Molecular cloning of a cytosolic ascorbate peroxidase cDNA from cell cultures of sweetpotato and its expression in response to stress

A cDNA encoding a cytosolic ascorbate peroxidase (APX), swAPX1 , was isolated from cell cultures of sweetpotato (Ipomoea batatas) by cDNA library screening, and its expression in the context of various environmental stresses was investigated. swAPX1 contains an ORF of 250 amino acids (27.5 kDa) encoding a protein with a pI value of 5.32. The swAPX1 ORF does not code for a transit peptide, suggesting that the product is a cytosolic isoform. RNA blot analysis showed that swAPX1 gene is expressed in cultured cells and mature leaves, but not in stems, non-storage or storage roots of sweetpotato. The level of swAPX1 RNA progressively increased during cell growth in suspension cultures. In leaf tissues, the gene responded differentially to various abiotic stresses, as revealed by RT-PCR analysis. swAPX1 was highly induced in leaves by wounding, and treatment with methyl viologen (50 M), hydrogen peroxide (440 mM), abscisic acid (ABA; 100 M) or exposure to high temperature (37°C). In addition, the gene was strongly induced in the leaves following inoculation with a bacterial pathogen (Pectobacterium chrysanthemi). These results indicate that swAPX1 may be involved in hydrogen peroxide-detoxification and thus help to overcome the oxidative stress induced by abiotic and biotic stresses.Communicated by G. Jürgens     

A bound water molecule is crucial in initiating autocatalytic precursor activation in an N-terminal hydrolase

Cephalosporin acylase is a member of the N-terminal hydrolase family, which is activated from an inactive precursor by autoproteolytic processing to generate a new N-terminal nucleophile Ser or Thr. The gene structure of the precursor cephalosporin acylases generally consists of a signal peptide that is followed by an alpha-subunit, a spacer sequence, and a beta-subunit. The cephalosporin acylase precursor is post-translationally modified into an active heterodimeric enzyme with alpha- and beta-subunits, first by intramolecular cleavage and, second, by intermolecular cleavage. Intramolecular autocatalytic proteolysis is initiated by nucleophilic attack of the residue Ser-1beta onto the adjacent scissile carbonyl carbon. This study determined the precursor structure after disabling the intramolecular cleavage. This study also provides experimental evidence showing that a conserved water molecule plays an important role in assisting the polarization of the OG atom of Ser-1beta to generate a strong nucleophile and to direct the OG atom of the Ser-1beta to a target carbonyl carbon. Intramolecular proteolysis is disabled as a result of a mutation of the residues causing conformational distortion to the active site. This is because distortion affects the existence of the catalytically crucial water at the proper position. This study provides the first evidence showing that a bound water molecule plays a critical role in initiating intramolecular cleavage in the post-translational modification of the precursor enzyme.     

Induction of a cytosolic pyruvate kinase 1 gene during the resistance response to Tobacco mosaic virus in Capsicum annuum

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P₀. Previously, to elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P₀ and genes specifically up-regulated during the HR were isolated by microarray analysis. One of the clones, Capsicum annuum cytosolic pyruvate kinase 1 (CaPK _c 1) gene was increased specifically in the incompatible interaction with TMV-P₀. The expression of CaPK _c 1 gene was also triggered not only by various hormones such as salicylic acid (SA), ethylene, and methyl jasmonate (MeJA), but also NaCl and wounding. These results suggest that CaPK _c 1 responds to several defense-related abiotic stresses in addition to TMV infection. The nucleotide sequence data reported in this paper were submitted to the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number DQ114474.     

cAMP differentially regulates gamma-globin gene expression in erythroleukemic cells and primary erythroblasts through c-Myb expression

Our previous studies demonstrated roles of cyclic nucleotides in gamma-globin gene expression. We recently found that, upon activation of the cAMP pathway, expression of the gamma-globin gene is inhibited in K562 cells but induced in adult erythroblasts. Here we show that c-Myb, a proto-oncogene product that plays a role in cell growth and differentiation, is involved in the cAMP-mediated differential regulation of gamma-globin gene expression in K562 cells and primary erythroblasts. Our studies found that c-Myb is expressed at a high level in K562 cells compared to primary erythroblasts, and that c-Myb expression is further increased following the treatment with forskolin, an adenylate cyclase activator. The induction of gamma-globin gene expression was also inhibited in K562 cells by raising the levels of c-Myb expression. Importantly, forskolin-induced erythroid differentiation in K562 cells, as determined by the expression of glycophorins and CD71, suggesting that high-level expression of c-Myb may not be sufficient to inhibit the differentiation of erythroid cells. In contrast, c-Myb was not expressed in adult erythroblasts treated with forskolin and primary erythroblasts may lack the c-Myb-mediated inhibitory mechanism for gamma-globin gene expression. Together, these results show that the cAMP pathway blocks gamma-globin gene expression in K562 cells by increasing c-Myb expression and c-Myb plays a role in defining the mode of response of the gamma-globin gene to fetal hemoglobin inducers in erythroid cells.     

Intraspecific molecular phylogeny, genetic variation and phylogeography of Reticulitermes speratus (Isoptera: Rhinotermitidae)

Population structure was investigated in Reticulitermes speratus populations in the Korean Peninsula and the Japanese Archipelago. All trees derived from analyses of the combined sequence dataset of two mitochondrial genes, COII and COIII, showed that R. speratus populations cluster into two major clades comprising the Korean/southern Japanese populations and the north-ern Japanese populations. Analysis of population ge-netic structure showed strong genetic partitioning between populations of the two clades. To understand historical migration routes and current distributions, the phylogeographic history of R. speratus was inferred from intra-/interspecific phylogeny and diver-gence times estimated between the clades of the phylogenetic tree. The estimated migration route and divergence time of ancestral R. speratus are congruent with recent paleogeographic hypotheses involving land-bridge connections between the Asian continent and the Japanese Archipelago. We suggest that ancestral R. speratus separated into northern and southern Japanese populations after its migration into the Japanese main islands from East China during the early Pleistocene via the East China Sea basin, which may have been exposed during that period. The Korean populations seem to have diverged recently from southern Japanese populations; this may explain the current distribution of R. speratus in the Japanese Arachipelago, and account for why it is restricted to northern areas of the Tokara Strait.     

An intracellular antibody can suppress tumorigenicity in Hepatitis B virus X-expressing cells

Although the hepatitis B virus X protein (HBx) is thought to play a causative role in the development of hepatocellular carcinoma, it is not yet known whether interfering with HBx function may affect the cellular transformation of HBx-expressing tumor cells. To address this question, we adopted an intracellular antibody fragment expression approach to block the function of HBx. Expression of a single-chain variable fragment (scFv) specific to HBx (designated as H7scFv) inhibited HBx-dependent cellular transactivation. Furthermore, H7scFv suppressed the growth of HBx-expressing tumor cells in both soft agar and nude mice. The suppressive effect of H7scFv on tumorigenicity appeared not to be mediated by inhibition of HBx-induced growth stimulation since the growth rate of these cells was not affected significantly by H7scFv expression. In conclusion, these data suggest that the HBx-dependent transformed phenotype is reversible and that HBx may be a good molecular target for the treatment of HBV-related tumors.This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health& Welfare, Republic of Korea (03-PJ1-PG3-20200–0023)     

Enzymatic preparation of maltohexaose, maltoheptaose, and maltooctaose by the preferential cyclomaltooligosaccharide (cyclodextrin) ring-opening reaction of Pyrococcus furiosus thermostable amylase

Specific-length maltooligosaccharides, particularly maltohexaose, maltoheptaose, and maltooctaose, were prepared from cyclomaltooligosaccharides (cyclodextrins, CDs) by the preferential cyclodextrin ring-opening reaction of an amylolytic enzyme from Pyrococcus furiosus. The enzyme primarily produces maltohexaose, maltoheptaose, and maltooctaose by hydrolyzing alpha-, beta-, and gamma-CD, respectively. This study aims to develop a high-efficiency synthesis of specific maltooligosaccharides at high-purity. [formula: see text]     

Xanthorrhizol, a natural sesquiterpenoid from Curcuma xanthorrhiza, has an anti-metastatic potential in experimental mouse lung metastasis model

Xanthorrhizol is a sesquiterpenoid compound isolated from the rhizome of Curcuma xanthorrhiza. In this study, the anti-metastatic activity of xanthorrhizol was evaluated by using an in vivo mouse lung metastasis model and a tumor mass formation assay. Interestingly, xanthorrhizol dramatically inhibited the formation of tumor nodules in the lung tissue and the intra-abdominal tumor mass formation. Next, to examine the mechanism of the anti-metastatic action of xanthorrhizol in the mouse lung metastasis, expression patterns of the several intracellular signaling molecules were evaluated using the lung tissues with tumor nodules. Higher expression levels of cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and phosphorylated extracellular signal-regulated kinase (ERK) were observed in the metastatic group compared with control, but these were attenuated by the treatment of xanthorrhizol. In conclusion, xanthorrhizol exerts anti-metastatic activity in vivo and this effect could be highly linked to the metastasis-related multiplex signal pathway including ERK, COX-2, and MMP-9.     

EGF-induced inhibition of glucose transport is mediated by PKC and MAPK signal pathways in primary cultured chicken hepatocytes

EGF is a regulator of a wide variety of processes in various cell systems. Hepatocytes are important sites in the body's metabolism and function. Glucose transporter 2 (GLUT2) is a major transporter that is expressed strongly in hepatocytes. Therefore, this study examined the effect of EGF on GLUT2 and its related signal cascades in primary cultured chicken hepatocytes. EGF decreased [(3)H]deoxyglucose uptake in a dose- and time-dependent manner (>10 ng/ml, 2 h). AG-1478 (an EGF receptor antagonist) and genistein and herbimycin A (tyrosine kinase inhibitors) blocked the EGF-induced decrease in [(3)H]deoxyglucose uptake, which correlated with the GLUT2 expression level. In addition, the EGF-induced decrease in GLUT2 protein expression was inhibited by staurosporine, H-7, or bisindolylmaleimide I (PKC inhibitors), PD-98059 (a MEK inhibitor), SB-203580 (a p38 MAPK inhibitor), and SP-600125 (a JNK inhibitor), suggesting a role of both PKC and MAPKs (p44/42 MAPK, p38 MAPK, and JNK). In particular, EGF increased the translocation of PKC isoforms (PKC-alpha, -beta(1), -gamma, -delta, and -zeta) from the cytosol to the membrane fraction and increased the activation of p44/42 MAPK, p38 MAPK, and JNK. Moreover, PKC inhibitors blocked the EGF-induced phosphorylation of three MAPKs. In conclusion, EGF decreases the GLUT2 expression level via the PKC-MAPK signal cascade in chicken hepatocytes.