Carbocisteine normalizes the viscous property of mucus through regulation of fucosylated and sialylated sugar chain on airway mucins.

Almost all of fucose and sialic acid in mucus are found on the mucus glycoproteins (mucins), and these sugar components on mucins are known to be associated with the viscous property of mucus. We have reported some aspects of carbocisteine, a mucoregulatory drug, correcting fucose and sialic acid contents in mucus. At present, carbocisteine's expectorant action of airway mucus is postulated to involve - the regulation of fucose and sialic acid contents on mucins. However little information is available about the relationship between the viscosity and sugar contents on mucins when treated with carbocisteine. To investigate further the mechanism behind the action of carbocisteine, the present study prepared MUC5AC fusion protein which has tandem repeat regions associated with MUC5AC, and evaluated the effects of carbocisteine on tumor necrosis factor (TNF)-alpha-induced increases of mucus viscosity and sialyl-Lewis x-epitopes antigen, an antigen which consists of fucosylated and sialylated sugar chains on the MUC5AC fusion proteins. Carbocisteine inhibited the TNF-alpha-induced increases of the viscosity and sialyl-Lewis x-epitopes on MUC5AC fusion protein. These findings suggest that carbocisteine may normalize the viscosity of mucus through "balancing" of fucose and sialic acid contents on airway mucins.

Identification of differentially expressed genes in hepatocyte/endothelial cell co-culture system.

Cell-cell communication between multiple-cell populations is believed to be important for the activation of many cellular functions. Previously, we showed that, in the rat hepatoma cell line Fao, grown in a layered co-culture system with a human umbilical vein endothelial cell (HUVEC) sheet, the expression of albumin and apolipoprotein A-I increased time-dependently for 10 days and was maintained at a significantly higher level than Fao without the HUVEC sheet. Because the gene-expression profile of hepatocytes and HUVECs under double-layered co-culture has not previously been elucidated, in the present study, we examined the difference in messenger ribonucleic acid expression between Fao or HUVEC monolayer cells and double-layered co-cultured Fao cells/HUVECs using suppression subtractive hybridization. More than 200 transcripts were differentially screened to ensure unique expression. The expression levels of genes in the co-cultured and monolayer cells were determined using SYBR Green I real-time reverse transcription polymerase chain reaction with species-specific primers. We found 23 genes that showed at least a 2-fold difference in expression level. Five hepatocyte-specific genes--alpha1-acidglycoprotein, alpha2-microglobulin, hepcidin, transferrin, and transthyretin--were identified from the Fao cells. Two cell-surface protein genes--bone morphogenetic protein receptor type II and CD82--which may be related to cell-cell communication, showed greater expression in the HUVECs co-cultured with Fao cells. These results indicate that many hepatocyte and endothelial cell functions increase in intensity upon layered co-culture.

Nucleotide sequence of the cryptic plasmid pTT8 from Thermus thermophilus HB8 and isolation and characterization of its high-copy-number mutant.

The complete nucleotide sequence of pTT8, a cryptic plasmid from Thermus thermophilus HB8, was determined. pTT8 was 9328bp long and its G+C content was 69%. pTT8 contained eight putative open reading frames, three of which showed extensive similarities to the plasmid addiction proteins PasA and PasB of pTC-F14 and pAM10.6, and the RepA protein of the ColE2-related plasmids, respectively. During the analysis of pTT8-based plasmid pPP442, which had been obtained during a promoter-screening experiment, we occasionally isolated a plasmid with a relatively high-copy-number. This plasmid, pPP442m, contained a 1025 bp fragment derived from the genome of the HB27 host strain immediately upstream of the putative repA gene. Using the ori region of pPP442m, we constructed an expression vector, pTEV131m, with an estimated high-copy-number of 30-40. This plasmid was stably maintained in T. thermophilus HB27 under nonselective conditions for at least 100 generations. Cloning of the alpha-amylase gene of Bacillus stearothermophilus DY-5 into pTEV131m gave more than twofold production of the enzyme compared with pTEV131, the parental plasmid.