine the effects of β-TCP particles on NLR family pyrin domain containing 3 (NLRP3) inflammasome complexes. We found that β-TCP activates NLRP3 inflammasomes, and increases interleukin (IL)-1β production in primary cultured mouse dendritic cells (DCs) and macrophages, and human THP-1 cells in caspase-1 dependent manner. In THP-1 cells, β-TCP increased also IL-18 production, and NLRP3 inflammasome activation by β-TCP depended on phagocytosis, potassium efflux, and reactive oxygen species (ROS) generation. We also investigated the effects of β-TCP in wild-type and NLRP3-deficient mice in vivo. Immune cell migration around subcutaneously injected β-TCP particles was reduced in NLRP3-deficient mice. These findings suggest that the effects of β-TCP particles in vivo are at least partly mediated by NLRP3 inflammasome complexes.

A high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI) procedure for the simultaneous determination of nine local anesthetic drugs (procaine, mepivacaine, lidocaine, ropivacaine, oxybuprocaine, tetracaine, bupivacaine, T-caine and dibucaine) in human serum is described. The chromatographic separation was performed on a Mightysil-RP-18 GP II column (2.0mm×150mm, particle size 5μm). The mobile phase consisted of 10mM acetic ammonium buffer (pH 5.4) and acetonitrile and was delivered at a flow rate of 0.20mL/min. The triple quadrupole mass spectrometer was operated in positive ion mode, and multiple reaction monitoring was used for drug quantification. Solid-phase extraction of the nine local anesthetic drugs added to the human serum was performed with an Oasis(®) HLB extraction cartridges column. The method was linear for the investigated drugs over the concentration range of 10-100ng/mL. The recoveries of these drugs were in the range of 81.4-144%. The standard deviation (SD) values for all analytes were

The peroxisome proliferator-activated receptor (PPAR) gamma is regarded as a "master regulator" of adipocyte differentiation and is abundantly expressed in adipose. To understand the biological role of PPARgamma in adipose, RNA interference (RNAi) of PPARgamma should be a powerful tool. 3T3-L1 cell line serves an excellent model to investigate the mechanism of preadipocyte-to-adipocyte differentiation. However, this cell line is difficult to transfect by plasmid vectors and viral vectors. We optimized the transduction of both 3T3-L1 preadipocytes and adipocytes by means of fiber-modified adenovirus (Ad) vectors. Among the various vectors tested, polylysine modification of the C-terminal of the fiber knob most markedly improved the transduction efficiency in both 3T3-L1 preadipocytes and adipocytes. Then, we examined whether fiber-modified Ad vectors with polylysine peptides expressing the small interfering RNA (siRNA) for PPARgamma inhibit the differentiation of 3T3-L1 preadipocytes into adipocytes. Oil red O staining and measurement of glycerol-3-phosphate dehydrogenase (GPDH) activity indicated that the vectors effectively suppressed the differentiation of 3T3-L1 preadipocytes to adipocytes. These results suggested that the combination of fiber-modified Ad vectors containing polylysine peptides and RNAi is an effective tool for the study of the biological and physiological mechanism of adipogenesis in adiposity and diabetes using 3T3-L1 models. Ad vector-mediated RNAi for PPARgamma should also be useful to clarify the biological role of the PPARgamma pathway in various tissues in addition to adipose and for therapeutic application to a variety of diseases, including adiposity and diabetes.

Targeted gene delivery to the tissue of interest by recombinant adenovirus (Ad) vectors is limited by the relatively broad expression of the primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, alphav integrin. This problem could be overcome by mutating the fiber and penton base, which bind with CAR and alphav integrin, respectively. In this study, we constructed CAR-binding ablated Ad vectors and alphav integrin-binding ablated Ad vectors by mutation in the FG loop of fiber knob and in the RGD motif of penton base, respectively, and compared the gene transfer properties of their vectors into various types of cultured cells and mice with conventional Ad vectors. We also generated Ad vectors containing RGD peptide in the HI loop of the fiber knob. CAR-binding ablated Ad vectors mediated about 1% of gene transfer activity into CAR-positive cultured cells, compared with conventional Ad vectors, while alphav integrin-binding ablated Ad vectors maintained at least 76% of gene transfer activity into cultured CAR-positive cells. Inclusion of the RGD peptide into the HI loop of the fiber knob of CAR-binding ablated Ad vectors restored gene transfer activity in vitro. On the other hand, systemically administered CAR-binding ablated Ad vectors, as well as alphav integrin-binding ablated Ad vectors mediated similar levels of gene transfer into mouse liver with the conventional Ad vectors. These results suggest that continued interaction of either the fiber with CAR or the penton base with alphav integrin offers an effective route of virus entry into mouse liver in vivo. Inhibition of the interaction of both the fiber with CAR and the penton base with alphav integrin is likely to be crucial to the development of targeted Ad vectors.

The use of recombinant adenovirus (Ad) vectors containing genetically modified capsid proteins is an attractive strategy for achieving targeted gene transfer. The HI loop of the fiber knob is a promising candidate location for the incorporation of foreign ligands for achieving this goal. However, the method of constructing an Ad vector containing a foreign ligand in the HI loop of the fiber knob has proved difficult. In this study, we developed a simple system to construct fiber-modified vectors. To do this, a vector plasmid containing a complete E1/E3-deleted Ad type 5 genome and a unique Csp45I and/or ClaI site between positions 32679 and 32680 of the Ad genome (residues threonine-546 and proline-547 of the fiber protein) was constructed. Oligonucleotides corresponding to the Arg-Gly-Asp (RGD) or Asn-Gly-Arg (NGR)-containing peptide motif (as a model) and containing a Csp45I and/or ClaI recognition site, were ligated into the Csp45I and/or ClaI-digested plasmid. The foreign transgene expression cassette was inserted into the E1 deletion site of the vector plasmid and the fiber-mutant Ad vector was produced by transfection of the PacI-digested plasmid into 293 cells. The virus containing the RGD or NGR peptide on the fiber knob was able to infect human glioma cells, which do not express coxsackievirus and adenovirus receptor (CAR), one of the Ad virus receptors, about 100-1000 times more efficient than the virus containing wild-type fiber. This suggested that the mutant virus mediated CAR-independent cell entry pathway. The simplicity of this method allows not only for easy construction of fiber-mutant Ad vectors, but also for screening of the peptides that target the vector to the desired cells and tissues.

The steroid/thyroid receptor superfamily comprises a group of structurally related transcription factors that program developmental, physiological, and behavioral responses to a variety of signals. NGFI-B, Nurr1 and NOR-1 are members of this superfamily of ligand-inducible transcription factors, and constitute one subgroup called the NGFI-B family. However, ligands have not yet been identified for the family members, and therefore they also belong to the category of “orphan receptors.” Although our knowledge of the NGFI-B family is limited, it is clear that these transcription factors have important functions in mediating responses to various signals and play critical roles in biological processes including cell growth, differentiation and apoptosis. In this review, we summarize their structural features, genomic organization, gene expression and transcriptional regulation, and illustrate their biological functions inferred from recent reports.

The role of protein kinase C in the interleukin 1 (IL-1)-mediated production of pro-matrix metalloproteinases (proMMPs) and tissue inhibitor-1 of metalloproteinases (TIMP-1) in human uterine cervical fibro-blasts has been investigated. IL-1 and a protein kinase C activator, 12- O -tetradecanoylphorbol 13-acetate (TPA) augmented the production of proMMP-1 (interstitial procollagenase), proMMP-3 (prostromelysin-1) and TIMP-1, but their effects were inhibited by the protein kinase C inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine in a dose-dependent manner. The suppressive effect of H-7 and staurosporine on the IL-1-induced production of proMMPs-1 and -3 and TIMP-1 resulted from the decrease in the steady-state levels of their mRNAs. When protein kinase C was down-regulated by treating the cells with a high level of TPA, the inductive effect of IL-1 upon proMMP-3 production was reduced considerably. These results indicate that protein kinase C mediates the IL-1-induced production of proMMPs-1 and -3 and TIMP-1 at the pretranslational level in human uterine cervical fibroblasts. On the other hand, neither IL-1 nor TPA modulated the production of proMMP-2 (progelatinase A). Both IL-1 and TPA also accelerated the productoin of prostaglandin E 2 (PGE 2 ) by cervical fibroblasts. However, the treatment of the cells with staurosporine in the presence of IL-1 or TPA further augmented PGE 2 synthesis, suggesting that the increased synthesis of PGE 2 by IL-1 treatment is mediated via signalling pathways distinct from those of proMMPs-1 and -3 and TIMP-1.

RNA interference (RNAi) is a powerful tool for the knockdown of gene expression. Here, we report on the development of an adenovirus (Ad) vector-mediated doxycycline (Dox)-inducible small interfering RNA (siRNA) expression system. We used this siRNA system to control the expression of p53 and c-Myc in human cancer cells. Coinfection of Ad vectors containing the siRNA expression system under the control of the Dox-inducible H1 promoter and Ad vectors expressing a tetracycline repressor inhibited the expression levels of p53 and c-Myc in a dose-dependent manner with both Dox and viral dose. Regulated silencing of p53 and c-Myc expression was obtained. Because an Ad vector-mediated inducible RNAi system can efficiently transduce a variety of cell types in vitro and in vivo, and the degree of loss of gene expression can be modulated according to the dose of Dox, this expression system should be a useful tool for both basic research on the analysis of gene function and therapeutic applications of RNAi.

Plasmids containing the origin of replication, oriP, of the Epstein-Barr virus (EBV) and EBV nuclear antigen-1 genes replicate extrachromosomally in primate cells. However, these plasmids have been believed not to replicate in rodent cells. We demonstrate here that these plasmids can replicate in some types of rodent cells over a long period. This result should offer not only the new insight into the mechanisms of species-specific replication of EBV, but also the possibility that an EBV-based vector can be used for gene transfer experiments in non-primate cells and an animal experiment regarding human gene therapy.

In the conventional method for constructing an adenoviral (Ad) vector expressing small interfering RNA (siRNA), short hairpin RNA (shRNA)-coding oligonucleotides are introduced downstream of a polymerase III (or polymerase II)-based promoter cloned into a shuttle plasmid. An siRNA expression cassette, which is cloned into the shuttle plasmid, is then introduced into the E1 deletion region of the Ad vector plasmid by in vitro ligation or homologous recombination in Escherichia coli, and the linearized plasmid is transfected into 293 cells, generating an Ad vector expressing siRNA. Therefore, two-step plasmid manipulation is required. In this study, we developed a method by which shRNA-coding oligonucleotides can be introduced directly into the Ad vector plasmid. To do this, we constructed a new vector plasmid into which the human U6 promoter sequence was cloned in advance. Unique restriction enzyme sites were introduced at the transcription start site of the U6 promoter sequence in the vector plasmid. Luciferase and p53 genes were efficiently knocked down by Ad vectors generated by the new method and expressing siRNA against the target gene. This method should be useful for RNA interference-based experiments, and should make it easy to construct an siRNA-expressing Ad vector library for functional screening.

The mechanisms by which epidermal growth factor (EGF) enhances the production of pro-matrix metalloproteinase 3 (proMMP-3/prostromelysin 1) and tissue inhibitor of metalloproteinases (TIMP)-1 were investigated using human uterine cervical fibroblasts. The treatment of the cells with EGF for 24 h resulted in about 5–6-fold increase in the production of proMMP-3 and TIMP-1 compared with the untreated control cells. This increase was accompanied by an increase of proMMP-3 and TIMP-1 mRNAs. However, an about 3- and 2-fold increase in the production of proMMP-3 and TIMP-1, respectively, was observed as early as 1 h after the treatment of the cells with EGF, and it was not accompanied by any apparent increase in proMMP-3 and TIMP-1 mRNAs. This early effect of EGF on the enhanced production of proMMP-3 and TIMP-1 was not inhibited by actinomycin D, even though actinomycin D inhibited the synthesis of the total RNA in both the EGF-treated and untreated cells. These results indicate that EGF enhances the apparent production of proMMP-3 and TIMP-1 by two mechanisms: one by the accelerated translation of their mRNAs; and the other by the enhanced transcription of their genes. The former event takes place much earlier than the latter.