[USF1 Suppresses Expression of Fibrillar Type I, II, and III Collagen and pNP Adamts-3 in Osteosarcoma Cells].

Collagens are the main components of human tissues. Various regulatory factors and cytokines may influence expression levels for collagen-encoding genes, and, therefore, contrubite to some collagen-associated pathologies. In this study, we demonstrate regulatory effects of USF1 on expression of genes encoding fibrillar collagen types I, II, and III in osteoblastic Saos-2 and MG-63 cells. An ectopic expression of the human USF1 led to a decrease in both mRNA and protein expression levels of the collagen-encoding genes mentioned above. ADAMTS-3 is a proteinase primarily responsible for the amino-terminal cleavage of type I and type II collagen precursors. The ADAMTS-3 promoter region contains potential binding sites for USF1. Here we show that an overexpression of USF1 lead to a decrease in ADAMTS-3 mRNA and protein expression levels. In co-transfection studies, USF1 negatively regulated ADAMTS-3 promoter activity. Further, in EMSA studies, we showed that USF1 binds to the ADAMTS-3 promoter region. In conclusion, it seems that ADAMTS-3 and USF1 contribute to the regulation of collagen encoding genes in osteosarcoma.

Differential in vitro inhibitory effects of anticancer drugs on tumor-associated carbonic anhydrase isozymes CA IX and CA XII.

Carbonic anhydrase IX (CA IX) and, to a lesser extent, carbonic anhydrase XII (CA XII) are highly overexpressed in hypoxic tumors. In this study, the inhibitory effects of 11 different anticancer drugs including paclitaxel, amethopterin, etoposide, irinotecan, gemcitabine, 5-fluorouracil, oxaliplatin, epirubicin, cisplatin and carboplatin on the tumor-associated carbonic anhydrase isozymes CA IX and CA XII and cytosolic carbonic anhydrases I and II have been investigated. SX.18MV-R Applied Photophysics stopped-flow instrument was used for measuring the initial velocities for the CO2 hydration reaction catalyzed by different CA isozymes, by following the change in the absorbance of a pH indicator. CA IX and CA XII were the most affected by carboplatin and cisplatin amongst the panel of anticancer drugs. Moreover, the cytosolic carbonic anhydrases I and II can also be affected. Consequently, CA IX and CA XII are interesting targets for anticancer drug development, although more selective and powerful CA inhibitors could prove useful for elucidating the role of the protein in hypoxic cancers, for controlling the pH imbalance in tumor cells and for developing diagnostic or therapeutic applications for the management of hypoxic tumors, generally unresponsive to classical chemo- and radiotherapy.

Analysis of the Xenopus laevis CCAAT-enhancer binding protein alpha gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1.

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the regulation of gene expression during differentiation, development and disease. Autoregulation is relatively common in the modulation of C/EBP gene expression and the murine and human C/EBPalpha genes have been shown to be auto-activated by different mechanisms. In the light of this finding, it is essential that autoregulation of C/EBPalpha genes from a wider range of different species be investigated in order to gauge the degree of commonality, or otherwise, that may exist. We report here studies that investigate the regulation of the Xenopus laevis C/EBPalpha gene (xC/EBPalpha). The -1131/+41 promoter region was capable of directing high levels of expression in both the human hepatoma Hep3B and the Xenopus kidney epithelial A6 cell lines, and was auto-activated by expression vectors specifying for xC/EBPalpha or xC/EBPss. Deletion analysis showed that the -321/+41 sequence was sufficient for both the constitutive promoter activity and auto-activation and electrophoretic mobility shift assays identified the interaction of C/EBPs and Sp1 to this region. Although deletion of either the C/EBP or the Sp1 site drastically reduced the xC/EBPalpha promoter activity, multimers of only the C/EBP site could confer autoregulation to a heterologous SV40 promoter. These results indicate that, in contrast to the human promoter and in common with the murine gene, the xC/EBPalpha promoter was subject to direct autoregulation. In addition, we demonstrate a novel species-specific action of Sp1 in the regulation of C/EBPalpha expression, with the factor able to repress the murine promoter but activate the Xenopus gene.

Characterisation and developmental regulation of the Xenopus laevis CCAAT-enhancer binding protein beta gene.

We report here the cloning, characterisation and developmental expression profile of the Xenopus laevis CCAAT-enhancer binding protein beta (xC/EBPbeta) gene. The protein synthesised from the xC/EBPbeta gene interacts specifically with a C/EBP-recognition sequence and acts as a transcriptional activator. Several conserved regions are present in the xC/EBPbeta sequence, including the basic region, leucine zipper, activation domains, three in-frame AUG codons, and a consensus site for mitogen activated protein kinase. The corresponding mRNA is present at high levels in the kidney, liver, lung, muscle and adipose tissue, and at low levels in the ovary, brain and heart. Although the xC/EBPbeta mRNA and protein are present throughout embryogenesis, there is a biphasic increase in their expression levels during development. Whole-mount in situ hybridisation shows a restricted spatial expression profile of the xC/EBPbeta gene during early embryogenesis, with transcripts present around the blastopore lip and in the endodermal cells at the mid-gastrula stage, and, the whole dorsal side at the neurula and early tailbud stage. The expression domain becomes almost ubiquitous during later embryonic development, and includes the brain, spinal cord, somites and regions that give rise to the liver and the heart.

Molecular characterization of zeta class glutathione S-transferases from Pinus brutia Ten.

Glutathione transferases (GSTs; EC 2.5.1.18) play important roles in stress tolerance and metabolic detoxification in plants.In higher plants, studies on GSTs have focussed largely on agricultural plants. There is restricted information about molecular characterization of GSTs in gymnosperms. To date, only tau class GST enzymes have been characterized from some pinus species. For the first time, the present study reports cloning and molecular characterization of two zeta class GST genes, namely PbGSTZ1 and PbGSTZ2 from Pinus brutia Ten., which is an economically important pine native to the eastern Mediterranean region and have to cope with several environmental stress conditions. The PbGSTZ1 gene was isolated from cDNA, whereas PbGSTZ2 was isolated from genomic DNA. Sequence analysis of PbGSTZ1 and PbGSTZ2 revealed the presence of an open reading frame of 226 amino acids with typical consensus sequences of the zeta class plant GSTs. Protein and secondary structure prediction analysis of two zeta class PbGSTZs have shared common features of other plant zeta class GSTs. Genomic clone, PbGSTZ2 gene, is unexpectedly intronless. Extensive sequence analysis of PbGSTZ2, with cDNA clone, PbGSTZ1, revealed 87% identity at nucleotide and 81% identity at amino acid levels with 41 amino acids differences suggesting that genomic PbGSTZ2 gene might be an allelic or a paralogue version of PbGSTZ1.

Sequence and expression analysis of a novel Xenopus laevis cDNA that encodes a protein similar to bacterial and chloroplast ribosomal protein L24.

We report here the cloning and the characterization of a Xenopus laevis cDNA that encodes a basic protein of 276 amino acids with a central core region, which shows a substantial degree of homology to bacterial and chloroplast ribosomal protein L24, and additional diverged N- and C-terminal polypeptide extensions. The N-terminal extension displays similarities to the mitochondrial targetting sequence, thereby suggesting that the cDNA probably codes for a mitochondrial ribosomal protein. Although the gene was expressed ubiquitously, at fairly constant levels, during embryogenesis, the abundance of the transcripts in the different tissues varies with the mRNA levels in the kidney, adipose tissue, muscle and liver being greater than that present in the brain, heart, ovary and lung.

Amphenicol and macrolide derived antibiotics inhibit paraoxonase enzyme activity in human serum and human hepatoma cells (HepG2) in vitro.

Human serum paraoxonase (hPON1) was separately purified by ammonium sulfate precipitation and hydrophobic interaction chromatography. The in vitro effects of commonly used antibiotics, namely clarithromycin and chloramphenicol, on purified human serum paraoxonase enzyme activity (serum hPON1) and human hepatoma (HepG2) cell paraoxonase enzyme activity (liver hPON1) were determined. Serum hPON1 and liver hPON1 were determined using paraoxon as a substrate and IC(50) values of these drugs exhibiting inhibition effects were found from graphs of hydratase activity (%) by plotting concentration of the drugs. We determined that chloramphenicol and clarithromycin were effective inhibitors of serum hPON1.