Evaluation of a -Octaarginine-linked polymer as a transfection tool for transient and stable transgene expression in human and murine cell lines.

Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression.

Cloning of Hynobius lichenatus (Tohoku hynobiid salamander) p53 and analysis of its expression in response to radiation.

Background: Caudata species such as salamanders are easily affected by environmental changes, which can drastically reduce their population. The effects of acute X-rays and chronic γ-irradiation on Hynobius lichenatus, the Japanese Tohoku hynobiid salamander, are known. However, the expression of radiation-inducible genes, such as the DNA-damage checkpoint response gene p53, has not been analyzed in H.

Functional comparison of the human epidermal growth factor receptor and telomerase reverse transcriptase promoters in canine tumor cells.

We previously showed that the promoter region of the human epidermal growth factor receptor (hEGFR) gene elicits high transduction efficiency, with transgene expression restricted to canine breast tumor cells. However, it was unclear whether this promoter induces tumor cell-specific transgene expression in canine urothelial carcinoma cells. Furthermore, compared with studies in human cancer cells, the utility of the telomerase reverse transcriptase (TERT) gene promoter for therapeutic transgene expression in canine cancer cells has not been evaluated thus far.

The membrane-type estrogen receptor G-protein-coupled estrogen receptor suppresses lipopolysaccharide-induced interleukin 6 via inhibition of nuclear factor-kappa B pathway in murine macrophage cells.

The female sex hormone estrogen exerts anti-inflammatory effects. The G-protein-coupled estrogen receptor (GPER) has been recently identified as a novel membrane-type estrogen receptor that can mediate non-genomic estrogenic effects on many cell types. We previously demonstrated that GPER inhibits tumor necrosis factor alpha-induced expression of interleukin 6 (IL-6) through repression of nuclear factor-kappa B (NF-κB) promoter activity using human breast cancer cells.

Expression of HIF-1α ODD domain fused canine caspase 3 by EGFR promoter-driven adenovirus vector induces cytotoxicity in canine breast tumor cells under hypoxia.

Adenovirus (Ad) vectors are widely used in cancer gene therapies. However, compared to human patients, relatively limited information is available on gene transduction efficiency or cell-specific cytotoxicity in canine tumor cells transduced with Ad vectors. Since epidermal growth factor receptor (EGFR) is highly expressed on canine breast tumor cells, we sought to develop an Ad vector based on the RGD fiber-mutant adenovirus vector (AdRGD) that expresses canine caspase 3 under the control of EGFR promoter.

Detection of Bcl-2 mRNA and its product in the glomerular podocytes of the normal rat kidney.

Podocyte apoptosis underlies podocytepenia leading to glomerulosclerosis. An apoptosis inhibitory protein Bcl-2 is expressed in the podocytes in the early stage of nephrogenesis and downregulated in the maturing stage of human fetal kidneys. Recent studies reported changed localization and expression of Bcl-2 in the renal glomeruli under the pathologic conditions.

First case report of histoplasmosis in a cat in Japan.

Cytological, histopathological and immunohistochemical examinations were carried out on a presumed 10-year-old Japanese cat showing vomiting and emaciation. On cytologic examination of the mass of the upper abdominal cavity, many yeast-like organisms were detected in the macrophages. At necropsy, the upper part of colon was markedly dilated with a thickened wall.

Identification of tocopherol-associated protein as an activin/TGF-beta-inducible gene in mast cells.

Previous studies have demonstrated that treatment with activin A and TGF-beta(1), members of the TGF-beta family, stimulated maturation of mouse bone marrow-derived cultured mast cells (BMMC), which was characterized by morphology and gene expression of mouse mast cell proteases (mmcps). In order to gain a better understanding of activin A- and TGF-beta(1)-induced maturation in mast cells, we investigated the genes that were up-regulated in response to treatment with these two members of the TGF-beta family. The cDNA microarray analyses indicated that in BMMC, five genes were induced by treatment with 4 nM activin A for 2 h.

Requirement of Smad3 for mast cell growth.

The involvement of the TGF-beta family in cell growth of bone marrow-derived mast cells (BMMC) cultured with medium containing pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) was examined. Doubling time of BMMC from Smad3-null mice was longer than that from wild-type (WT) mice, and the differences tended to be larger with time of culture. Consistent with the results, uptake and reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was lower in Smad3-deficient BMMC.

Involvement of p38 MAP kinase and Smad3 in TGF-beta-mediated mast cell functions.

Transforming growth factor-beta (TGF-beta) modulates functions of bone marrow-derived cultured mast cells (BMMCs); cell maturation (up-regulation of mouse mast cell proteases (mmcps)), growth arrest and migration. We investigated the roles of p38 MAP kinase and Smad3 in TGF-beta-mediated cell responses in BMMCs. Treating BMMCs with TGF-beta induced the phosphorylation of p38 within 2 h and persisted for 24 h.

Transcriptional regulation of mouse mast cell protease-7 by TGF-beta.

Mouse mast cell protease-7 (mmcp-7) is a tryptase predominantly expressed in differentiated connective tissue-type mast cells. Previous study revealed that transforming growth factor-beta (TGF-beta) increases gene transcript of mmcp-7 in mast cells. The present study explored molecular mechanism of the up-regulation of mmcp-7 by TGF-beta.

Responses during cell preparation for functional analyses in mouse bone marrow-derived cultured mast cells.

Murine bone marrow-derived cultured mast cells (BMMCs) are most widely used in in vitro experiments for evaluation of mast cell functions. The present study has shown that cell preparation procedure, i.e.

Transcriptional regulation of plasminogen activator inhibitor-1 by transforming growth factor-beta, activin A and microphthalmia-associated transcription factor.

Plasminogen activator inhibitor-1 (PAI-1) is a key molecule that regulates turnover of the extracellular matrix. In the present study, we characterized PAI-1 gene expression in mast cells and melanocytes. In bone marrow-derived cultured mast cells, up-regulation of the PAI-1 gene was observed upon treatment with TGF-beta1, and was regulated at the transcriptional level.

Expression of tocopherol-associated protein in mast cells.

Tocopherol-associated protein (TAP) was expressed in mouse mast cells. TAP was predominantly localized in the cytoplasm, and the subcellular localization was not changed by alpha-tocopherol. The results suggest that the physiological role of TAP in mast cells is not regulation of tocopherol function but an as-yet-unidentified activity.

Effect of PCB-126 on intracellular accumulation and transepithelial transport of vinblastine in LLC-PK1 and its transformant cells expressing human P-glycoprotein.

The effects of 3, 3', 4, 4', 5-pentachlorobiphenyl (PCB-126), which is the most toxic congener of coplanar polychlorinated biphenyls (Co-PCBs), on intracellular accumulation and transepithelial transport of vinblastine were examined in porcine kidney cells, LLC-PK1, and its transformant cells expressing human P-glycoprotein (LLC-MDR1). The accumulation decreased less than one-tenth in LLC-MDR1 compared to LLC-PK1. In both cells, the accumulation increased with the addition of PCB-126 and cyclosporine A (CYA), which are P-glycoprotein modulators, though the magnitudes were different in these two cell groups as well as for these two chemicals.

Up-regulation of mouse mast cell protease-6 gene by transforming growth factor-beta and activin in mast cell progenitors.

Previous studies have revealed that members of the transforming growth factor-beta (TGF-beta) including TGF-beta1 and activin A modulate the function of mast cells. Here we show the up-regulation of mouse mast cell protease-6 (mMCP-6), which is expressed in differentiated mast cells, by TGF-beta1 and activin A in bone marrow-derived cultured mast cell progenitors (BMCMCs). Quantitative real time RT-PCR analyses revealed that the mRNA level of mMCP-6 was slightly but reproducibly increased by treatment with TGF-beta1 or activin A, which was regulated at the transcription level.

Reliability of RT-PCR methods for measuring relative gene expression in mast cells.

Three methods to quantify gene transcript levels in mast cells, real-time RT-PCR, competitive RT-PCR and conventional RT-PCR analyses, were compared. Linear regression analysis on five gene transcripts revealed that the mRNA levels measured by real-time RT-PCR analysis were minimally correlated with those by conventional RT-PCR analysis. In addition, differences in the mRNA level between samples measured by conventional RT-PCR analysis were smaller than those by real-time RT-PCR analysis, suggesting that conventional RT-PCR analysis is less sensitive at measuring mRNA levels.

Contribution of mast cells to bacterial clearance and their proliferation during experimental cystitis induced by type 1 fimbriated E. coli.

Bacterial infections of the urinary bladder are very common, and the role of mast cells in these infections is invariably thought of as a detrimental one. However, recent studies have shown that mast cells play a key role in host defense against various enterobacterial infections. In this manuscript, using mast cell-deficient (WBB6F1 - W/Wv) and mast cell-sufficient (WBB6F1 - +/+) mice we have investigated the protective role of mast cells in urinary bladder infections in vivo.

Transcriptional activation of mouse mast cell protease-9 by microphthalmia-associated transcription factor.

We explored transcriptional regulation of mouse mast cell protease-9 (mMCP-9), which is implicated in inflammation of the jejunum during helminth infections and tissue remodeling of the uterus during pregnancy. Transcription was positively regulated by microphthalmia-associated transcription factor (MITF), a member of the basic helix-loop-helix-leucine zipper family that binds to the E-box, a CANNTG sequence. The most significant segment for positive regulation by MITF was nt -183 to -177 of the mMCP-9 promoter, CATCATG, which bound MITF-M.

Transcriptional activation of mouse mast cell Protease-7 by activin and transforming growth factor-beta is inhibited by microphthalmia-associated transcription factor.

Previous studies have revealed that activin A and transforming growth factor-beta1 (TGF-beta1) induced migration and morphological changes toward differentiation in bone marrow-derived cultured mast cell progenitors (BMCMCs). Here we show up-regulation of mouse mast cell protease-7 (mMCP-7), which is expressed in differentiated mast cells, by activin A and TGF-beta1 in BMCMCs, and the molecular mechanism of the gene induction of mmcp-7. Smad3, a signal mediator of the activin/TGF-beta pathway, transcriptionally activated mmcp-7.

Degranulation in RBL-2H3 cells: regulation by calmodulin pathway.

Involvement of the calmodulin pathway in Ca2+-induced degranulation was evaluated in RBL-2H3 mast cells. Pretreatment of RBL-2H3 cells with a calmodulin antagonist, W-13, blocked ionomycin-dependent release of beta-hexosaminidase into the supernatant, although W-13 treatment alone slightly but significantly increased the release. Ca2+/calmodulin activates various protein kinases and phosphatases including myosin-light chain kinase (MLCK), calmodulin-dependent protein kinases (CaMKs), and calcineurin.

Altered function of murine mast cells in response to lipopolysaccharide and peptidoglycan.

Toll-like receptors (TLRs) recognize and signal the presence of bacterial components such as lipopolysaccharide (LPS) and peptidoglycan (PG) as a part of innate immunity. Our previous studies revealed that mast cells function as effector cells in the protection of mice against lethal enterobacterial infections. In this study, we examined both the gene expression of molecules involved in TLR signaling and the effects of LPS and PG in bone marrow-derived cultured mast cells (BMCMCs).

Role of activin A in murine mast cells: modulation of cell growth, differentiation, and migration.

Activins, members of the transforming growth factor-beta (TGF-beta) superfamily, are potent growth and differentiation factors. Our previous studies revealed that activin A, a homodimer of inhibin/activin beta(A), was induced in mast cells and peritoneal macrophages in response to their activation. In the present study, we examined the roles of activin A in murine bone marrow-derived, cultured mast cell progenitors (BMCMCs), which expressed gene transcripts for molecules involved in activin signaling, suggesting that BMCMCs could be target cells of activin A.