Takuo Yamamoto

Cloning and Sequencing of the Genes Encoding Cyclic Tetrasaccharide-synthesizing Enzymes from Bacillus globisporus C11

The genes for isomaltosyltransferase (CtsY) and 6-glucosyltransferase (CtsZ), involved in synthesis of a cyclic tetrasaccharide from α-glucan, have been cloned from the genome of Bacillus globisporus C11. The amino-acid sequence deduced from the ctsY gene is composed of 1093 residues having a signal sequence of 29 residues in its N-terminus. The ctsZ gene encodes a protein consisting of 1284 residues with a signal sequence of 35 residues. Both of the gene products show similarities to α-glucosidases belonging to glycoside hydrolase family 31 and conserve two aspartic acids corresponding to the putative catalytic residues of these enzymes. The two genes are linked together, forming ctsYZ. The DNA sequence of 16,515 bp analyzed in this study contains four open reading frames (ORFs) upstream of ctsYZ and one ORF downstream. The first six ORFs, including ctsYZ, form a gene cluster, ctsUVWXYZ. The amino-acid sequences deduced from ctsUV are similar in to a sequence permease and a sugar-binding protein for the sugar transport system from Thermococcus sp. B1001. The third ctsW encodes a protein similar to CtsY, suggested to be another isomaltosyltransferase preferring panose to high-molecular-mass substrates.

Protein N-Myristoylation Is Required for Cellular Morphological Changes Induced by Two Formin Family Proteins, FMNL2 and FMNL3

The subcellular localization of 13 recently identified N-myristoylated proteins and the effects of overexpression of these proteins on cellular morphology were examined with the aim of understanding the physiological roles of the protein N-myristoylation that occurs on these proteins. Immunofluorescence staining of HEK293T cells transfected with cDNAs coding for the proteins revealed that most of them were associated with the plasma membrane or the membranes of intracellular compartments, and did not affect cellular morphology. However, two proteins, formin-like2 (FMNL2) and formin-like3 (FMNL3), both of them are members of the formin family of proteins, were associated mainly with the plasma membrane and induced significant cellular morphological changes. Inhibition of protein N-myristoylation by replacement of Gly2 with Ala or by the use of N-myristoylation inhibitor significantly inhibited membrane localization and the induction of cellular morphological changes, indicating that protein N-myristoylation plays critical roles in the cellular morphological changes induced by FMNL2 and FMNL3.

Structure of a novel highly branched α-glucan enzymatically produced from maltodextrin

The bacterial strain PP710, isolated from soil and identified as Paenibacillus species, produced a low-digestibility alpha-glucan containing a large amylase-resistant portion. This alpha-glucan was obtained in high yields from maltodextrin (dextrose equivalent 3) by using the condensed culture supernatant of the strain as the enzyme preparation. The water-soluble dietary fiber content of the low-digestibility alpha-glucan was 80.2%, and showed resistance to a rat intestinal enzyme preparation. The alpha-glucan was found to be a novel highly branched alpha-glucan by acid hydrolysis, NMR analysis, gel permeation chromatography, methylation analysis, and enzymatic digestion.

Cloning and sequencing of kojibiose phosphorylase gene from Thermoanaerobacter brockii ATCC35047

A gene encoding kojibiose phosphorylase was cloned from Thermoanaerobacter brockii ATCC35047. The kojP gene encodes a polypeptide of 775 amino acid residues. The deduced amino acid sequence was homologous to those of trehalose phosphorylase from T. brockii and maltose phosphorylases from Bacillus sp. and Lactobacillus brevis with 35%, 29% and 28% identities, respectively. Kojibiose phosphorylase was efficiently overexpressed in Escherichia coli JM109. The DNA sequence of 3956 bp analyzed in this study contains three open reading frames (ORFs) downstream of kojP. The four ORFs, kojP, kojE, kojF, and kojG, form a gene cluster. The amino acid sequences deduced from kojE and kojF are similar to those of the N-terminal and C-terminal regions of a sugar-binding periplasmic protein from Thermoanaerobacter tengcongensis MB4. Furthermore, the amino acid sequence deduced from kojG is similar to that of a permease of the ABC-type sugar transport systems from T. tengcongensis MB4. Each of three amino acid substitutions, D362N, K614Q and E642Q, caused a complete loss of kojibiose phosphorylase activity. These results suggest that D362, K614 and E642 play an important role in catalysis. Another mutation, D459N, increased K(m) values for kojibiose (7-fold that for the wild type), beta-G1P (11-fold) and glucose (7-fold), whereas K(m) for inorganic phosphate was minimally affected by this mutation, suggesting that D459 may be involved in the binding to saccharides.

Purification and Characterization of Highly Branched α-Glucan–Producing Enzymes from Paenibacillus sp. PP710

Highly branched α-glucan molecules exhibit low digestibility for α-amylase and glucoamylase, and abundant in α-(1→3)-, α-(1→6)-glucosidic linkages and α-(1→6)-linked branch points where another glucosyl chain is initiated through an α-(1→3)-linkage. From a culture supernatant of Paenibacillus sp. PP710, we purified α-glucosidase (AGL) and α-amylase (AMY), which were involved in the production of highly branched α-glucan from maltodextrin. AGL catalyzed the transglucosylation reaction of a glucosyl residue to a nonreducing-end glucosyl residue by α-1,6-, α-1,4-, and α-1,3-linkages. AMY catalyzed the hydrolysis of the α-1,4-linkage and the intermolecular or intramolecular transfer of maltooligosaccharide like cyclodextrin glucanotransferase (CGTase). It also catalyzed the transfer of an α-1,4-glucosyl chain to a C3- or C4-hydroxyl group in the α-1,4- or α-1,6-linked nonreducing-end residue or the α-1,6-linked residue located in the other chains. Hence AMY was regarded as a novel enzyme. We think that the mechanism of formation of highly branched α-glucan from maltodextrin is as follows: α-1,6- and α-1,3-linked residues are generated by the transglucosylation of AGL at the nonreducing ends of glucosyl chains. Then AMY catalyzes the transfer of α-1,4-chains to C3- or C4-hydroxyl groups in the α-1,4- or α-1,6-linked residues generated by AGL. Thus the concerted reactions of both AGL and AMY are necessary to produce the highly branched α-glucan from maltodextrin.

Enzymatic properties of recombinant kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus ATCC43494.

One kojibiose phoshorylase (KP) homolog gene was cloned from Caldicellulosiruptor saccharolyticus ATCC43494. Recombinant KP from C. saccharolyticus (Cs-KP) expressed in Escherichia coli showed highest activity at pH 6.0 at 85 °C, and was stable from pH 3.5 to 10.0 and up to 85 °C for phosphorolysis. Cs-KP showed higher productivity of kojioligosaccharides of DP≧4 than KP from Thermoanaerobacter brockii ATCC35047.

High coupling performance of JT-60U ICRF antennas

Sufficient coupling of an ICRF antenna for high power experiments was obtained even for a wide gap between the separatrix and the antenna in JT-60U. The loading resistances for an out-of-phase mode are over 4 Omega for a gap of 13 cm between the separatrix and the Faraday shield over the wide range of electron density from 1 × 1019 to 5.5 × 1019 m-3. In particular, the loading resistances for an in-phase mode are about 5 Ω for a gap of 33 cm between the separatrix and the Faraday shield for the same plasma parameters. However, the heating response for the out-of-phase mode is much stronger than that for the in-phase mode

Usefulness of free-angle M-mode echocardiography in assessing left ventricular dimension and left ventricular systolic function

ObjectivesTo assess the usefulness of free-angle M-mode echocardiography in measuring left ventricular (LV) dimension and global systolic function.BackgroundThe validity of conventional M-mode echocardiography in assessing LV dimension and global systolic function is well known; the incidental angle between the M-mode cursor and true LV minor axis diameter, however is a potential cause of measurement error. Free-angle M-mode echocardiography may overcome the limitation of M-mode cursor arrangement in conventional M-mode echocardiography.MethodsThirteen normal volunteers and 10 patients in whom abnormal left ventricular wall motion was not detected by echocardiography (mean age, 53±17 years) were enrolled in this study. Conventional and free-angle M-mode echocardiographic images of the LV were obtained by echocardiography (ALOKA SSD-5500) using a 2.5-MHz transducer, and the LV end-diastolic (LVDd) and end-systolic (LVDs) dimensions were measured by the leading edge method. LV end-diastolic and end-systolic volumes were calculated using a formula by Teichholz, and the LV ejection fraction (LVEF) was obtained. Data from conventional M-mode echocardiography and free-angle M-mode echocardiography were then compared.ResultsMeasurements obtained with conventional M-mode and free-angle M-mode echocaardiography were strongly correlated. Correlation coefficients for LVDd, LVDs, and LVEF were 0.98, 0.98, and 0.96, respectively (p<0.001 in each case).ConclusionsAssessment of left ventricular dimension, and global systolic function with free-angle M-mode can be as accurate as conventional M-mode in subjects in whom left ventricular wall motion abnormality is not detectable by echocardiogram. Moreover, when there is improper M-mode cursor direction in conventional echocardiography, free-angle M-mode echocardiography can assess global left ventricular systolic function more accurately and conveniently than conventional M-mode echocardiography.