Transcriptional activation of the human claudin-18 gene promoter through two AP-1 motifs in PMA-stimulated MKN45 gastric cancer cells

Claudin-18 (CLDN18), a member of the claudin family of proteins that are structural components of tight junctions, has two alternatively spliced variants, claudin-18a1 and claudin-18a2, which are highly expressed in lung and stomach, respectively. Downregulation of claudin-18a2 is associated with gastric cancers of an intestinal phenotype; however, the mechanisms regulating its expression have not been defined. Here, we found that phorbol 12-myristate 13-acetate (PMA) treatment of MKN45 human gastric cancer cell line increased claudin-18a2 expression. In addition, this study aimed to characterize the human CLDN18a2 promoter. Using reporter gene assays and deletion analysis, we mapped the critical promoter region of the PMA-stimulated claudin-18a2 expression to the -923/-286 region. Electrophoretic mobility shift assays and mutational analyses revealed that two activator protein (AP)-1 binding sites played an important role in the expression of claudin-18a2 in PMA-stimulated MKN45 cells. Protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors suppressed the upregulation of claudin-18a2. These results indicate that the PKC/MAPK/AP-1 dependent pathway regulates claudin-18a2 expression in gastric cells.     

Lyme Disease Borrelia spp. in Ticks and Rodents from Northwestern China

In May 1999, field surveys of Lyme disease spirochetes were conducted around the Tianshan Mountains in Xinjiang Uygur Autonomous Region in northwestern People's Republic of China. Ixodes persulcatus ticks were obtained in a Tianchi Lake valley with primary forest, while the tick fauna was poor in the semidesert or at higher altitudes in this region. Species identities were confirmed by molecular analysis in which an internal transcribed spacer sequence was used. Of 55 adult ticks, 22 (40%) were positive for spirochetes as determined by Barbour-Stoenner-Kelly culture passages. In addition, some rodents, including Apodemus uralensis (5 of 14 animals) and Cricetulus longicaudatus (the only animal examined), and some immature stages of I. persulcatus (4 of 11 ticks) that had fed on A. uralensis were positive for spirochetes. Based on 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis and reactivity with monoclonal antibodies, 35 cultures (including double isolation cultures) were identified as Borrelia garinii (20 isolates, including 9 Eurasian pattern B isolates and 11 Asian pattern C isolates), Borrelia afzelii (10 pattern D isolates), and mixed cultures (5 cultures, including isolates that produced B. garinii patterns B and C plus B. afzelii pattern D). These findings revealed that Lyme disease pathogens are distributed in the mountainous areas in northwestern China even though it is an arid region, and they also confirmed the specific relationship between I. persulcatus and genetic patterns of Borrelia spp. on the Asian continent.     

Dissociation of Bax from a Bcl-2/Bax heterodimer triggered by phosphorylation of serine 70 of Bcl-2.

Serine 70 in the loop region of Bcl-2 is specifically phosphorylated by paclitaxel-treatment in tumor cells and BHK cells expressing Bcl-2. The phosphorylation of serine 70 of Bcl-2 (pS70-Bcl-2) peaks 24 to 48 h after paclitaxel treatment and accelerates apoptosis. Phosphorylation is effectively inhibited in the presence of actinomycin D or cycloheximide, which restore cell viability to the same level as control cells not expressing Bcl-2. These results indicate that paclitaxel-induced kinase(s) and/or its activator(s) are synthesized de novo and play an important role in paclitaxel-induced apoptosis by phosphorylating Bcl-2. In binding assays using the phosphorylation-specific antibody against pS70-Bcl-2, the induction of serine 70 phosphorylation 70 results in a loss of the binding ability of Bcl-2 to Bax, a pro-apoptotic partner, and induces subsequent cell death. When the pS70-Bcl-2 antibody was added to human breast cancer tissue, serine 70 phosphorylation was also detected, even prior to treatment with anticancer agents. Further study of breast cancers revealed 83% of tumors with high pS70-Bcl-2 expression responded to paclitaxel or docetaxel treatment, whereas 57% of those with low expression not respond. These findings suggest that pS70-Bcl-2 might be a predictive factor for prognosis and sensitivity to paclitaxel treatment for breast cancer.     

Development of free radical products during the greening reaction of caffeic acid esters (or chlorogenic acid) and a primary amino compound.

ESR spectra were measured directly on a marked greening reaction mixture of Et-caffeate and a primary amino compound in alkali solution under aeration. A clear hyperfine structure was commonly detected early in the greening reaction with different amino compounds. Its hyperfine spectrum split into seven peaks was analyzed and found to be due to the oxidized free radical product of the Et-caffeate using an authentic sample system. Another type of hyperfine ESR spectrum was observed later in the reaction, and was altered with different amino compounds. The hyperfine structure for n-butylamine split into 12 lines. The latter type of free radical products were assumed to be a semiquinone type radical compound of the trihydroxy benzacridine derivative, which was identified as the principal structure of the green and yellow pigments formed by this greening reaction system. A formation mechanism of the green pigment and related products involving these free radical products is proposed.     

Determination of a new thymidine phosphorylase inhibitor, TPI, in dog and rat plasma by reversed-phase ion-pair high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of a new thymidine phosphorylase inhibitor, TPI, in dog and rat plasma is described. TPI was isolated from biological samples by solid-phase extraction on Bond Elut PRS columns. Chromatographic separation was achieved on a C₁₈ column using a mobile phase consisting of acetonitrile–10 mM acetate buffer (pH 4.3) including hexanesulfonate, with UV detection at 276 nm. This method has been validated across the range of 50–50 000 ng/ml using a 0.1-ml plasma volume. The mean recoveries from spiked plasma were 93% for dog and 94% for rat, respectively. The accuracy, precision and specificity of the method were demonstrated to be acceptable, and it was applied to the toxicokinetic study of TPI in rats.     

Increase of ceramides and its inhibition by catalase during chemically induced apoptosis of HL-60 cells determined by electrospray ionization tandem mass spectrometry.

A change in all ceramide species during chemically induced apoptosis of HL-60 cells was determined using electrospray tandem mass spectrometry. Ceramides of C16:0, C24:1 and C26:1 increased significantly 4 h after the addition of actinomycin D, when the activation of caspase-3 was maximal. Addition of catalase, which inhibited apoptosis, the activation of caspase-3-like protease, and the release of cytochrome c from mitochondria to cytosol caused by actinomycin D or daunorubicin, significantly inhibited the increase of these ceramides at all time points. Ceramides of C16:0, C24:1, C18:0, C22:1 and C26:1 increased significantly 4 h after the addition of daunorubicin to HL-60 cells. Catalase also significantly inhibited the increase of these ceramides induced by daunorubicin. Based on time courses of events and inhibition studies, it is concluded that the increase of ceramides is downstream from both generation of hydrogen peroxide and cytochrome c release from mitochondria and takes place almost simultaneously with the activation of caspase-3.     

Expression of the Pib rice-blast-resistance gene family is up-regulated by environmental conditions favouring infection and by chemical signals that trigger secondary plant defences

The rice blast resistance gene Pib is a member of the nucleotide binding site (NBS) and leucine-rich repeat (LRR) class of plant disease resistance (R) genes and belongs to a small gene family. We describe here the isolation and characterization of a Pib homologue (PibH8), and extensive investigation of the expression of the Pib gene family (Pib, PibH8, HPibH8-1, HPibH8-2) under various environmental and chemical treatments. PibH8 shows 42% identity and 60% similarity to Pib and, like Pib, has a duplication of the kinase 1a, 2, and 3a motifs of the NBS region in the N-terminal half of the protein. Interestingly, genes of the Pib family exhibit a diurnal rhythm of expression. RNA gel blot analysis revealed that their expression was regulated dramatically by environmental signals. such as temperature, light and water availability. Their expression was also induced by chemical treatments, such as jasmonic acid, salicylic acid, ethylene and probenazole. Our findings suggest that expression of the Pib gene family is up-regulated by environmental conditions that would favour pathogen infection. This may reflect the evolution of anticipatory control of R gene expression.     

Transformation of <Emphasis Type="Italic">Spirulina platensis </Emphasis>Strain C1 (<Emphasis Type="Italic">Arthrospira</Emphasis> sp. PCC9438) with Tn5 Transposase–Transposon DNA–Cation Liposome Complex

Spirulina platensis is one of the most commercially important species of microalgae. Thus, it is an attractive candidate for genetic manipulation and the development of novel practical applications. However, this process is hampered by the absence of a stable gene transfer system, specifically the limited number of suitable vectors and transformation methods available for this organism. Artificial transposon systems developed by extracting the essential elements from natural transposons have been extensively studied, and recently a mutated transposase and transposon system was reported to improve transformation efficiency by electroporation. We applied a modified transformation strategy using a natural Tn5 transposon, transposase, and cation liposome complex by electroporation to improve the transformation efficiency for Spirulina platensis strain C1 (Arthrospira sp. PCC9438). Aggregation of cells became visible after 3 weeks during 2.0 g/ml chloramphenicol selection, and growth continued for more than 12 months. Transfected chloramphenicol acetyltransferase (CAT) genes were detected in the genomic DNA by Southern hybridization. Transformed cells demonstrated CAT activity, but non-transformed cells did not.     

The Origin of Cluster N2 of the Energy-Transducing NADH–Quinone Oxidoreductase: Comparisons of Phylogenetically Related Enzymes

NADH–quinone (Q) oxidoreductase is a large and complex redox proton pump, which utilizes the free energy derived from oxidation of NADH with lipophilic electron/proton carrier Q to translocate protons across the membrane to generate an electrochemical proton gradient ( ). Although its molecular mechanism is largely unknown, recent biochemical, biophysical, and molecular biological studies have revealed that particular subunits and cofactors play an essential role in the energy-coupling reaction. Based on these latest experimental data, we exhaustively analyzed the sequence information available from evolutionarily related enzymes such as [NiFe] hydrogenases. We found significant and conserved sequence differences in the PSST/Nqo6/NuoB, 49kDa/Nqo4/NuoD, and ND1/Nqo8/NuoH subunit homologs between complex I/NDH-1 and [NiFe] hydrogenases. The alterations, especially in the postulated ligand motif for cluster N2 in the PSST/Nqo6/NuoB subunits, appear to be evolutionarily important in determining the physiological function of complex I/NDH-1. These observations led us to propose a hypothetical evolutionary scheme: during the course of evolution, drastic changes have occurred in the putative cluster N2 binding site in the PSST/Nqo6/NuoB subunit and the progenitors of complex I/NDH-1 have concurrently become to utilize a lipophilic electron/proton carrier such as Q as its physiological substrate. This scheme provides new insights into the structure and function relationship of complex I/NDH-1 and may help us understand its energy-coupling mechanism.