Construction of an expressible BAC library of the unculturable insect microorganism, stink bug Plautia stali symbiont, for the search of biologically active and useful symbiont products.

While gene products and metabolites of insect symbiotic bacteria may act as useful resources for insect-microbe studies and medicinal use, it is usually difficult to obtain the insect symbionts to some extent in quantity because most of them are unculturable. In this study, the possibility of using bacterial artificial chromosome (BAC) libraries as a heterologous gene expression tool for the discovery of novel symbiont metabolites was evaluated. A BAC library was constructed from the symbiont purified from the posterior midgut cecum of the stink bug Plautia stali. The BAC library, which consisted of 513 clones with an average insert size of 41 kb, represented greater than five-fold coverage of the genome. The ability of the BAC clones to express plural genes from large-sized insert DNA in Escherichia coli was examined by the growth of BAC-transformed leu operon-deficient DH10B cells on M9 minimal medium supplemented with glucose. Two BAC clones complemented leucine deficiency in DH10B cells; the clones contained the leu operon of the symbiont chromosome. The P. stali symbiont genes introduced into the BAC vector are functional in E. coli, and these genes are expressed in an operon unit. BAC libraries can be used to generate gene product- and metabolite-libraries, facilitating to characterize potential metabolites of the P. stali symbiont.

Expression of genes derived from the genomic DNA fragments of the brown-winged green bug (Plautia stali) symbiont in Escherichia coli.

Many insect species harbour symbiotic microorganisms (symbionts) that are generally unculturable in media. To utilize symbionts as genome resources, we examined whether insect symbiont genes can be expressed in Escherichia coli. 144 plasmid clones were isolated from gene libraries, which were constructed from the genomic DNA of the intestinal bacterial symbiont in the brown-winged green bug, Plautia stali, using an E. coli system. Proteins prepared from a culture of each clone were analysed using SDS-PAGE. A discrete symbiont-specific band was detected in six clones. From the structural analyses of the insert in each clone, the candidate gene encoding the symbiont-specific protein was predicted and the amino acid sequence of the protein was deduced. The amino acid sequence in the N-terminal region of each protein was identical to that deduced from the genomic DNA sequence of the symbiont, but not of the host. The promoter sequences of the symbiont genes, very similar to those of the corresponding E. coli genes, were found in the insert DNA. These findings clearly indicate that genes derived from genomic DNA fragments of the P. stali symbiont can be expressed in E. coli.

Symbiont of the stink bug Plautia stali synthesizes rough-type lipopolysaccharide.

The structures and biosynthesis of lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, have been studied extensively in cultured bacteria such as Escherichia coli. In contrast, little is known about the structures and biosynthesis of the LPS of unculturable bacteria, including insect symbionts, many of which are Gram-negative bacteria. A brown-winged green bug, Plautia stali, is known to harbor a single species of gamma-proteobacterium in the posterior mid-gut caeca. To characterize the features of its LPS, we analyzed the genome sequence of the symbiont, and identified the putative genes involved in LPS synthesis. Genes involved in the synthesis of lipid A and the core oligosaccharide were found in the genome, but waaL, which encodes the O-antigen ligase, was not. Furthermore, we characterized the LPS of this symbiont using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Toll-like receptor 4 (TLR4) stimulation assays. Consistent with the genomic analysis, the SDS-PAGE analysis suggested that the symbiont had rough-type LPS, which lacked the O-antigen. The TLR4 stimulation assay demonstrated that LPS purified from the symbionts activated NF-κB-dependent reporter expression, indicating the existence of a bioactive lipid A portion in the LPS. These results suggest that the P. stali symbiont produces rough-type LPS.

Synergistic effect of green tea catechins on cell growth and apoptosis induction in gastric carcinoma cells.

(-)-Epigallocatechin gallate (EGCG), a major component of green tea catechins, is known to inhibit cell growth and to induce apoptosis in a variety of cultured cells. We examined effects of green tea catechins in cultured cells derived from human gastric carcinoma. The proliferation of four cell lines (MKN-1, MKN-45, MKN-74 and KATO-III) was inhibited with EGCG in a dose-dependent manner. The growth of MKN-45 cells was most efficiently inhibited by the treatment (IC(50): 40 muM EGCG) among the four cell lines, while KATO-III cells were most insensitive (IC(50): 80-150 muM) to the EGCG treatment. In addition, (-)-epicatechin (EC) had a major synergistic effect on the induction of apoptosis in MKN-45 cells treated with EGCG; however it had little effect on the inhibition of cell growth induced by EGCG. To study the molecular mechanisms behind the induction of apoptosis by EGCG, the activity of caspases in MKN-45 cells treated with EGCG was examined. Activity levels of caspases-3, -8 and -9 were elevated in EGCG-treated cells, suggesting that these caspases are involved in the apoptosis induced by EGCG. Furthermore, the synergistic effect of EC with EGCG on the induction of apoptosis was specifically canceled by catalase treatment, suggesting that the synergism involves the extracellular production of reactive oxygen species.

Implication of CDK inhibitors p21 and p27 in the differentiation of HL-60 cells.

All-trans retinoic acid (ATRA) differentiates HL-60 cells into granulocyte-like cells and cellular proliferation is repressed markedly along with the morphological and physiological changes specific for cellular differentiation. To elucidate the implication of cyclin-dependent kinase (CDK) inhibitors during differentiation, we examined the expression of CDK inhibitors during the differentiation of HL-60 cells. The expression of p21 and p27 among the CDK inhibitors we examined increased during the differentiation induced with ATRA. Then, we established stable transformants of HL-60 cells expressing antisense RNA for p21 and p27 and examined the ability of these cells to differentiate into granulocyte-like cells. The extents of fully differentiated HL-60 cells transfected with genes for antisense RNA of p21 and p27 were only 53% and 60%, respectively, whereas 90% of the parental HL-60 cells differentiated by the ATRA treatment. These results suggest that increased expression of CDK inhibitors, p21 and p27, is necessary for the differentiation of HL-60 cells induced with ATRA.

Length polymorphism of thymidylate synthase regulatory region in Chinese populations and evolution of the novel alleles.

The tandemly repeated 28-bp sequence in the 5'-terminal regulatory region of human thymidylate synthase (TSER), which has been reported to be polymorphic in different populations, was surveyed in 668 Chinese from 9 Han groups, 8 ethnic populations, and 36 individuals representing a three-generation pedigree. Amplified fragments were separated by electrophoresis on 4% agarose gel. In addition to the reported double and triple repeats of the 28-bp sequence in TSER, we also detected a novel quintuple repeat in this region. The transient expression activity of TSER with the quintuple repeat is almost the same as that of the reported TSER with the triple repeat. All three alleles of the repeat type (2, 3, and 5) were further confined by sequencing. The frequencies of the TSER allele 2 and 3 were 18.82 and 81% in totally unrelated Chinese samples, respectively, while the frequency of allele 3 was variable in different Chinese populations with a range from 62 to 95%. On the basis of the sequences of the different alleles, the existence of the tandem repeats in each allele might be explained by slipped-strand mispairing during DNA replication.