Biomedical subjects

K Takeo

Publications and source records attributed to K Takeo.

At least 55 records · Page 3Linked to original sources

PubMed1995

A structural model for the mechanisms of elicitor release from fungal cell walls by plant beta-1,3-endoglucanase.

The release of elicitor-active carbohydrates from fungal cell walls by beta-1,3-endoglucanase contained in host tissues has been implicated as one of the earliest processes in the interaction between soybean (Glycine max) and the fungal pathogen Phytophthora megasperma f. sp. glycinea leading to host defense responses such as phytoalexin production. The present study was conducted to evaluate the primary structure of the glucanase-released elicitor (RE). Gel-filtration chromatography of carbohydrates released from mycelial walls by purified soybean beta-1,3-endoglucanase resolved them into the four fractions (elicitor-active RE-I, -II, and -III and elicitor-inactive RE-IV). Sugar composition analysis indicated that all of the fractions were composed almost entirely of glucose. 1H- and 13C-nuclear magnetic resonance analysis indicated the presence of both beta-1,3- and beta-1,6-linkages for the elicitor-active RE-I, -II, and -III fractions and only beta-1,3 linkage for the elicitor-inactive RE-IV fraction. Methylation analysis and degradation studies employing beta-1,3-endo- and beta-1,3-exoglucanase further suggested that the basic structure of elicitor-active RE consists of beta-1,6-linked glucan backbone chains of various lengths with frequent side branches composed of beta-1,3-linked one or two glucose moieties. From these structural analyses of RE, a structural model of how RE is originally present in fungal cell walls and released by host beta-1,3-endoglucanase is also proposed.

Carbohydrate Sequence↗

PubMed1994

An antitumor, branched (1-->3)-beta-D-glucan from a water extract of fruiting bodies of Cryptoporus volvatus.

A water-soluble, (1-->6)-branched (1-->3)-beta-D-glucan (H-3-B) was isolated from a hot-water extract of the fruiting bodies of the fungus, Cryptoporus volvatus (Basidiomycetes). Enzymatic analysis using exo-(1-->3)-beta-D-glucanase and methylation analysis indicated that this polysaccharide has a main chain composed of beta-(1-->3)-linked D-glucopyranosyl residues, and single, beta-(1-->6)-linked D-glucopyranosyl residues attached as side chains to, on average, every fourth sugar residue of the main chain. This structure was confirmed by 13C NMR spectra of the glucan in Me2SO-d6. The weight-average molecular weight (Mw) of H-3-B was determined to be 44.0 x 10(4) by gel permeation chromatography equipped with a low-angle laser-light-scattering photometer. The electron microscopic observations showed that H-3-B and its sonicated sample (S-H-3-B, Mw = 13.7 x 10(4)) can be described as linear worm-like chains. The mass per unit length for native and sonicated H-3-B was determined to be 1750 and 1780 g mol-1 nm-1, respectively, from the contour lengths obtained by electron microscopy and the molecular weights. These values are in good agreement with that expected for the triple stranded structure. A sample denatured in 0.1 M NaOH and subsequently renatured by neutralization showed a mixture of linear and cyclic structures, and larger aggregates with less well-defined morphology. The H-3-B and S-H-3-B had antitumor activity against the Sarcoma 180 tumor.

Animals↗

PubMed1994

Molecular and crystal structure of (2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-(1-->3)- [2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl-(1-->6)]- (2,4-di-O-acetyl-beta-D-glucopyranosyl)- (1-->3)-1,2,4,6-tetra-O-acetyl-beta-D-glucopyranose.

Crystals of the tetrasaccharide, (2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-(1-->3)- [2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl-(1-->6)]- (2,4-di-O-acetyl-beta-D-glucopyranosyl)-(1-->3)- 1,2,4,6-tetra-O-acetyl-beta-D-glucopyranose, belong to the monoclinic system, space group P2(1), with a = 12.709(4), b = 27.767(9), c = 9.567(4) A, beta = 105.07(2) degrees, and Z = 2. The crystal structure was solved by the direct method and refined by the full-matrix least-squares procedure to an R-value of 0.071 for 302- observed reflections in the X-ray data. All the four D-glucopyranose residues have the usual 4C1 chair conformations. The torsional angles at two (1-->3)-beta-linkages were phi(O-5-1-O-1-C-3) = -74 degrees and psi (C-1-O-1-C-3-C-2) = -122 degrees between the nonreducing and the middle residues, and phi = -67 degrees, psi = -111 degrees between the middle and the reducing residues. The orientation about the (1-->6)-beta-linkage was found to be phi(O5-C1-O-1-C-6) = -77 degrees, theta(C1-O-1-C-6-C-5) = 160 degrees, and chi(O-1-C-6-C-5-O-5) = 63 degrees (gt conformation). The primary acetate groups at C-6 of the reducing and the (1-->6)-branched residues were in the gt conformations (O-5-C-5-C-6-O-6 = 81 degrees and 84 degrees, respectively). On the other hand, the gg conformation was observed for the primary acetate group in the nonreducing residue (O-5-C-5-C-6-O-6 = 78 degrees).

Carbohydrate Conformation↗

PubMed1994

Pleoanamorphic life cycle of Exophiala (Wangiella) dermatitidis.

The anamorph life cycle of the black yeast Exophiala (Wangiella) dermatitidis is described. The fungus is dimorphic, yeast cells being the prevalent form of propagation. The fungus is strongly hydrophilic, probably completing its anamorph life cycle in submersion. Adaptation to dry conditions is slow. Types of conidiogenesis comprise annellidic, phialidic and sympodial reproduction, in addition to isotropic development. Phialoconidia fail to germinate under the conditions tested, and thus may have a function other than dispersal. Sterile, multicellular bodies resembling a Capronia teleomorph are described.

Exophiala↗

PubMed1994

Conformation of (2-->1)-beta-D-fructan in aqueous solution.

The conformation and dilute solution properties of (2-->1)-beta-D-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (Mw) from 1.49 x 10(4) to 5.29 x 10(6) were obtained from a native sample by ultrasonic degradation and fractional precipitation. For Mw < 4 x 10(4), the intrinsic viscosity [eta] varies with Mw0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For Mw > 10(5), [eta] exhibits an unusually weak dependence on Mw and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [eta] observed.

Aspergillus↗

PubMed1993

The existence of a dispensable fibrillar layer on the wall surface of mycelial but not yeast cells of Aureobasidium pullulans.

Wall surface ultrastructure of Aureobasidium pullulans was studied by freeze-etching. Yeast cells had a smooth wall surface as in typical yeast species. Mycelial cells and chlamydospores had an extra layer on the wall surface made mostly of fibrils. The fibrils were 20 nm in diameter, and thicker than typical major fungal wall skeletal fibrils of beta-glucan and chitin. This layer was apparently easily detached from the wall proper, presumably as a result of enzymic activity or by physical means, suggesting that it is a physiologically dispensable wall component.

Candida albicans↗

PubMed1993

Synthesis of the laminara-oligosaccharide methyl beta-glycosides of dp 3-8.

Ethyl 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-glucopyranoside has been prepared in a good yield by anomerization of the corresponding beta-thioglucoside with tin(IV) chloride and transformed, in three steps, into ethyl 2-O-benzoyl-4,6-O-benzylidene-1-thio-alpha-D-glucopyranoside (18). Chloroacetylation of 18, followed by treatment of the product with chlorine gave crystalline 2-O-benzoyl-4,6-O-benzylidene-3-O-chloroacetyl-beta-D-glucopyranosyl chloride (20). This was coupled with methanol in the presence of silver carbonate-silver perchlorate and the product was O-dechloroacetylated to afford methyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranoside (22). Silver triflate-promoted glucosylation of 18 with 20 gave a beta-(1-->3)-linked disaccharide derivative, reaction of which with chlorine yielded crystalline O-(2-O-benzoyl-4,6-O-benzylidene-3-O-chloroacetyl-beta-D-glucopyranosyl) - (1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl chloride (24). Likewise, condensation of 22 with 20 gave a beta-(1-->3)-linked disaccharide glycoside, which was partially deprotected to give methyl O-(2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl)-(1-->3)-2-O-benz oyl- 4,6-O-benzylidene-beta-D-glucopyranoside (26). The methyl beta-glycosides of a homologous series of (1-->3)-linked beta-D-gluco-oligosaccharides from the tri- to the octa-saccharide have been synthesized in a blockwise manner by using 22 and 26 as the glycosyl acceptors, 24 as the disaccharide donor, and silver triflate as the promoter.

Carbohydrate Conformation↗

PubMed1993

Rapid, extensive and reversible vacuolation of Schizosaccharomyces pombe induced by amphotericin B.

The fission yeast Schizosaccharomyces pombe has no large vacuoles under normal growth conditions, although budding yeasts usually have large central vacuoles. The minimum inhibitory concentration of amphotericin B to S. pombe was 0.5 microgram ml-1; treatment with 0.2 microgram ml-1 for 20 min induced rapid and extensive vacuolation in S. pombe exponential phase cells. Growth rate of the cells with 0.2 microgram ml-1 amphotericin B was much reduced for 6 h, showing extensive vacuolation. Vacuolation in itself was not fatal: on removal of the drug, most cells recovered gradually and eventually multiplied.

Amphotericin B↗

PubMed1993

Temperature-induced conformational transition in xanthans with partially hydrolyzed side chains.

The conformational properties of xanthans with partially hydrolyzed side chains were investigated by optical rotation, CD, and differential scanning calorimetry (DSC). All variants displayed the well-known temperature-driven, cooperative order-disorder transition, and both optical rotation and DSC showed that the transition temperature was essentially independent of the content of terminal beta-mannose. It was found that up to 80% of the changes in the specific optical rotation accompanying the transition reflects conformational changes linked to the terminal beta-mannose in the side chains. Modification of the side chains also affected the CD when xanthan was in the ordered state, but in this case the data suggest that the glucuronic acid is the major component determining the magnitude of the CD signal. DSC measurements showed that the transition enthalpy (delta Hcal) increased linearly with the fraction of beta-mannose, again indicating that a significant part (up to 80%) of delta Hcal reflects conformational changes in the side chains. The conformational transition of the xanthan variants generally showed a higher degree of cooperativity (sharper transition) than unmodified, pyruvated xanthan. Calculation of the cooperativity parameter sigma by means of the Zimm-Bragg theory (OR data) or from the ratio between delta Hcal and the van't Hoff enthalpy (delta HvH) using DSC data showed a correlation between sigma and the content of beta-mannose, but the two methods gave different results when the content of beta-mannose approached 100%. The ionic strength dependence of the transition temperature, expressed as d (log I)/d(T-1m), was nearly identical for intact xanthan and a sample containing only 6% of the terminal beta-mannose. Application of the Manning polyelectrolyte theory does not readily account for the observed delta Hcal values, neither does it provide new information on the nature of the ordered and disordered conformations in xanthan.

Calorimetry, Differential Scanning↗

PubMed1993

Physicochemical properties of (1-->6)-branched (1-->3)-beta-D-glucans. 1. Physical dimensions estimated from hydrodynamic and electron microscopic data.

The physical dimensions of several (1-->6) branched (1-->3)-beta-D-glucan samples obtained from different organisms and their derivatives have been studied by electron microscopy, light scattering measurements, viscometry, and gel permeation chromatography. The electron micrographs indicate that in most samples these biopolymers are adequately described as linear worm-like coils. A sample reconstituted from alkaline media appeared as a blend of the linear, circular, and aggregated polymer morphologies. The average mass per unit length, ML = Mw/Lw for the macroscopically linear samples, was estimated to be 2100 +/- 200 g mol-1 nm-1. The parameter ML was determined from the contour lengths obtained by electron microscopy and the molecular weight by light scattering measurements. The observed ML was consistent with the triple-helical structure reported from x-ray diffraction studies and observed degree of side-chain substitution. From the molecular snapshots shown in the electron micrographs, the persistence lengths of these beta-D-glucans were determined to be 140 +/- 30 nm. The experimentally determined intrinsic viscosities were consistent with these estimates of ML and persistence length. Comparison of the molecular weight distributions obtained from gel permeation chromatography and those deduced from the electron micrographs indicates that number and weight average contour lengths are more reliable than z and z + 1 averages.

Carbohydrate Conformation↗

PubMed1993

Heterogeneity of carbonic anhydrase and 68 kDa neurofilament in nerve roots analyzed by two-dimensional electrophoresis.

The heterogeneity of carbonic anhydrase (CA) and 68 kDa neurofilament (NF) in nerve roots of Japanese monkeys (Macaca Macacus) was analyzed by two-dimensional electrophoresis combined with immunoblotting. CA was detected as heterogeneous spots of type I and II having the same molecular weight (M(r)) but different isoelectric point (pI), in the range of pH 4.5-5.5. CA had much higher concentrations in soluble fractions of dorsal roots than ventral roots but not in membrane-bound fractions of either root. NF was detected as a single spot at the position of 68 kDa and pI 5.2 in soluble fractions of both roots, however, it was detected in the membrane-bound fractions as heterogeneous spots having different molecular masses and pI in the range of 60-68 kDa and 5.0-5.5, respectively. The presence of heterogeneous CA type I and II at high concentrations in dorsal roots enables us to distinguish the sensory nerves from the motor nerves. Furthermore, the heterogeneity of NF in membrane-bound fractions gives us information for understanding the dynamic assembly and the degradation of NF in nerve roots.

Animals↗

PubMed1993

Affinity maturation of anti-hapten antibodies in a single mouse analyzed by two-dimensional affinity electrophoresis.

Affinity maturation of anti-hapten antibodies in an inbred mouse (BALB/c) was analyzed by two-dimensional affinity electrophoresis (2D-AEP) in which the antibodies specific to a hapten of dinitrophenyl (DNP) group were separated into a large number of IgG families according to the differences in their isoelectric points (pI) and in their affinities for the ligand of DNP immobilized in the gel matrices. Each of the IgG families consisted of several spots showing an identical affinity for the ligand but different pI, and the spots were specifically stained by immunostaining with an anti-murine IgG subclass antibody. These results lead us to the conclusion that each of the IgG families is derived from a single clone of antibody-producing cells specific to DNP. The mass and affinity of the IgG families varied in the course of immunization with DNP-conjugated chicken serum albumin (DNP-CSA). The first injection of DNP-CSA induced a small amount of IgG families showing a wide variety in their affinity for DNP. The second injection induced a large amount of IgG families, especially of families having a medium affinity for DNP. Additional injections, however, did not change the mass or affinity of the IgG families significantly. This suggests that an antigen-induced somatic mutation of the immunoglobulin gene does not occur frequently to mature the affinity of antibodies by the additional injections after the second injection. 2D-AEP enables us to analyze the affinity maturation of antibodies in vivo in a single mouse and to document the subclass switch of the antibody in the course of immunization.

Animals↗

PubMed1993

N-linked sugar chain structure of recombinant human lymphotoxin produced by CHO cells: the functional role of carbohydrate as to its lectin-like character and clearance velocity.

Recombinant human lymphotoxin (rhLT) produced by CHO cells transfected with human LT genomic DNA was purified to homogeneity, but approximately 5% of the molecules were devoid of the last two amino terminal residues. A peptide N-glycosylated at Asn62 (Tr-45) and one partially O-glycosylated at Thr7 (Tr-14) on cleavage with trypsin were separated by reverse phase HPLC. The N-linked sugar chains of Tr-45 were released quantitatively as oligosaccharides on hydrazinolysis (100 degrees C, 8 h), followed by N-acetylation. After being reduced with either NaB3H4 or NaB2H4, their structures were determined by a combination of serial lectin affinity chromatography, exoglycosidase digestion, and methylation analysis: 82.7% of the sugar chains occur as biantennary complex-type sugar chains, the remainder being C-2 and C-2,4/C-2,6 branched triantennary, and C-2,4 and C-2,6 branched tetraantennary complex-type sugar chains with a fucosylated mannose core. Their sialic acid residues occur only as the Neu5Ac alpha 2-->3Gal group. The clearance velocity from the bloodstream dramatically increased with desialylation, and rhLT tends to have accumulated in the kidney, indicating that there may exist other mechanisms for clearance from the circulation besides the galactose-binding protein in hepatocytes and the filtration system of the kidney. Desialylated rhLT showed a lectin-like binding character to uromodulin similar to that of tumor necrosis factor, although intact rhLT did not. The interaction between desialylated rhLT and uromodulin was inhibited by N,N'-diacetylchitobiose and [Man alpha 1-->6(Man alpha 1-->3)Man alpha 1-->6](Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc-->Asn. These results indicate that the lectin-like domain of rhLT is exposed on its desialylation.

Amino Acid Sequence↗

PubMed1993

A comparative study of four different staining methods for estimation of live yeast form cells of Paracoccidioides brasiliensis.

A comparative study of four different staining methods for estimation of live yeast form cells of Paracoccidioides brasiliensis was carried out. The staining methods used were fluorescent staining, vital dye exclusion tests with erythrosin B and by Janus green and lactophenol cotton blue staining. Colony forming units (cfu) of the yeast form of eight P. brasiliensis isolates on brain heart infusion agar (BHIA) supplemented with 4% horse serum plus 5% P. brasiliensis cell extract (BHIA + HS + EXT) were examined for reliability of staining in determining the number of live fungal units in eight different isolates. Cfu on BHIA + HS + EXT plates showed an excellent plating efficiency over 96% in all isolates tested. The percentage of the live cells indicated by fluorescent staining (FL) or vital dye exclusion test with erythrosin B (EB) or Janus green (JG-1) was lower than that of cfu. By contrast, the percentage due to modified dye exclusion test with Janus green (JG-2) and that due to lactophenol cotton blue staining (LPCB) showed a close correration to that of cfu. Our results indicate that the modified dye exclusion test with Janus green and lactophenol cotton blue staining are useful for estimating cell viability of yeast form cells of P. brasiliensis.

Azo Compounds↗

PubMed1993

An improved culture medium for detecting live yeast phase cells of Paracoccidioides brasiliensis.

The plating efficiency of standard mycological media such as brain heart infusion (BHI) agar is poor for Paracoccidioides brasiliensis. We prepared a water-extract of yeast phase cells of P. brasiliensis and examined it for growth-enhancing activity for the fungus. The water-extract, when added to BHI agar to a concentration of 5%, improved the plating efficiency of the medium for the fungus to some extent, but the degree of improvement was considerably varied among P. brasiliensis isolates. By contrast, when the water-extract was added in combination with horse serum (4%), the plating efficiency was highly improved (to 94-99%) for all the P. brasiliensis isolates employed. The growth-enhancing factor(s) in the water-extract was heat-stable and heating at 120 degrees C for 15 min had little, if any, effect on growth-enhancing activity.

Culture Media↗

PubMed1993

Analysis of ploidy and sexual characteristics of natural isolates of Cryptococcus neoformans.

We report here an extensive examination of the ploidy of a basidiomycetous yeast. The ploidy of 34 strains of Cryptococcus neoformans var. neoformans, isolated mostly from Japanese patients, was determined by means of quantitative fluorescence microscopy after DNA staining with propidium iodide. The sexual characteristics of the strains including the two tester strains were as follows. Twenty-four strains were of the heterothallic alpha type, 1 was of the heterothallic a type, and 2 were self fertile; the remaining 7 showed no sexual response. The following strains were found to be haploid: the heterothallic a strain, 20 of the 24 heterothallic alpha strains, and 3 of the 7 asexual strains. Thus, the C. neoformans strains were mostly haploid, as the sexual response of this organism suggests. Both of the self-fertile strains and four of the seven asexual strains were diploid. Unexpectedly, 4 of the 24 heterothallic alpha strains were diploid.

Cryptococcus neoformans↗

PubMed1993

Determination of cardiac ejection fraction and left ventricular volume: contrast-enhanced ultrafast cine MR imaging vs IV digital subtraction ventriculography.

OBJECTIVE: To assess the accuracy of contrast-enhanced, single breath-hold cine MR imaging in the calculation of left ventricular volume and ejection fraction, we compared values obtained by using this method with those obtained by using IV digital subtraction angiography (IV-DSA). SUBJECTS AND METHODS: All patients (n = 28) had conventional cine and contrast-enhanced ultrafast cine MR imaging. For ultrafast cine MR imaging, a phase-rewind gradient-echo (rewind-SMASH) sequence was used: TR, 8 msec (standard excitation and acquisition block of 6 msec with phase rewind pulse of 2 msec); TE, 3.2 msec; a 128 x 96 matrix (pile encode factor, 6; k-space segment, 16); a 200-mm field of view; and one excitation. RESULTS: Values for left ventricular volume and ejection fraction obtained with ultrafast cine MR imaging correlated well with those obtained with IV-DSA (end-diastolic volume, y = 0.986x - 7.79, r = .985; end-systolic volume, y = 0.863x + 0.71, r = .984; ejection fraction, y = 0.877x + 6.44, r = .887). In the calculation of left ventricular volume by the area-length method, manual tracing of the left ventricular cavity was more difficult when the conventional cine method was used than when the enhanced ultrafast cine method was used. CONCLUSION: Our results show that cardiac multiphase study with horizontal long-axis, first-pass, contrast-enhanced, single breath-hold, cine MR imaging is an accurate and highly reproducible method of evaluating left ventricular volume and ejection fraction.

Angiography, Digital Subtraction↗

PubMed1993

[The study of senile symptoms and home care on the elderly living in Korea].

The purpose of this study is to investigate the daily life and care condition of elderly people living in Korea, and to observe the symptom of senile dementia among them. In the first survey, 713 subjects were screened and 42 subjects were diagnosed as suffering from senile dementia. In the second survey, 26 elderly and their caregivers who agreed to participate in this program, were interviewed. The results were as follows. 1. About 25% of the 713 subjects needed some kind of help in their daily life. 2. 85% of the subjects could go out and about their houses without help. Only 5% of them had a complete or a partial loss of activity. 3. Of all the subjects, 42 were diagnosed as suffering from senile dementia. The prevalence of this disease was calculated at 5.6% in the survey. 4. Each demented elderly person had 5.3 mental symptoms on the average. Depression was observed more among women and violence was observed more among men. 5. The elderly who had a lower CPR score (Caretaker-Patient Relationship Score), indicating a poor relationship between the patient and caretaker, had a higher prevalence of mental symptoms than those who had a higher CPR score.

Aged↗