Complete Amino Acid Sequence of a Copper/Zinc-Superoxide Dismutase from Ginger Rhizome

Superoxide dismutase (SOD) is an antioxidant enzyme protecting cells from oxidative stress. Ginger (Zingiber officinale) is known for its antioxidant properties, however, there are no data on SODs from ginger rhizomes. In this study, we purified SOD from the rhizome of Z. officinale (Zo-SOD) and determined its complete amino acid sequence using N terminal sequencing, amino acid analysis, and de novo sequencing by tandem mass spectrometry. Zo-SOD consists of 151 amino acids with two signature Cu/Zn-SOD motifs and has high similarity to other plant Cu/Zn-SODs. Multiple sequence alignment showed that Cu/Zn-binding residues and cysteines forming a disulfide bond, which are highly conserved in Cu/Zn-SODs, are also present in Zo-SOD. Phylogenetic analysis revealed that plant Cu/Zn-SODs clustered into distinct chloroplastic, cytoplasmic, and intermediate groups. Among them, only chloroplastic enzymes carried amino acid substitutions in the region functionally important for enzymatic activity, suggesting that chloroplastic SODs may have a function distinct from those of SODs localized in other subcellular compartments. The nucleotide sequence of the Zo-SOD coding region was obtained by reverse-translation, and the gene was synthesized, cloned, and expressed. The recombinant Zo-SOD demonstrated pH stability in the range of 5–10, which is similar to other reported Cu/Zn-SODs, and thermal stability in the range of 10–60?°C, which is higher than that for most plant Cu/Zn-SODs but lower compared to the enzyme from a Z. officinale relative Curcuma aromatica.     

Characterization of resistance mechanism in transgenic Nicotiana benthamiana containing Turnip crinkle virus coat protein

Two transgenic lines, of Nicotiana benthamiana expressing Turnip crinkle virus (TCV)-coat protein (CP) gene with contrasting phenotype, the highest (#3) and the lowest (#18) CP expressers, were selected and challenged with the homologous TCV. The former, the highest expresser, showed nearly five times more CP expression than the latter. Progenies of #3 and #18 lines showed 30 and 100% infection rates, respectively. The infected progenies of #3 line showed mild and delayed symptom with TCV. This is a coat protein-mediated resistance (CP-MR), and its resistance level is directly proportional to CP transgene expression. However, CP-MR of the transgenic plants was specific only for TCV but not for heterologous viruses. Newly growing leaves of those infected progenies of #3 line did not show any visible symptoms at 4-week post-inoculation (wpi) with TCV, suggesting a reversal from infection. This was confirmed by RT-PCR analysis with the disappearance of the target at 4 wpi. This is a case of RNA-mediated resistance, and a threshold level of transgene expression may be needed to achieve the silent state. To confirm the RNA silencing, we infiltrated Agrobacterium carrying TCV-CP into leaves of progenies of #3 and performed RT-PCR analysis. The results indicate that TCV-CP’s suppressor activity against RNA silencing itself can be silenced by the homologous expression of TCV-CP in the transgenic plants. The transgenic plants containing TCV-CP seem to be a model system to study viral protection mediated by a combination of protein and RNA silencing. Ayyappan Vasudevan and Tae-Kyun Oh have contributed equally in this study.     

Specific detection of buckwheat residues in processed foods by polymerase chain reaction

A specific and qualitative detection method for buckwheat in foods using the polymerase chain reaction (PCR) was developed. Trace amounts of buckwheat in commercial food products were qualitatively detected by this method. It should be reliable for detecting buckwheat residues in processed foods and practical for monitoring the labeling system for allergenic food materials.     

Real-time PCR method using capturing oligo-immobilized PCR tubes to determine the specific gene for soybean and genetically modified soybean in food matrices

A new real-time PCR method using capturing oligo-immobilized PCR tubes is described. This method was used to detect specific genes for soybean and genetically modified (GM) soybean in food matrices. In a standard reaction using soybean genomic DNA and a capturing oligo for the lectin gene (Le1) immobilized on the tube, we examined the effects of such hybridization conditions as the location, length, and amount of the capturing oligo, and the incubation time and temperature. Under optimized conditions, the copy number of Le1 was determined in a concentration-dependent manner from soybean genomic DNA and soybean lysate (DNA 10-1000 ng, r=0.99; lysate 1-100%, r=0.99). The copy number of a Roundup Ready soybean (RRS) gene was also successfully detected in a concentration-dependent manner (1-100%, r=0.99) from GM soybean lysate, using PCR tubes with an immobilized capturing oligo for the transgene. Our data indicate that this is a rapid and simple method to determine specific genes for soybean and GM soybean in food matrices.     

Absolute configuration of (+)-pinoresinol 4-O-[6″-O-galloyl]-β-d-glucopyranoside, macarangiosides E, and F isolated from the leaves of Macaranga tanarius

A lignan glucoside, (+)-pinoresinol 4-O-[6″-O-galloyl]-β-d-glucopyranoside (1), and two megastigmane glucosides, named macarangiosides E and F (2, 3), together with 15 known compounds (4–18) were isolated from leaves of Macaranga tanarius (L.) Müll.-Arg. (Euphorbiaceae). Their structures were elucidated by spectroscopic and chemical analyses. In addition, the absolute stereochemistry of macarangiosides B and C isolated previously from the same plant was also determined for the first time. Compounds 1 and 2 were galloylated on glucose and possessed potent DPPH radical-scavenging activity.     

Association between antibody response against cytomegalovirus strain-specific glycoprotein H epitopes and HLA-DR

The gH of CMV is a major target for strain-specific neutralizing antibodies. To verify whether there is a correlation between HLA-DR type and strain-specific antibodies, antibodies against CMV gH in potential donors and recipients for renal transplantation were investigated. Among 471 subjects, 404 (86%) showed reactivity to CMV gH, but no antibodies against gH were detected in 67 (14%) subjects. The positive rates were over 80% in most HLA subpopulations. Fewer subjects with HLA-DR10 and DR11 had antibodies to CMV gH than did those without HLA-DR10 and DR11. HLA-DR10 and DR11 may be associated with fewer/non-responders for strain-specific neutralizing antibodies.     

Structural Basis for the Kexin-like Serine Protease from Aeromonas sobria as Sepsis-causing Factor

The anaerobic bacterium Aeromonas sobria is known to cause potentially lethal septic shock. We recently proposed that A. sobria serine protease (ASP) is a sepsis-related factor that induces vascular leakage, reductions in blood pressure via kinin release, and clotting via activation of prothrombin. ASP preferentially cleaves peptide bonds that follow dibasic amino acid residues, as do Kex2 (Saccharomyces cerevisiae serine protease) and furin, which are representative kexin family proteases. Here, we revealed the crystal structure of ASP at 1.65 Å resolution using the multiple isomorphous replacement method with anomalous scattering. Although the overall structure of ASP resembles that of Kex2, it has a unique extra occluding region close to its active site. Moreover, we found that a nicked ASP variant is cleaved within the occluding region. Nicked ASP shows a greater ability to cleave small peptide substrates than the native enzyme. On the other hand, the cleavage pattern for prekallikrein differs from that of ASP, suggesting the occluding region is important for substrate recognition. The extra occluding region of ASP is unique and could serve as a useful target to facilitate development of novel antisepsis drugs.Aeromonas species are Gram-negative facultative anaerobic bacteria found ubiquitously in a variety of aquatic environments (1). The main syndrome caused by infection with Aeromonas is gastroenteritis (2, 3), although, in severe cases, sepsis is induced as a deuteropathy (4, 5). Two species, Aeromonas hydrophila and Aeromonas sobria, are associated with human disease (6, 7). Factors thought to play important roles in the pathogenesis include fimbrial and afimbrial adherence factors; a variety of exotoxins, including hemolysin, cytotonic enterotoxin, heat-stable enterotoxin, and heat-labile enterotoxin; and several secreted proteases and lipases (8–12). Recently, we purified a 65-kDa A. sobria serine protease (ASP)² from the culture supernatant of A. sobria and found that the enzyme induces vascular leakage, reduces blood pressure through activation of the kallikrein/kinin system (13), promotes human plasma coagulation through activation of prothrombin (14), and causes the formation of pus and edema through the action of anaphylatoxin C5a (15). From these observations, we concluded that ASP mediates the induction of disseminated intravascular coagulation through α-thrombin production, which is a common and deadly consequence of sepsis (14).ASP is a kexin-like serine protease belonging to the subtilisin family (subtilases) (16), which can be subdivided into six groups: the subtilisins, thermitases, proteinase K, lantibiotic peptidases, pyrolysins, and kexins. Among the kexins, the first identified was Kex2 (17), which is expressed by Saccharomyces cerevisiae; subsequently, the mammalian kexin-like protease furin was identified (18). Furin processes the precursors of biologically active peptides and proteins via limited proteolysis at paired basic amino acids to generate biologically active molecules (19). The domain structures of kexins and furins include a signal peptide, a partially conserved propeptide, a highly conserved subtilisin-like domain containing the characteristic Asp, His, and Ser catalytic residues, a relatively well conserved region called the P-domain, and a transmembrane domain followed by a cytoplasmic tail (20–22). Kex2 usually shows a high degree of specificity for cleavage after dibasic (P2-P1: Lys-Arg or Arg-Arg) or multiple basic residues (23). Among the kexins, which are nearly all eukaryotic and share a substantial degree of sequence homology (>40%), ASP is positioned as the most distant member of this family on the phylogenetic tree (16). The domain structure of ASP consists of the propeptide, the catalytic subtilisin-like domain, and the P-domain. For maturation of ASP, the first 24 residues of the propeptide are cleaved, and although a functional P-domain is reportedly necessary for maturation of the subtilisin domain in kexins (24, 25), the function of the P-domain in ASP remains unknown. Notably, in an earlier study of ASP expression, we found that for the maturation of the ASP subtilisin domain, another gene product, encoded by open reading frame 2, is required to serve as a chaperone in the periplasmic space (26).Here, we report the crystal structure of wild-type ASP as a sepsis-related factor at 1.65 Å resolution. We found that ASP has a unique occluding region at the active site within the subtilisin domain and that a different form of ASP that is cleaved within the occluding region shows a different pattern of proteolysis from the native enzyme. Our findings suggested that the novel occluding region plays an important role in determining substrate specificity and that because it is unique, it could facilitate development of novel antisepsis drugs that have no inhibitory effect on furin-like human proteases.     

Abnormalities in aggression and anxiety in transgenic mice overexpressing activin E

To study the function of activin E, a TGF-β superfamily member, in the regulation of affective behavior, we investigated the behavior of transgenic mice overexpressing activin E (TgActβE mice). Male TgActβE mice showed aggressive behavior in resident-intruder tests. In elevated plus-maze tests, the percentage of open arm entries was significantly increased in female TgActβE mice compared with that in wild-type mice. Furthermore, female TgActβE mice stayed in the central area for a significantly longer time than wild-type mice in open field tests. These results indicated that TgActβE mice had less anxiety-like behavior. The number of restraint-stress-evoked c-Fos-positive cells in the hypothalamic paraventricular nucleus in TgActβE mice was significantly decreased compared with that in wild-type mice. This suggests that synthesis of corticotrophin-releasing hormone induced by stress was decreased in TgActβE mice. Taking these results together, activin E may act as a regulator of the hypothalamic-pituitary-adrenal axis.     

Functional phenotypes and gene expression profiles of peripheral blood mononuclear cells in chronic hepatitis C patients who developed non-Hodgkin’s B-cell lymphoma

Epidemiological data have indicated a close relationship between chronic HCV infection and non-Hodgkin’s B-cell lymphoma (B-NHL). In this study, functional phenotypes and gene expression profiles of PBMCs were analyzed in chronic hepatitis C (CHC) patients who developed B-NHL. The frequencies of effector CD8⁺ T cells and cytotoxic natural killer cells increased in CHC patients with B-NHL compared to those in CHC patients without B-NHL. These phenotypic changes may reflect the host’s immune response to neoplasia. The mRNA expression levels of several oncogenes increased in CHC patients without B-NHL, but were much higher in CHC patients with B-NHL, while mRNA levels of type I IFNs were decreased in CHC patients without B-NHL and were nearly negligible in CHC patients with B-NHL. Interestingly, the mRNA expression levels of activation-induced cytidine deaminase and caspase recruitment domain-containing proteins markedly increased in CHC patients without B-NHL but decreased in CHC patients with B-NHL. These results are discussed in view of the possible involvement of HCV infection in B-cell lymphomagenesis.     

Drosophila big brain does not act as a water channel, but mediates cell adhesion

The neurogenic gene Drosophilabig brain (bib) has a high sequence homology to aquaporin-4. However, its cellular functions in Drosophila neurogenesis have remained elusive. Here we investigated cell adhesion, and the ion and water permeability of Bib. The adhesive function was examined by a cell aggregation assay using L cells. Bib-transfected L cells formed aggregated clusters, while control-L cells remained as a single cell suspension. Ion permeation was not confirmed in L cells stably expressing Bib. When expressed in COS7 cells, Bib exhibited limited water permeability. This newly found cell adhesive function of Bib may be important for Drosophila neurogenesis.