Biomedical subjects

A Yokota

Publications and source records attributed to A Yokota.

At least 55 records · Page 3Linked to original sources

PubMed2000

Molecular cloning and high-level expression in Escherichia coli of fungal alpha-galactosidase from Absidia corymbifera IFO 8084.

A cDNA encoding the alpha-galactosidase of Absidia corymbifera IFO 8084 was cloned and sequenced. The cloned DNA has a single open-reading frame consisting of 2190 base pairs, and the deduced amino acid sequence revealed that the mature enzyme consisted of 730 amino acid residues with a molecular mass of 82,712 Da. The native structure of the alpha-galactosidase of A. corymbifera IFO 8084 was determined to be a tetramer. Comparison with amino acid sequences of other alpha-galactosidase showed high homology with sequences of members of family 36. An expression vector, pET32Trx/galalpha, was constructed by introducing the cDNA coding region into a thioredoxin fusion system, pET32-Ek/LIC. The resulting transformant, pET32Trx/galalpha, overproduced the active enzyme as a thioredoxin fused form in the host Escherichia coli. By using His-binding metal affinity chromatography, recombinant alpha-galactosidase was purified to homogeneity in a single step. The purified recombinant fusion alpha-galactosidase showed properties very similar to the native alpha-galactosidase from A. corymbifera IFO 8084.

Journal Article↗

PubMed2000

Microbial demetallization of crude oil: nickel protoporphyrin disodium as a model organo-metallic substrate.

A soil isolate designated as YA-1 strain was selected for its ability to degrade nickel protoporphyrin disodium (NiPPDS). The strain was capable of utilizing NiPPDS as the sole source of carbon. This strain, a gram-negative aerobic rod, was identified as Pseudomonas azelaica YA-1 based on the result of its 16S rRNA analysis. Product analyses by HPLC showed that this strain can decompose the porphyrin ring to which a metal ion is bound. However, the use of whole bacterial cells cannot result in extensive NiPPDS degradation; therefore, the YA-1 enzyme was extracted and purified. This NiPPDS-degrading enzyme named as protoporphyrinase was purified from P. azelaica YA-1 by ammonium sulfate fractionation and sequential chromatographies using DEAE Toyopearl 650 M, CM Toyopearl 650 M and Biogel P-60 columns, with a yield of 11.3% based on the enzyme activity and an overall purification of 498-fold. The molecular weight of this enzyme is estimated to be 39,000 Da by SDS-PAGE and 34,000 Da by gel filtration. The optimum pH and temperature for the enzyme were 7.0 and 30 degrees C, respectively. The activity was stable at pH 2.0-11.0 and at temperatures below 50 degrees C. The enzyme activity was inactivated by ferric chloride, potassium ferricyanide, ZnCl2 and CdCl2.

Journal Article↗

PubMed2000

Preservation of tumour oxygen after hyperbaric oxygenation monitored by magnetic resonance imaging.

Hyperbaric oxygen (HBO) has been proposed to reduce tumour hypoxia by increasing the dissolved molecular oxygen in tissue. Using a non-invasive magnetic resonance imaging (MRI) technique, we monitored the changes in MRI signal intensity after HBO exposure because dissolved paramagnetic molecular oxygen itself shortens the T1 relation time. SCCVII tumour cells transplanted in mice were used. The molecular oxygen-enhanced MR images were acquired using an inversion recovery-preparation fast low angle shot (IR-FLASH) sequence sensitizing the paramagnetic effects of molecular oxygen using a 4.7 tesla MR system. MR signal of muscles decreased rapidly and returned to the control level within 40 min after decompression, whereas that of tumours decreased gradually and remained at a high level 60 min after HBO exposure. In contrast, the signal from the tumours in the normobaric oxygen group showed no significant change. Our data suggested that MR signal changes of tumours and muscles represent an alternation of extravascular oxygenation. The preserving tumour oxygen concentration after HBO exposure may be important regarding adjuvant therapy for cancer patients.

Animals↗

PubMed2000

The role of structural intersubunit microheterogeneity in the regulation of the activity in hysteresis of ribulose 1, 5-bisphosphate carboxylase/oxygenase.

Many enzymes are composed of subunits with the identical primary structure. It has been believed that the protein structure of these subunits is the same. Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) comprises eight large subunits with the identical amino acid sequence and eight small subunits. Rotation of the side chains of the lysine residues, Lys-21 and Lys-305, in each of the eight large subunits in spinach RuBisCO in two ways produces microheterogeneity among the subunits. These structures are stabilized through hydrogen bonds by water molecules incorporated into the large subunits. This may cause different effects upon catalysis and a hysteretic, time-dependent decrease in activity in spinach RuBisCO. Changing the amino acid residues corresponding to Lys-21 and Lys-305 in non-hysteretic Chromatium vinosum RuBisCO to lysine induces hysteresis and increases the catalytic activity from 8.8 to 15.8 per site per second. This rate is approximately five times higher than that of the higher-plant enzyme.

Catalysis↗

PubMed2000

Determination of the rate of photoreduction of O2 in the water-water cycle in watermelon leaves and enhancement of the rate by limitation of photosynthesis.

A study was performed to determine how the electron fluxes for the photosynthetic carbon reduction (PCR) and the photorespiratory carbon oxidation (PCO) cycles affect the photoreduction of O2 at PSI, which is the limiting step in the water-water cycle. Simultaneous measurements were made of CO2-gas exchange, transpiration and quantum yield of PSII [phi(PSII)] using leaves of watermelon (Citrullus lanatus). The total electron flux in PSII[Je(PSII)], as estimated from phi(PSII), was always larger than the total electron flux required for the PCR and PCO cycles at various partial pressures of CO2 and O2 and 1,100 micromol photons m(-2)s(-1). This observation suggested the existence of an alternative electron flux (Ja). Ja was divided into O2-dependent [Ja(O2-depend)] and O2-independent [Ja(O2-independ)] components. The magnitude of half Ja(O2-depend), 7.5 to 9.5 micromol e- m(-2)s(-1), and its apparent Km for O2, about 8.0 kPa, could be accounted for by the photoreduction of O2 at PSI either mediated by ferredoxin or catalyzed by monodehydroascorbate reductase. The results indicated that Ja(O2-depend) was driven by the water-water cycle. A decrease in the intercellular partial pressure of CO2 from 23 to 5.0 Pa at 21 kPa O2 enhanced Ja(O2-depend) by a factor of 1.3. Saturation of the activities of both the PCR and PCO cycles by increasing the photon flux density induced Ja. These results indicate the electron flux in PSII that exceeds the flux required for the PCR and PCO cycles induces the photoreduction of O2 in the water-water cycle.

Carbon Dioxide↗

PubMed2000

Responses of wild watermelon to drought stress: accumulation of an ArgE homologue and citrulline in leaves during water deficits.

Wild watermelon from the Botswana desert had an ability to survive under severe drought conditions by maintaining its water status (water content and water potential). In the analysis by two-dimensional electrophoresis of leaf proteins, seven spots were newly induced after watering stopped. One with the molecular mass of 40 kilodaltons of the spots was accumulated abundantly. The cDNA encoding for the protein was cloned based on its amino-terminal sequence and the amino acid sequence deduced from the determined nucleotide sequences of the cDNA exhibited homology to the enzymes belong to the ArgE/DapE/Acy1/Cpg2/YscS protein family (including acetylornithine deacetylase, carboxypeptidase and aminoacylase-1). This suggests that the protein is involved in the release of free amino acid by hydrolyzing a peptidic bond. As the drought stress progressed, citrulline became one of the major components in the total free amino acids. Eight days after withholding watering, although the lower leaves wilted significantly, the upper leaves still maintained their water status and the content of citrulline reached about 50% in the total free amino acids. The accumulation of citrulline during the drought stress in wild watermelon is an unique phenomenon in C3-plants. These results suggest that the drought tolerance of wild watermelon is related to (1) the maintenance of the water status and (2) a metabolic change to accumulate citrulline.

Amidohydrolases↗

PubMed2000

Purification and characterization of chloroplast dehydroascorbate reductase from spinach leaves.

Green leaves of plants require the high-level activity that can regenerate ascorbate during photosynthesis. One of such enzyme is dehydroascorbate reductase (DHAR), but the molecular and enzymological properties of the enzyme remain to be fully characterized. In this study, we showed that two major DHAR existed in spinach leaves. The two DHARs occupied at least over 90% of total DHAR activity. The amount of the two DHARs was almost the same. We purified both DHARs from spinach leaves. One form of DHAR originated in chloroplasts; the other occurred in the subcellular compartment other than chloroplasts. The chloroplast DHAR had Km values of 70 microM and 1.1 mM for dehydroascorbate and reduced glutathione, respectively. The specific activity of the purified enzyme corresponded to 360 micromol of ascorbate formed per milligram of protein per minute. These properties were quite different from those of trypsin inhibitor, which has been reported to be the plastid DHAR. The other DHAR had the very similar properties to those of chloroplast DHAR. Chloroplast and the other DHARs functioned as a monomer with molecular masses of 26 kDa and 25 kDa, respectively. cDNA for the chloroplast DHAR was cloned with the determined amino-terminal amino acid sequence. The primary sequence predicted from the cDNA included the plastid-targeting sequence. Finally, the significance of chloroplast DHAR in the regeneration of ascorbate is discussed.

Amino Acid Sequence↗

PubMed2000

Secondary mitochondrial dysfunction after transient intrauterine ischemia in the fetal rat brain.

OBJECTIVE: Recirculation following transient intrauterine ischemia has previously been found to cause partial recovery and secondary deterioration of cellular bioenergetic states in the fetal rat brain. Our objective was to assess whether secondary bioenergetic failure is due to mitochondrial dysfunction. STUDY DESIGN: Cerebral cortical tissues were obtained from 20-day-old fetal rats at the end of 30 minutes of intrauterine ischemia (n = 6) and after 1 hour (n = 6), 2 hours (n = 6), and 4 hours (n = 6) of recirculation. Mitochondrial respiratory activities were measured polarographically using homogenates. RESULTS: Mitochondrial activities decreased to 45% of sham-operated controls at the end of ischemia. Recirculation (1 hour) brought about partial recovery, but continued reflow (2 hours and 4 hours) was associated with a secondary deterioration of mitochondrial activities. CONCLUSION: The results indicate that although during the early time period after ischemia mitochondrial respiratory capacity is restored, secondary mitochondrial dysfunction develops in the immature rat brain.

Animals↗

PubMed2000

Changes in uterine and ovarian arterial impedance during the periovulatory period in conception and nonconception cycles.

OBJECTIVE: To evaluate whether the Doppler velocimetry of uterine and ovarian arteries during the periovulatory period in conception cycles differs from that in nonconception cycles. DESIGN: Transvaginal color Doppler sonography was used to assess uterine and ovarian perfusion in 63 infertile women during the periovulatory period. RESULTS: In the conception cycles (n = 16), the PI values of uterine arteries at the day of ovulation were significantly less than those in nonconception cycles (n = 47). There were no significant differences in the PI values of ovarian arteries between the conception and nonconception cycles. CONCLUSIONS: In the present study, the conception cycles showed lower vascular impedance in the uterine during the periovulatory period than did the nonconception cycles. This endometrial perfusion presents an accurate noninvasive assay of uterine receptivity that can be used to predict the successful outcome of implantation and to reveal unexplained infertility problems.

Adult↗

PubMed2000

Purification and characterization of 2,6-beta-D-fructan 6-levanbiohydrolase from Streptomyces exfoliatus F3-2.

Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a beta-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60 degrees C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50 degrees C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only beta-2,6-linkage of levan, but also beta-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-beta-D-fructan 6-levanbiohydrolase (EC 3.2.1.64).

Disaccharides↗

PubMed2000

Cholate resistance in Lactococcus lactis is mediated by an ATP-dependent multispecific organic anion transporter.

The cholate-resistant Lactococcus lactis strain C41-2, derived from wild-type L. lactis MG1363 through selection for growth on cholate-containing medium, displayed a reduced accumulation of cholate due to an enhanced active efflux. However, L. lactis C41-2 was not cross resistant to deoxycholate or cationic drugs, such as ethidium and rhodamine 6G, which are typical substrates of the multidrug transporters LmrP and LmrA in L. lactis MG1363. The cholate efflux activity in L. lactis C41-2 was not affected by the presence of valinomycin plus nigericin, which dissipated the proton motive force. In contrast, cholate efflux in L. lactis C41-2 was inhibited by ortho-vanadate, an inhibitor of P-type ATPases and ATP-binding cassette transporters. Besides ATP-dependent drug extrusion by LmrA, two other ATP-dependent efflux activities have previously been detected in L. lactis, one for the artificial pH probe 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) and the other for the artificial pH probe N-(fluorescein thio-ureanyl)-glutamate (FTUG). Surprisingly, the efflux rate of BCECF, but not that of FTUG, was significantly enhanced in L. lactis C41-2. Further experiments with L. lactis C41-2 cells and inside out membrane vesicles revealed that cholate and BCECF inhibit the transport of each other. These data demonstrate the role of an ATP-dependent multispecific organic anion transporter in cholate resistance in L. lactis.

Adenosine Triphosphate↗

PubMed2000

Cholic acid is accumulated spontaneously, driven by membrane deltapH, in many lactobacilli.

Many lactobacilli from various origins were found to apparently lack cholic acid extrusion activity. Cholic acid was accumulated spontaneously, driven by the transmembrane proton gradient. Accumulation is a newly identified kind of interaction between intestinal microbes and unconjugated bile acids and is different from extrusion and modification, which have been described previously.

Biological Transport↗

PubMed2000

Characterization of a novel gene encoding a putative single zinc-finger protein, ZIM, expressed during the reproductive phase in Arabidopsis thaliana.

By differential screening of an arrayed normalized cDNA library from the inflorescence apex in Arabidopsis, a cDNA clone having a deduced amino acid sequence with a motif for a zinc finger was isolated as one of the genes expressed specifically in the reproductive phase. The deduced protein has a modular structure with a putative single C2-C2 zinc-finger motif distantly related to a GATA-1-type finger, a basic region with a sequence resembling a nuclear localization signal, and an acidic region. The gene seemed to have been formed by the exon-shuffling during its molecular evolution, since individual domains are encoded by discrete exons. RNA gel blot analysis showed its expression in shoot apex and flowers in the reproductive phase. The gene was named ZIM for Zinc-finger protein expressed in Inflorescence Meristem. The nuclear localization of ZIM was detected using GFP as a reporter. These results suggest that ZIM is a putative transcription factor involved in inflorescence and flower development.

Amino Acid Sequence↗

PubMed2000

Systematic isolation of highly transcribed genes in inflorescence apices in Arabidopsis thaliana from an equalized cDNA library.

To analyze plant meristem functions, we systematically isolated thirteen cDNA clones for transcripts that are highly expressed in the inflorescence apices of Arabidopsis by differential screening, using an equalized cDNA library with the clones arrayed on membranes. Their deduced amino acid sequences indicate important functions for proteins in determining the cell wall architecture, translation capacity and mitotic activity in the shoot apex during reproductive growth.

Arabidopsis↗

PubMed2000

Gordonan, an acidic polysaccharide with cell aggregation-inducing activity in insect BM-N4 cells, produced by Gordonia sp.

An acidic polysaccharide, termed gordonan, was isolated from the culture medium of Gordonia sp. as an inducer of cell aggregation in an insect cell line, BM-N4. Gordonan had an average molecular weight of 5 x 10(6) and its structure was identified as -->3)-4-O-(1-carboxyethyl)-beta-D-Manp-(1-->4)-beta-D-GlcAp-(1-->4)-beta-D-Glcp-(1--> mainly by acid hydrolysis experiments and NMR analysis. It induces cell aggregation at the concentration of 4 microg/ml. A partially hydrolyzed polysaccharide derived from gordonan with a molecular weight of 5 x 10(5) showed weak activity, while any fragment molecules with lower molecular weights prepared from gordonan showed no activity.

Actinomycetales↗

PubMed2000

Group I intron located in PR protein homologue gene in Youngia japonica.

A Youngia japonica strain had a group I intron that was suggested to have been transferred from Protomyces inouyei, a pathogenic fungus of Y. japonica. It was located in the miraculin homologue coding gene by reverse complementation. The deduced amino acid sequence of this miraculin homologue of Y. japonica was similar to the amino acid sequences of tobacco and tomato pathogenesis-related proteins.

Amino Acid Sequence↗

PubMed2000

Changes in maternal lipid peroxidation levels and antioxidant enzymatic activities before and after delivery.

OBJECTIVE: Our goal was to characterize the changes in maternal lipid peroxidation levels and antioxidant enzymatic activities before and after delivery. METHODS: Predelivery and 1, 24, and 48 hours post-partum plasma concentrations of malondialdehyde, erythrocyte enzyme superoxide dismutase, glutathione peroxidase and catalase were measured in uncomplicated pregnancies. RESULTS: Malondialdehyde levels increased slightly from predelivery to 24 hours post-partum and then decreased significantly at 48 hours post-partum. At one hour post-partum superoxide dismutase and catalase levels increased significantly to about 125% and 170% of predelivery levels, respectively. Thereafter, these values decreased significantly from one hour to 48 hours post-partum. The relative changes in superoxide dismutase and catalase levels at one hour post-partum compared to predelivery values correlated significantly with the duration of labor. CONCLUSION: The results suggest that the uncontrolled lipid peroxidation caused by reactive oxygen species, which are produced in consequence of tissue reoxygenation, may occur during labor and that prolonged labor, may cause maternal oxidative stress.

Adult↗

PubMed2000

Development of the cavernous sinus in the fetal period: a morphological study.

The development and morphological structure of the lateral sellar compartment (LSC), an interdural space containing the cavernous sinus (CS), cranial nerves, and internal carotid artery (ICA), was investigated by histological examination of sections of the LSC and cerebral venograms from human fetal specimens. Twenty-eight LSC coronal sections were obtained from 14 fetuses of 13-32 weeks' gestation. Venograms of 11 other fetuses of 13-32 weeks' gestation were studied to observe changes in venous drainage. The CS appeared as a collection of small venous canals with an endothelial layer. These venous canals gradually became much larger through expansion and unification. The CS and basilar venous plexus were demonstrated as a faint cluster of small vessels on venograms obtained after 13 weeks' gestation. The dura mater increased in thickness and collagen fiber networks developed around all the components in the LSC after 23 weeks' gestation. The LSC lateral wall could not be histologically differentiated as separate multiple layers. Branching and joining of the cranial nerve fascicles were completed with the envelopment of collagen fibers after 23 weeks' gestation. The ICA at 13-15 weeks' gestation ran straight within the LSC, becoming tortuous before birth. CS formation occurs through the development of venous canals without smooth muscle layers, followed by web-formation by collagen fibers in the mesenchymal interstices. LSC formation, including the dense dura mater and an internal structure like that seen in the adult, is largely completed before birth.

Adult↗