Role of IRAK1 on TNF-induced proliferation and NF-ĸB activation in human bone marrow mesenchymal stem cells.

In this study, we determined the effect of TNF-α on hBMSCs proliferation as well as the role of IL-1 receptor-associated kinase 1 (IRAK1) on TNF-α signaling. Western blot analysis revealed that TNF-α treatment increased the phosphorylation of IRAK1 in hBMSCs. The downregulation of IRAK1 inhibited TNF-α-induced NF-ĸB activation and COX-2 expression. TNF-α treatment increased hBMSCs proliferation in a dose-dependent manner and increased ERK, JNK, and NF-ĸB activity. U0126, an ERK inhibitor, decreased hBMSCs proliferation and significantly blocked TNF-α -induced hBMSCs proliferation. In cells with IRAK1 or TRADD downregulation, the U0126 treatment inhibited hBMSCs proliferation and significantly suppressed TNF-α-induced hBMSCs proliferation. The downregulation of IRAK1 or TRADD inhibited TNF-α-induced ERK and JNK activation, and hBMSCs proliferation. Inhibition of NF-ĸB by decoy oligonucleotides reduced the TNF-α-induced hBMSCs proliferation. Immunoprecipitation analysis showed that IRAK1 does not physically interact with TNF receptor 1 (TNFR1) even in the presence of TNF-α. Suppression of IRAK1 binding protein (IRAK1BP1) inhibited TNF-α-induced increase of the proliferation and ERK1 phosphorylation of hBMSCs in the presence of TNF-α. Our data indicate that TNF-α modulates hBMSCs proliferation through ERK signaling pathways, and that IRAK1 plays an important role in TNF-α-induced NF-ĸB activation in hBMSCs.

Gold nanoparticle-assisted delivery of small, highly structured RNA into the nuclei of human cells.

Previous studies have shown that functionalized gold nanoparticles (AuNPs) can be used as a general platform for loading and delivering DNA oligonucleotides and short hairpin RNA to living systems. Here, we report the ability of functionalized AuNP to deliver RNA aptamers into the nuclei of human cells. An in vitro-synthesized RNA aptamer specific to the ß-catenin protein was delivered into the HepG2 human cell line more efficiently via functionalized AuNP than liposome-based delivery, and resulted in nearly complete inhibition of ß-catenin binding to the p50 subunit of NF-κB in the nucleus. This inhibition led to repression of NF-κB p50-dependent transcription of CRP. Also, the ß-catenin aptamer in the nucleus led to down-regulation of ß-catenin-mediated transcriptional activity through the TCF complex and resulted in decrease in the levels of cyclin D, and c-myc mRNA by ~47% and ~57%, respectively. In addition, we used functionalized AuNP to deliver another RNA aptamer targeted to the p50 subunit of NF-κB into the A549 human cell line, and this was sufficient to induce apoptosis of the cells. Our findings demonstrate that AuNP GDS can be used to deliver small, highly structured RNA aptamers into the nucleus of human cells where they modulate the activity of transactivators by interacting with target proteins.

Effect of the modulation of leucine zipper tumor suppressor 2 expression on proliferation of various cancer cells functions as a tumor suppressor.

ß-catenin is a component of the adhesion complex linking cadherin and actin cytoskeleton, as well as a major mediator of the Wnt pathway, which is a critical signal cascade regulating embryonic development, cell polarity, carcinogenesis, and stem cell function. NF-κB functions as a key regulator of immune responses and apoptosis, and mutations in NF-κB signaling can lead to immune diseases and cancers. We previously showed that NF-κB-mediated modulation of ß-catenin/Tcf signaling is mediated by leucine zipper tumor suppressor 2 (Lzts2) and that lzts2 expression is differentially regulated in various cancer cells. Its functional significances, however, are poorly understood. We showed that NF-κB-induced modulation of ß-catenin/Tcf pathway is regulated by lzts2 expression in mesenchymal stem cells (MSCs) and several cancer cells, and that NF-κB-induced lzts2 expression is differentially regulated among cancer cell types. Here, using a promoter-reporter assay and EMSA, we demonstrate that NF-κB regulates lzts2 transcription by directly binding to the lzts2 promoter, and that NF-κB-induced lzts2 transcription differs by cell types. Modulation of lzts2 expression by lentiviral techniques affected proliferation and tumorigenicity of several cancer cell lines such as breast, colon, prostate cancer, and glioma, but did not affect cisplatin sensitivity or cell migration. Our data indicate that lzts2 expression is transcriptionally regulated by NF-κB activities, and the modulation of lzts2 expression affects cell proliferation and tumor growth through the Wnt/ß-catenin pathway in various cancer cell lines.

NF-kappaB activation stimulates osteogenic differentiation of mesenchymal stem cells derived from human adipose tissue by increasing TAZ expression.

Tumor necrosis factor-alpha (TNF-alpha) is a skeletal catabolic agent that stimulates osteoclastogenesis and inhibits osteoblast function. Although TNF-alpha inhibits the mineralization of osteoblasts, the effect of TNF-alpha on mesenchymal stem cells (MSC) is not clear. In this study, we determined the effect of TNF-alpha on osteogenic differentiation of stromal cells derived from human adipose tissue (hADSC) and the role of NF-kappaB activation on TNF-alpha activity. TNF-alpha treatment dose-dependently increased osteogenic differentiation over the first 3 days of treatment. TNF-alpha activated ERK and increased NF-kappaB promoter activity. PDTC, an NF-kappaB inhibitor, blocked the osteogenic differentiation induced by TNF-alpha and TLR-ligands, but U102, an ERK inhibitor, did not. Overexpression of miR-146a induced the inhibition of IRAK1 expression and inhibited basal and TNF-alpha- and TLR ligand-induced osteogenic differentiation. TNF-alpha and TLR ligands increased the expression of transcriptional coactivator with PDZ-binding motif (TAZ), which was inhibited by the addition of PDTC. A ChIP assay showed that p65 was bound to the TAZ promoter. TNF-alpha also increased osteogenic differentiation of human gastroepiploic artery smooth muscle cells. Our data indicate that TNF-alpha enhances osteogenic differentiation of hADSC via the activation of NF-kappaB and a subsequent increase of TAZ expression.

Comparison of in vivo nephrotoxicity in the rabbit by a pyrrolidinyl-thio Carbapenem CW-270031.

CW-270031 is a novel synthesized carbapenem antibiotic with a broad antimicrobial activity. Carbapenem antibiotics are well known for their nephrotoxicity. In this study, we evaluated the nephrotoxicity potential of this compound in rabbits, which are known for being more sensitive than other animals to renal insult. CW-270031 was administered to NZW male rabbits via an ear vein (200 mg/kg, single injection). Blood samples were collected on 2, 3, and 4 days after treatment. Urea nitrogen and creatinine in plasma were quantified. Four days after the treatment, all animals were autopsied and histopathological examinations were performed on their kidneys, revealing that cephaloridine and imipenem were highly nephrotoxic, and cefazolin had mild renal toxicity, whereas CW-270031 as well as meropenem and tienam had no toxicity to the kidney. The present findings suggest that CW-270031 is a potential carbapenem antibiotic with no nephrotoxicity.

Enhancement of biomass, lipids, and polyunsaturated fatty acid (PUFA) production in Nannochloropsis oceanica with a combination of single wavelength light emitting diodes (LEDs) and low temperature in a three-phase culture system.

A three-phase culture system combining blue (465 nm) light-emitting diode (LED) wavelength as the first phase, green (550 nm) as the second phase, and temperature stress as the third phase was applied to a Nannochloropsis oceanica culture in 14-L photobioreactors. Microalgal growth promotion parameters were optimized in the first phase, followed by green LED stress for lipid production in the second phase. Maximum biomass and lipid production values of 0.75 gdcw L-1 and 57.6% (w/w) were obtained at an aeration rate of 0.50 vvm, with a light intensity of 250 µmol m-2 s-1 and 24:0 h light/dark cycle. Culture temperatures of 15, 10 and 5 °C were applied in the third phase, where temperature stress induced the production of monounsaturated and polyunsaturated fatty acid synthesis in N. oceanica. The production of α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid increased by 52% (w/w), 96% (w/w), and 77% (w/w), respectively, at 5 °C in the third phase.

A pseudoknot improves selection efficiency in ribosome display.

The size and diversity of ribosome display libraries depends upon stability of the complex formed between the ribosome, mRNA and translated protein. To investigate if mRNA secondary structure improves stability of the complex, we tested a pseudoknot, originating from the genomic RNA of infectious bronchitis virus (IBV), a member of the positive-stranded coronavirus group. We used the previously-isolated anti-DNA scFv, 3D8, as a target protein. During in vitro translation in rabbit reticulocyte lysate, we observed that incorporation of the pseudoknot into the mRNA resulted in production of a translational intermediate that corresponded to the expected size for ribosomal arrest at the pseudoknot. Complexes containing the mRNA pseudoknot exhibited a higher efficiency of affinity selection than that those without, indicating that the pseudoknot improves stability of the mRNA-ribosome-antibody complex in a eukaryotic translation system. Thus, in order to improve the efficiency of selection, this relatively short pseudoknot sequence could be incorporated into ribosome display.

Functional analysis of the residues C770 and G771 of E. coli 16S rRNA implicated in forming the intersubunit bridge B2c of the ribosome.

Structural analyses have shown that nucleotides at the positions 770 and 771 of Escherichia coli 16S rRNA are implicated in forming one of highly conserved intersubunit bridges of the ribosome, B2c. To examine a functional role of these residues, base substitutions were introduced at these positions and mutant ribosomes were analyzed for their protein synthesis ability using a specialized ribosome system. The results showed requirement of a pyrimidine at the position 770 for ribosome function regardless of the nucleotide identity at the position 771. Sucrose gradient profiles of ribosomes revealed that the loss of protein-synthesis ability of mutant ribosome bearing a base substitution from C to G at the position 770 stems from its inability to form 70S ribosomes. These findings indicate involvement of nucleotide at the position 770, not 771, in ribosomal subunit association and provide a useful rRNA mutation that can be used as a target to investigate the physical interaction between 16S and 23S rRNA.

Cinnamomum cassia bark produced by solid-state fermentation with Phellinus baumii has the potential to alleviate atopic dermatitis-related symptoms.

In order to evaluate whether the aqueous fraction of Cinnamomum cassia produced by solid-state fermentation with Phellinus baumii (afCc/Pb) inhibits atopic symptoms in vivo, its efficacy was evaluated in an animal model of 2,4-dinitrofluorobenzene (DNFB)-induced atopic dermatitis. Immune-related cells were quantified using hematoxylin and eosin staining, and phenotypic cytokines, enzymes and the expression of other proteins in the animal model were evaluated. The data revealed that afCc/Pb (100 µg/ml) exhibited strong anti-atopic activity, causing a significant 40% reduction in immune response, as shown by the extent of ear swelling, resulting from a decrease in the number of eosinophils in the skin tissues due to decreased matrix metalloproteinase-2 and interleukin-31 expression. These results collectively suggest that afCc/Pb has the potential to alleviate the symptoms of atopic dermatitis in a mouse model of DNFB-induced atopic dermatitis, and that it may be a valuable bioresource for the cosmetic/cosmeceutical industry.

Comparison of the toxicity of aqueous and ethanol fractions of Angelica keiskei leaf using the eye irritancy test.

To determine whether aqueous and ethanol fractions of the Angelica keiskei leaf exert toxicity when used for cosmetic purposes, we performed the acute eye irritancy test. Animals were treated with sample fractions (100 mg/dose) according to standard procedure guidelines. No significant changes or damage was detected in the fraction-treated groups in terms of ocular lesions in the cornea, the size of the cornea with turbidity, swelling of the eyelid and emission discharge. However, sodium dioctyl sulfosuccinate, a positive control, induced severe toxic symptoms. Thus, aqueous and ethanol fractions of Angelica keiskei do not appear to induce acute toxicity in the eye lens, as assessed from anatomical and pathological observations in the rabbit eye. Our results collectively suggest that aqueous and ethanol fractions show promise as cosmetic ingredients that do not cause eye toxicity.

Inhibition of xenograft tumor growth in mice by gold nanoparticle-assisted delivery of short hairpin RNAs against Mcl-1L.

A prerequisite for the therapeutic use of small RNAs is the development of a method that can deliver them into animals. Previous studies have shown the capability of functionalized gold nanoparticles to serve as a general platform for loading and delivering DNA oligonucleotides and short hairpin RNAs (shRNAs) into cultured human cells. Here, we report the ability of the gold nanoparticle-assisted gene delivery system (AuNP-GDS) to deliver shRNA to a xenograft tumor in a mouse model. AuNP-GDS delivery of in vitro synthesized shRNA targeted to the Mcl-1L gene knocked down levels of Mcl-1L mRNA and protein by ~36% and ~26%, respectively, which were sufficient to induce apoptosis of the xenograft tumor cells and consequently inhibited the development of the tumor. We demonstrated that our lego-like AuNP-GDS, which can easily load and deliver shRNAs targeted to any gene of interest into living systems, can deliver shRNAs into xenograft tumors, leading to antitumor activity in an animal model.

Functional investigation of residue G791 of Escherichia coli 16S rRNA: implication of initiation factor 1 in the restoration of P-site function.

Using a specialized ribosome system, previous studies have identified G791 in Escherichia coli 16S rRNA as an invariant and essential residue for ribosome function. To investigate the functional role of G791, we searched for multicopy suppressors that partially restored the protein synthesis ability of mutant ribosomes bearing a G to U substitution at position 791 (U791 ribosomes). Analyses of isolated multicopy suppressors showed that overexpression of initiation factor 1 (IF1) enhanced the protein synthesis ability of U791 ribosomes. In contrast, overexpression of initiation factor 2 (IF2) or IF3 did not enhance the protein synthesis ability of wild-type or U791 ribosomes, and overexpression of IF1 did not affect the function of wild-type or mutant ribosomes bearing nucleotide substitutions in other regions of 16S rRNA. Analyses of sucrose gradient profiles of ribosomes showed that overexpression of IF1 marginally enhanced the subunit association of U791 ribosomes and indicated lower binding affinity of U791 ribosomes to IF1. Our findings suggest the involvement of IF1 in the restoration of the P-site function that was impaired by a nucleotide substitution at residue G791.