A diphtheria toxin receptor deficient in epidermal growth factor-like biological activity

Targeted cell ablation in animals is a powerful method for analyzing the physiological function of cell populations and generating various animal models of organ dysfunction. To achieve more specific and conditional ablation of target cells, we have developed a method termed Toxin Receptor mediated Cell Knockout (TRECK). A potential shortcoming of this method, however, is that overexpression of human heparin-binding epidermal growth factor-like growth factor (hHB-EGF) as a diphtheria toxin (DT) receptor in target cells or tissues may cause abnormalities in transgenic mice, since hHB-EGF is a member of the EGF growth factor family. To create novel DT receptors that are defective in growth factor activity and resistant to metalloprotease-cleavage, we mutated five amino acids in the extracellular EGF-like domain of hHB-EGF, which contains both DT-binding and protease-cleavage sites. Two of the resultant hHB-EGF mutants, I117A/L148V and I117V/L148V, possessed little growth factor activity but retained DT receptor activity. Furthermore, these mutants were resistant to metalloprotease-cleavage by 12-O-tetradecanoylphorbol-13-acetate stimulation, which is expected to enhance DT receptor activity. These novel DT receptors should be useful for the generation of transgenic mice by TRECK.     

Two type IV pili of Vibrio parahaemolyticus play different roles in biofilm formation

Thirty-eight Shiga toxin-producing Escherichia coli (STEC) O157:H7/H(-) strains isolated from human infections, cattle and foods in Brazil and in some other Latin American countries were compared with regard to several phenotypic and genotypic characteristics. The genetic relatedness of the strains was also determined by pulsed-field gel electrophoresis (PFGE). Similar biochemical behaviour was identified, regardless of the origin and country of the strains. Most (89.5%) strains were sensitive to the antimicrobial agents tested, but resistance to at least one drug was observed among bovine strains. Although a diversity of stx genotypes was identified, most (77.8%) of the human strains harboured stx(2) or stx(2)stx(2c(2vha)), whereas stx(2c(2vha)) prevailed (64.2%) among strains isolated from cattle. stx(1) and stx(1)stx(2c(2vha)) were the genotypes identified less frequently, and occurred exclusively among strains isolated from food and cattle, respectively. Despite differences in the stx genotypes, all strains carried eae-gamma, efa1, ehx, iha, lpf(O157) and toxB sequences. Many closely related subgroups (more than 80% of similarity) were identified by PFGE, and the presence of a particular O157:H7 STEC clone more related to human infections in Brazil, as well as a common origin for some strains isolated from different sources and countries in Latin America can be suggested.     

Isomer-specific anti-obese and hypolipidemic properties of conjugated linoleic acid in obese OLETF rats

Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of linoleic acid, has attracted considerable attention because of its potentially beneficial biologic effects both in vitro and in vivo. Our results clearly show the specific action of the 10trans,12cis-CLA isomer against hyperlipidemia and obesity in obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. After 2 weeks of feeding with 10t,12c-CLA, but not 9cis,11trans-CLA, abdominal adipose tissue weight and serum and hepatic lipid levels in OLETF rats were lower than those in linoleic acid-fed rats. These effects were attributable to suppressed fatty acid synthesis and enhanced fatty acid beta oxidation in the liver on a 10t,12c-CLA diet. Additionally, we showed that mRNA expression of fatty acid synthase, carnitine palmitoyltransferase, leptin, and sterol regulatory element binding protein-1 was also regulated by 10t,12c-CLA. We suppose that 10t,12c-CLA reveals hypolipidemic and anti-obese activity through the alteration of mRNA expressions in the liver and white adipose tissue.     

Symmetry of non-carious cervical lesions in canines and premolars

Objectives: Clinically non‐carious cervical lesions (NCCLs) are frequently seen. The aim of this study was to investigate the relationship between the shape and symmetry of NCCLs, wear of cuSPS and triangular ridge, and the curvature of the tooth root. Methods: One hundred and twenty‐nine extracted human upper canine teeth and 274 extracted human upper premolar teeth with NCCLs were used in this study. The specimens were studied using photographs and three‐dimensional scanning. Results: Asymmetric NCCL was observed in 69.0% of the canines and 44.5% of the premolars. Wear of cusp and lingual ridges was observed in 82.9% and 93.0% of the canines, respectively. Wear of the buccal cusp and buccal triangular ridge was observed in 85.4% and 89.8% of the premolars, respectively. On the other hand, the wear of lingual cusp and lingual triangular ridge was observed in 89.1% and 93.8% of the premolars, respectively. The curvature of the root was observed in 48.1% of the canines and 43.4% of the premolars. Conclusions: There was no relationship between the symmetry of NCCLs, and the wear of cuSPS and triangular ridges for either canines or premolars. Although there was a relationship (p < 0.05) between the symmetry of NCCL and the curvature of the root in the canines, no relationship was observed between the symmetry of NCCL and the curvature of the root in the premolars.     

Syntheses of naturally occurring terphenyls and related compounds

Naturally occurring terphenyls and related compounds such as terferol and its corresponding quinone and phlebiarubrone were synthesized from 2,5-diphenyl-1,4-benzoquinone. According to the proposed biosynthetic pathway, chemical conversion of phlebiarubrone to ustalic acid, a toxic compound isolated from the poisonous mushroom, Tricholoma ustale, was examined to find a low-yield conversion to the ustalic acid dimethyl ester.     

Isolation and characterization of a beta-primeverosidase-like enzyme from Penicillium multicolor

p-Nitrophenyl and eugenyl beta-primeveroside (6-O-beta-D-xylopyranosyl-beta-D-glucopyranoside) hydrolytic activity was found in culture filtrate from Penicillium multicolor IAM7153, and the enzyme was isolated. The enzyme was purified as a beta-primeverosidase-like enzyme by precipitation with ammonium sulfate followed by successive chromatographies on Phenyl Sepharose, Mono Q, and beta-galactosylamidine affinity columns. The molecular mass was estimated to be 50 kDa by SDS-PAGE and gel filtration. The purified enzyme was highly specific toward the substrate p-nitrophenyl beta-primeveroside, which was cleaved in an endo-manner into primeverose and p-nitrophenol, but a series of beta-primeveroside as aroma precursors were hydrolyzed only slightly as substrates for the enzyme. In analyses of its hydrolytic action and kinetics, the enzyme showed narrow substrate specificity with respect to the aglycon and glycon moieties of the diglycoside. We conclude that the present enzyme is a kind of beta-diglycosidase rather than beta-primeverosidase.     

Resonance Raman characterization of archaeal and bacterial Rieske protein variants with modified hydrogen bond network around the [2Fe-2S] center

The rate of quinol oxidation by cytochrome bc(1)/b(6)f complex is in part associated with the redox potential (E(m)) of its Rieske [2Fe-2S] center, for which an approximate correlation with the number of hydrogen bonds to the cluster has been proposed. Here we report comparative resonance Raman (RR) characterization of bacterial and archaeal high-potential Rieske proteins and their site-directed variants with a modified hydrogen bond network around the cluster. Major differences among their RR spectra appear to be associated in part with the presence or absence of Tyr-156 (in the Rhodobacter sphaeroides numbering) near one of the Cys ligands to the cluster. Elimination of the hydrogen bond between the terminal cysteinyl sulfur ligand (S(t)) and Tyr-Oeta (as with the Y156W variant, which has a modified histidine N(epsilon) pK(a,ox)) induces a small structural bias of the geometry of the cluster and the surrounding protein in the normal coordinate system, and significantly affects some Fe-S(b/t) stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (S(b)) and Ser-Ogamma, which is weak and/or unfavorably oriented for extensive coupling with the Fe-S(b/t) stretching vibrations.     

Mechanism of docosahexaenoic acid-induced inhibition of in vitro Aβ1–42 fibrillation and Aβ1–42-induced toxicity in SH-S5Y5 cells

The mechanism of the effect of docosahexaenoic acid (DHA; C22:6, n -3), one of the essential brain nutrients, on in vitro fibrillation of amyloid β (Aβ_1–42), Aβ_1–42-oligomers and its toxicity imparted to SH-S5Y5 cells was studied with the use of thioflavin T fluorospectroscopy, laser confocal microfluorescence, and transmission electron microscopy. The results clearly indicated that DHA inhibited Aβ_1–42-fibrill formation with a concomitant reduction in the levels of soluble Aβ_1–42 oligomers. The polymerization (into fibrils) of preformed oligomers treated with DHA was inhibited, indicating that DHA not only obstructs their formation but also inhibits their transformation into fibrils. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12.5%), Tris–Tricine gradient(4–20%) gel electrophoresis and western blot analyses revealed that DHA inhibited at least 2 species of Aβ_1–42 oligomers of 15–20 kDa, indicating that it hinders these on-pathway tri/tetrameric intermediates during fibrillation. DHA also reduced the levels of dityrosine and tyrosine intrinsic fluorescence intensity, indicating DHA interrupts the microenvironment of tyrosine in the Aβ_1–42 backbone. Furthermore, DHA protected the tyrosine from acrylamide collisional quenching, as indicated by decreases in Stern–Volmer constants. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction efficiency and immunohistochemical examination suggested that DHA inhibits Aβ_1–42-induced toxicity in SH-S5Y5 cells. Taken together, these data suggest that by restraining Aβ_1–42 toxic tri/tetrameric oligomers, DHA may limit amyloidogenic neurodegenerative diseases, Alzheimer's disease.     

Molecular Cloning and Characterization of a cDNA for Pterocarpan 4-Dimethylallyltransferase Catalyzing the Key Prenylation Step in the Biosynthesis of Glyceollin,a Soybean Phytoalexin

Glyceollins are soybean (Glycine max) phytoalexins possessing pterocarpanoid skeletons with cyclic ether decoration originating from a C₅ prenyl moiety. Enzymes involved in glyceollin biosynthesis have been thoroughly characterized during the early era of modern plant biochemistry, and many genes encoding enzymes of isoflavonoid biosynthesis have been cloned, but some genes for later biosynthetic steps are still unidentified. In particular, the prenyltransferase responsible for the addition of the dimethylallyl chain to pterocarpan has drawn a large amount of attention from many researchers due to the crucial coupling process of the polyphenol core and isoprenoid moiety. This study narrowed down the candidate genes to three soybean expressed sequence tag sequences homologous to genes encoding homogentisate phytyltransferase of the tocopherol biosynthetic pathway and identified among them a cDNA encoding dimethylallyl diphosphate: (6aS, 11aS)-3,9,6a-trihydroxypterocarpan [(−)-glycinol] 4-dimethylallyltransferase (G4DT) yielding the direct precursor of glyceollin I. The full-length cDNA encoding a protein led by a plastid targeting signal sequence was isolated from young soybean seedlings, and the catalytic function of the gene product was verified using recombinant yeast microsomes. Expression of the G4DT gene was strongly up-regulated in 5 to 24 h after elicitation of phytoalexin biosynthesis in cultured soybean cells similarly to genes associated with isoflavonoid pathway. The prenyl part of glyceollin I was demonstrated to originate from the methylerythritol pathway by a tracer experiment using [1-¹³C]Glc and nuclear magnetic resonance measurement, which coincided with the presumed plastid localization of G4DT. The first identification of a pterocarpan-specific prenyltransferase provides new insights into plant secondary metabolism and in particular those reactions involved in the disease resistance mechanism of soybean as the penultimate gene of glyceollin biosynthesis.Typical phytoalexins of the Leguminosae are isoflavonoid derivatives with characteristic species-specific modifications in both their skeletons and their decoration, e.g. prenylation (Dixon, 1999). Isoflavonoids are formed through an early branching pathway in flavonoid metabolism. The most abundantly found isoflavonoid skeleton of leguminous phytoalexins is pterocarpan, and more than one-half of these pterocarpanoids are decorated in a complex manner mainly by isoprenoid-derived substituents (Tahara and Ibrahim, 1995). Glyceollin is the collective name for soybean (Glycine max) phytoalexins with pterocarpanoid skeletons and cyclic ether decoration originating from C₅ prenyl substitutions (Fig. 1). The biosynthesis mechanism of soybean phytoalexins has been studied extensively during the 1970s to 1990s, most actively by Grisebach et al. (Ebel and Grisebach, 1988), and the pathway and biosynthetic enzymes involved have been characterized intensively at the biochemical level (Ebel, 1986; Dixon, 1999). More recent studies with leguminous plants such as alfalfa (Medicago sativa), licorice (Glycyrrhiza echinata), Lotus japonicus, and Medicago truncatula in addition to soybean have resulted in the identification of many genes encoding enzymes involved in isoflavonoid formation (Dixon, 1999; Shimada et al., 2007; Veitch, 2007). However, some genes encoding enzymes of the later stages of glyceollin biosynthesis, especially the crucial prenylation step, have remained uncharacterized until now.Open in a separate windowFigure 1.Biosynthesis of glyceollin isomers in soybean. Abbreviations not defined in the text: HID, 2-hydroxyisoflavanone dehydratase; IFS, 2-hydroxyisoflavanone synthase; P6aH, pterocarpan 6a-hydroxylase; G2DT, dimethylallyl diphosphate: (−)-glycinol 2-dimethylallyltransferase.During glyceollin biosynthesis, a dimethylallyl group is introduced at either C-4 or C-2 of the pterocarpan skeleton (C-8 or C-6 by isoflavone numbering, respectively). A prenyltransferase activity catalyzing the dimethylallylation of (6aS, 11aS)-3,9,6a-trihydroxypterocarpan, (−)-glycinol, has been demonstrated in microsomal fractions of soybean cotyledons and cell cultures treated with a glucan elicitor derived from the cell walls of Phytophthora sojae (Zähringer et al., 1979). An increased toxicity of the prenylated pterocarpans against a phytopathogenic fungus was also demonstrated (Zähringer et al., 1981). An important finding was that the prenylation activity was localized to the chloroplast fraction of cotyledon cells in contrast to the endoplasmic reticulum (ER) where many of the cytochrome P450s (P450s) for glyceollin formation are localized (Welle and Grisebach, 1988; Biggs et al., 1990; Ayabe and Akashi, 2006). Efficient solubilization of the activity and partial purification of the enzyme have also been reported (Welle and Grisebach, 1991), but no complete purification was achieved to sequence the amino acids, and thus the gene responsible remains unidentified.Recently, plant cDNAs of aromatic substrate prenyltransferases have been characterized, and their nucleotide sequence information has become available (Yazaki et al., 2002; Sasaki et al., 2008). In view of the potential benefits of understanding the molecular mechanism underlying the phytopathogen resistance of soybean for the future disease-resistance breeding, studies toward the complete identification of the enzymes involved in glyceollin biosynthesis are important. Thus, this study undertook the molecular cloning and biochemical characterization of a soybean prenyltransferase involved in the glyceollin biosynthetic pathway.