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Biochem Cell Biol [journal]
- Regulation of transforming growth factor beta 1 gene expression by dihydropteridine reductase in kidney 293T cells. [Journal Article]
- Biochem Cell Biol 2013 Jun; 91(5):187-93.
Quinoid dihydropteridine reductase (QDPR) is an enzyme involved in the metabolic pathway of tetrahydrobiopterin (BH4). BH4 is an essential cofactor of nitric oxide synthase (NOS) and can catalyze arginine to citrulline to release nitric oxide. Point mutations of QDPR have been found in the renal cortex of spontaneous Otsuka Long Evans Tokushima Fatty (OLETF) diabetic rats. However, the role of QDPR in DN is not clear. This study investigates the effects of QDPR overexpression and knockdown on gene expression in the kidney. Rat QDPR cDNA was cloned into pcDNA3.1 vector and transfected in human kidney cells (293T). The expression of NOS, transforming growth factor beta 1 (TGF-β1), Smad3, and NADPH oxidase were examined by RT-PCR and Western blot analyses. BH4 was assayed by using ELISA. Expression of QDPR was significantly decreased and TGF-β1 and Smad3 were increased in the renal cortex of diabetic rats. Transfection of QDPR into 293T cells increased the abundance of QDPR in cytoplasm and significantly reduced the expression of TGF-β1, Smad3, and the NADPH oxidases NOX1 and NOX4. Moreover, abundance of neuronal NOS (nNOS) mRNA and BH4 content were significantly increased. Furthermore, inhibition of QDPR resulted in a significant increase in TGF-β1 expression. In conclusion, QDPR might be an important factor mediating diabetic nephropathy through its regulation of TGF-β1/Smad3 signaling and NADPH oxidase.
- Raman microspectroscopy as a diagnostic tool to study single living nasopharyngeal carcinoma cell lines. [Journal Article]
- Biochem Cell Biol 2013 Jun; 91(5):182-6.
Raman spectroscopy can provide molecular-level fingerprint information about the biochemical composition and structure of cells and tissues with excellent spatial resolution. In this study, Raman spectroscopy of 3 different nasopharyngeal carcinoma cell lines C666-1, CNE1, and CNE2 and 1 nasopharyngeal normal cell line NP69 acquired on a piece of silica glass slide are presented to investigate the differences among them. The results show the ratio of I1657/I1449 (= 0.7) could provide good distinction between tumor and normal cell lines very easily, which coincides with existing reports about the study of different cell lines and bronchial tissue. In addition, several statistical analytical methods were used to classify these 4 different cell lines and then achieved an exciting result with great sensitivity and specificity of >90%, respectively. The findings of this work further support former work where cells' Raman spectra were acquired on a different substrate. All of these results indicate Raman spectroscopy has the potential to discriminate between normal and tumor cells and have potential use in early diagnosis of nasopharyngeal carcinoma.
- Nucleotides metabolizing ectoenzymes as possible markers of mesenchymal stem cell osteogenic differentiation. [Journal Article]
- Biochem Cell Biol 2013 Jun; 91(5):176-81.
Growing murine mesenchymal stem cells (mMSCs) from mouse bone marrow decreased their rate of proliferation in the presence of benzoylbenzoyl-ATP persistently, but the inhibitory effect of ATP was strong only in a concentration of 50 μmol·L(-1) and lasted for 48 h in culture. These results hinted at ATP hydrolysis by the cell surface enzymes at the lower concentrations and thus it may be not able to inhibit MSCs. By using ATP, ADP, or AMP as substrates, we tested the ectonucleotidase activity on the surface of undifferentiated MSCs and MSC-derived osteoblasts. Here, we report that although nucleoside triphosphate diphosphohydrolase (NTPDase)1 and NTPDase8 are engaged in the metabolism of ATP in MSC-derived osteoblasts, NTPDase3 is responsible for its metabolism in undifferentiated MSCs. In this study, we also realized that osteoblasts effectively metabolize ADP to ATP and AMP. The enzymatic activity of adenylate kinase (AK) is consistent with the high expression level of the AK gene. Therefore, it was tempting to suggest that this enzyme, together with NTPDase1 and NTPDase8, assume the role of specific markers that allowed distinction between differentiated osteoblasts and early undifferentiated MSCs. Additionally, unlike osteoblasts, undifferentiated MSCs demonstrated the activity of 5'-nucleotidase (CD73). However, the expression analysis of CD73 mRNA did not show any differences; CD73 mRNA was expressed in both kinds of cells to the same extent.
- Phenotypic alteration of limbal niche-associated limbal epithelial stem cell deficiency by ultraviolet-B exposure-induced phototoxicity in mice. [Journal Article]
- Biochem Cell Biol 2013 Jun; 91(5):165-75.
Good vision requires a healthy cornea, and a healthy cornea needs healthy stem cells. Limbal epithelial stem cells (LESCs) are a traditional source of corneal epithelial cells and are recruited for the continuous production of epithelium without seizing throughout an animal's life, which maintains corneal transparency. Like the maintenance of other adult somatic stem cells, the maintenance of LESCs depends on the specific microenvironmental niche in which they reside. The purpose of this study was to determine the microenvironmental damage associated with LESCs fate due to ultraviolet (UV)-B exposure in a mouse model. Structural alteration and deregulation of the stem cell and its neighboring niche components were observed by using clinical, morphological, explant culture study, and flowcytometric analysis, which demonstrated that the limbal microenvironment plays an important role in cornea-related disease development. In UV-exposed mice, overexpression of vascular endothelial growth factor receptor 2 indicated neovascularization, decreased CD38 expression signified the alteration of limbal epithelial superficial cells, and the loss of limbal stem cell marker p63 indicated limbal stem cell deficiency in the limbal vicinity. We concluded that LESC deficiency diseases (LESCDDs) are associated with pathophysiological changes in the LESC niche, with some inhibitory interception such as UV-B irradiation, which results in corneal defects.
- Identification of amino acids responsible for stop codon recognition for polypeptide chain release factor. [Journal Article]
- Biochem Cell Biol 2013 Jun; 91(5):155-64.
One factor involved in eukaryotic translation termination is class 1 release factor in eukaryotes (eRF1), which functions to decode stop codons. Variant code species, such as ciliates, frequently exhibit altered stop codon recognition. Studies revealed that some class-specific residues in the eRF1 N-terminal domain are responsible for stop codon reassignment in ciliates. Here, we investigated the effects on stop codon recognition of chimeric eRF1s containing the N-terminal domain of Euplotes octocarinatus and Blepharisma japonicum eRF1 fused to Saccharomyces cerevisiae M and C domains using dual luciferase read-through assays. Mutation of class-specific residues in different eRF1 classes was also studied to identify key residues and motifs involved in stop codon decoding. As expected, our results demonstrate that 3 pockets within the eRF1 N-terminal domain were involved in decoding stop codon nucleotides. However, allocation of residues to each pocket was revalued. Our data suggest that hydrophobic and class-specific surface residues participate in different functions: modulation of pocket conformation and interaction with stop codon nucleotides, respectively. Residues conserved across all eRF1s determine the relative orientation of the 3 pockets according to stop codon nucleotides. However, quantitative analysis of variant ciliate and yeast eRF1 point mutants did not reveal any correlation between evolutionary conservation of class-specific residues and termination-related functional specificity and was limited in elucidating a detailed mechanism for ciliate stop codon reassignment. Thus, based on isolation of suppressor tRNAs from Euplotes and Tetrahymena, we propose that stop codon reassignment in ciliates may be controlled by cooperation between eRF1 and suppressor tRNAs.
- Mechanism of cardiomyocyte PGC-1α gene regulation by ERRα. [Journal Article]
- Biochem Cell Biol 2013 Jun; 91(5):148-54.
Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α) regulates critical genes involved in cardiac mitochondrial biogenesis and fatty acid oxidation, and its loss is associated with impaired metabolism and various cardiac pathologies. Estrogen-related receptor α (ERRα) targets many of the same genes as PGC-1α, and extensive cross talk exists between these 2 regulators. Here we report the identification of an evolutionarily conserved ERRα binding site within the PGC-1α promoter. Using luciferase reporter assays and overexpression, inhibition, or knockdown of ERRα, we show that PGC-1α expression is critically dependent upon ERRα in primary cardiomyocytes. We demonstrate that short-term hypoxia results in reduced ERRα mRNA expression, which precedes a similar loss of PGC-1α mRNA. However, chromatin immunoprecipitation reveals that despite a key role for ERRα in regulating PGC-1α in normoxic cardiomyocytes, ERRα loss is not responsible for PGC-1α loss in hypoxia. Histone deacetylase 5 (HDAC5) has previously been demonstrated to strongly inhibit expression of PGC-1α, and we show that overexpression of ERRα is sufficient to overcome this repressive effect. Our data elucidates the mechanism by which ERRα regulates cardiac PGC-1α gene expression, and suggests that ERRα may provide a means to normalize PGC-1α expression that could be useful in the development of strategies aimed at improving cardiac metabolism in disease.
- The effects of human umbilical cord perivascular cells on rat hepatocyte structure and functional polarity. [Journal Article]
- Biochem Cell Biol 2013 Jun; 91(5):140-7.
Hepatocyte culture is a useful tool for the study of their biology and the development of bioartificial livers. However, many challenges have to be overcome since hepatocytes rapidly lose their normal phenotype in vitro. We have recently demonstrated that human umbilical cord perivascular cells (HUCPVCs) are able to provide support to hepatocytes. In the present study we go further into exploring the effects that HUCPVCs have in the functional polarization, and both the internal and external organization, of hepatocytes. Also, we investigate HUCPVC-hepatocyte crosstalk by tracking both the effects of HUCPVCs on hepatocyte transcription factors and those of hepatocytes on the expression of hepatotrophic factors in HUCPVCs. Our results show that HUCPVCs maintain the functional polarity of hepatocytes ex vivo, as judged by the secretion of fluorescein into bile canaliculi, for at least 40 days. Transmission electron microscopy revealed that hepatocytes in coculture organize in an organoid-like structure embedded in extracellular matrix surrounded by HUCPVCs. In coculture, hepatocytes displayed a higher expression of C/EBPα, implicated in maintenance of the mature hepatocyte phenotype, and HUCPVCs upregulated hepatocyte growth factor and Jagged1 indicating that these genes may play important roles in HUCPVC-hepatocyte interactions.
- Transcriptional and posttranscriptional inhibition of HMGCR and PC biosynthesis by geraniol in 2 Hep-G2 cell proliferation linked pathways. [Journal Article]
- Biochem Cell Biol 2013 Jun; 91(5):131-9.
Geraniol, present in the essential oils of many aromatic plants, has in vitro and in vivo antitumor activity against several cell lines. We investigated the effects of geraniol on lipid metabolic pathways involved in Hep-G2 cell proliferation and found that geraniol inhibits the mevalonate pathway, phosphatidylcholine biosynthesis, cell growth, and cell cycle progression (with an arrest occurring at the G0/G1 interphase) and increases apoptosis. The expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting step in cholesterol synthesis, was inhibited at the transcriptional and posttranscriptional levels, as assessed by real-time RT-PCR, Western blots, and [(14)C]HMG-CoA-conversion radioactivity assays. That geraniol decreased cholesterogenesis but increased the incorporation of [(14)C]acetate into other nonsaponifiable metabolites indicated the existence of a second control point between squalene and cholesterol involved in redirecting the flow of cholesterol-derived carbon toward other metabolites of the mevalonate pathway. That exogenous mevalonate failed to restore growth in geraniol-inhibited cells suggests that, in addition to the inhibition of HMGCR, other dose-dependent actions exist through which geraniol can impact the mevalonate pathway and consequently inhibit cell proliferation. These results suggest that geraniol, a nontoxic compound found in many fruits and herbs, exhibits notable potential as a natural agent for combatting cancer and (or) cardiovascular diseases.
- A functional autophagy pathway is required for rapamycin-induced degradation of the Sgs1 helicase in Saccharomyces cerevisiae. [Journal Article]
- Biochem Cell Biol 2013 Jun; 91(5):123-30.
In yeast Saccharomyces cerevisiae, the immunosuppressant rapamycin mimics starvation by inhibiting the kinase Tor1. We recently documented that this treatment triggers a rapid degradation of Sgs1, a helicase involved in several biological processes such as the prevention of genomic instability. Herein, we show that yeast strains deleted for genes ATG2, ATG9, and PEP4, encoding components of the autophagy pathway, prevent rapamycin-induced degradation of Sgs1. We propose that defects in the autophagy pathway prevent degradation of key proteins in the rapamycin response pathway and as a consequence cause resistance to the drug.
- Retinoic acid dependent histone 3 demethylation of the clustered HOX genes during neural differentiation of human embryonic stem cells. [Journal Article, Research Support, Non-U.S. Gov't]
- Biochem Cell Biol 2013 Apr; 91(2):116-22.
Gene activation of HOX clusters is an early event in embryonic development. These genes are highly expressed and active in the vertebrate nervous system. Based on the presence of retinoic acid response elements (RAREs) in the regulatory region of many of the HOX genes, it is deduced that retinoic acid (RA) can influence epigenetic regulation and consequently the expression pattern of HOX during RA-induced differentiation of embryonic model systems. In this investigation, the expression level as well as the epigenetic regulation of several HOX genes of the 4 A-D clusters was analyzed in human embryonic stem cells, and also through their neural induction, in the presence and absence of RA. Expression analysis data significantly showed increased mRNA levels of all examined HOX genes in the presence of RA. Epigenetic analysis of the HOX gene regulatory regions also showed a significant decrease in methylation of histone H3K27 parallel to an absolute preferential incorporation of the demethylase UTX rather than JMJD3 in RA-induced neural differentiated cells. This finding clearly showed the functional role of UTX in epigenetic alteration of HOX clusters during RA-induced neural differentiation; the activity could not be detectable for the demethylase JMJD3 during this developmental process.