Anti-GGH (aa 236-264) polyclonal antibody

The ggh-A gene, encoding a 1,4-beta-D-glucan glucohydrolase/beta-glucosidase, of Microbispora bispora (Mb) was subcloned and expressed from a 4.0-kb XhoI DNA fragment. The nucleotide sequence of this fragment was determined. Analysis of the sequence revealed one open reading frame (ORF) which encodes a 986-amino-acid (aa) protein with a calculated molecular weight of 107 510. The ggh-A ORF has features typical of an actinomycete gene including high GC content (70.5%) and corresponding biased codon usage. Comparison of the aa sequence of the Mb 1,4-beta-D-glucan glucohydrolase (Mbggh-A) with other glycosidases reveals high overall homology to several beta-glucosidases and a 1,4-beta-D-glucan glucohydrolase belonging to the glycosyl hydrolase family 3. The aa sequence alignments of Mbggh-A and beta-glucosidases show that the active site region potentially involves two Asp residues. The aa sequence homology studies revealed a potential two-domain structure for Mbggh-A and other beta-glucosidases. Furthermore, Mbggh-A has localized homology to a cellulose-binding domain present in some xylanases. This report is significant, as, to date, 1,4-beta-D-glucan glucohydrolases have rarely been reported, though they are assumed to have a critical role in cellulolysis.

The chemoresistance of non-small cell lung cancer (NSCLC) that occurs in docetaxel (DOC) chemotherapy substantially decreases the survival of patients. To overcome DOC-induced chemoresistance, we established DOC-selected A549 lung cancer sublines (A549/D16 and A549/D32) and revealed that both sublines were cross-resistant to vincristine (VCR) and doxorubicin (DXR). Notably, both sublines were more sensitive to pemetrexed (PEM) than parental cells according to MTT and clonogenic assays. The expression levels of thymidylate synthase (TS) and gamma-glutamyl hydrolase (GGH) were downregulated in DOC-resistant sublines. When exogenous TS was overexpressed in A549/D16 cells, PEM sensitivity was significantly decreased, however it was not decreased by overexpression of exogenous GGH. PEM treatment induced more apoptotic sub-G1 cells in both DOC-resistant sublines and in the in vivo PEM sensitivities of A549/D16 cells. These findings were further confirmed by a xenografted tumor model. To unmask the mediator of TS downregulation, we investigated human lung cancer cell lines that have various TP53 statuses using DOC treatment. The level of TS protein was significantly decreased in wild-type TP53-containing cells with DOC treatment; TS expression levels were not affected in mutant-TP53 and TP53-null cells under the same conditions. Furthermore, when the expression of TP53 was inhibited in A549 cells, the expression level of TS was increased. Our data indicated that DOC activated wild-type TP53 and suppressed TS expression under continuous DOC exposure. Therefore, the expression of TS remained at low levels in DOC-resistant A549 cancer cells. Our data revealed that for lung cancer with DOC resistance and wild-type TP53 status, the administration of PEM as a second-line agent to overcome DOC-resistance may benefit patients.