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Y Inoue

Publications and source records attributed to Y Inoue.

At least 721 records · Page 40Linked to original sources
PubMed1997

Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP.

To relate transients of force by single kinesin molecules with the elementary steps of the ATPase cycle, we measured the time to force generation by kinesin after photorelease of ATP from caged ATP. Kinesin-coated beads were trapped by an infrared laser and brought onto microtubules fixed to a coverslip. Tension was applied to a kinesin-microtubule rigor complex using the optical trap, and ATP was released by flash photolysis of caged ATP with a UV laser. Kinesin started to generate force and move stepwise with a step size of 8 nm at average times of 31, 45, and 79 ms after photorelease of 450, 90, and 18 microM ATP, respectively. The kinetics of force generation were consistent with a two-step reaction: ATP binding, with an apparent second-order rate constant of 0.7 microM-1.s-1, followed by force generation at 45 s-1 per kinesin molecule. The transient rate of force generation was close to the rate of the ATPase cycle in solution, suggesting that the rate-limiting step of ATPase cycle is involved with the force generation.

Adenosine Triphosphate↗
PubMed1997

Identification of sulfated oligosialic acid units in the O-linked glycan of the sea urchin egg receptor for sperm.

The Strongylocentrotus purpuratus sea urchin egg receptor for sperm is a cell surface glycoprotein with a molecular mass of 350 kDa. Recent studies indicate that the sulfated O-linked glycans isolated from the receptor bind to acrosome-reacted sperm. The purified receptor was analyzed with respect to amino acid and carbohydrate content and shown to be composed of 70% carbohydrate by weight. Compositional analysis indicated that both N- and O-linked oligosaccharide chains were present. After peptide:N-glycanase treatment of the receptor to remove most of the N-linked glycan chains, the majority of the sialic acid residues remained associated with the receptor and were shown by several types of experiments to be composed of sulfated oligosialic acid units attached to the O-linked glycan chains of the receptor. Chemical and physical studies on oligosialic chains discovered earlier in the Pronase-generated glycopeptide fraction isolated from the egg cell surface complex of another species of sea urchin, Hemicentrotus pulcherrimus, established that these molecules had the structure: (SO(4)-)-9Neu5Gc alpha2(-->5-O(glycolyl)Neu5Gc alpha2-->)n. Based on comparative and analytical studies, it was concluded that this sulfated oligosaccharide is a component of a GalNAc-containing chain that is O-linked to the polypeptide chain of the sea urchin egg receptor for sperm. Using a competitive inhibition of fertilization bioassay it was shown that the sulfated oligosialic acid chains derived from the S. purpuratus egg cell surface complex inhibited fertilization; the nonsulfated form of this oligosialic chain had little inhibitory activity.

Animals↗
PubMed1997

The mechanism of mutation induction by a hydrogen bond ambivalent, bicyclic N4-oxy-2'-deoxycytidine in Escherichia coli.

The triphosphate of the nucleoside deoxyribosyl dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is known to be incorporated into DNA efficiently by Taq polymerase and is a useful tool for polymerase-mediated in vitro mutagenesis. It is shown here that dP is a potent mutagen in Escherichia coli and Salmonella typhimurium . In E.coli , this deoxycytidine analog induces both GC-->AT and AT-->GC transitions. No induced transversions are observed. It is highly mutagenic in wild-type E.coli, but this is much reduced in a strain lacking thymidine kinase. Mutagenesis induced by dP is efficiently inhibited by the addition of thymidine. Partially purified thymidine kinase from E.coli catalyzes phosphorylation of dP to its 5'-monophosphate. When E.coli was grown in the presence of dP, the nucleoside analog was incorporated into its DNA. The content of dP in DNA was dependent on the concentration of dP added to the medium. The incorporation characteristics of the 5'-triphosphate of dP (dPTP) were also studied using E.coli DNA polymerase I large fragment. The results confirm that this triphosphate can be incorporated opposite A and G in the template with similar efficiencies. This indicates that dP is metabolized as a thymidine analog and that the resulting triphosphate induces a high rate of mutagenesis through replicational errors.

Base Sequence↗
PubMed1997

Catalysis by a new sialidase, deaminoneuraminic acid residue-cleaving enzyme (KDNase Sm), initially forms a less stable alpha-anomer of 3-deoxy-D-glycero-D-galacto-nonulosonic acid and is strongly inhibited by the transition state analogue, 2-deoxy-2, 3-didehydro-D-glycero-D-galacto-2-nonulopyranosonic acid, but not by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid.

Deaminoneuraminic acid residue-cleaving enzyme (KDNase Sm) is a new sialidase that has been induced and purified from Sphingobacterium multivorum. Catalysis by this new sialidase has been studied by enzyme kinetics and 1H NMR spectroscopy. Vmax/Km values determined for synthetic and natural substrates of KDNase Sm reveal that 4-methylumbelliferyl-KDN (KDNalpha2MeUmb, Vmax/Km = 0.033 min-1) is the best substrate for this sialidase, presumably because of its good leaving group properties. The transition state analogue, 2, 3-didehydro-2,3-dideoxy-D-galacto-D-glycero-nonulosonic acid, is a strong competitive inhibitor of KDNase Sm (Ki = 7.7 microM versus Km = 42 microM for KDNalpha2MeUmb). 2-Deoxy-2, 3-didehydro-N-acetylneuraminic acid and 2-deoxy-2, 3-didehydro-N-glycolylneuraminic acid are known to be strong competitive inhibitors for bacterial sialidases such as Arthrobacter ureafaciens sialidase; however, KDNase Sm activity is not significantly inhibited by these compounds. This observation suggests that the hydroxyl group at C-5 is important for recognition of the inhibitor by the enzyme. Reversible addition of water molecule (or hydroxide ion) to the reactive sialosyl cation, presumably formed at the catalytic site of KDNase Sm, eventually gives rise to two different adducts, the alpha- and beta-anomers of free 3-deoxy-D-glycero-D-galacto-nonulosonic acid. 1H NMR spectroscopic studies clearly demonstrate that the thermodynamically less stable alpha-form is preferentially formed as the first product of the cleavage reaction and that isomerization rapidly follows, leading to an equilibrium mixture of the two isomers, the beta-isomer being the major species at equilibrium. Therefore, we propose that KDNase Sm catalysis proceeds via a mechanism common to the known exosialidases, but the recognition of the substituent at C-5 by the enzyme differs.

Flavobacterium↗
PubMed1997

Studies on selectin blockers. 4. Structure-function relationships of sulfated sialyl Lewis X hexasaccharide ceramides toward E-, P-, and L-selectin binding.

In order to clarify the real ligand structure of L-selectin proposed by Rosen et al., we first synthesized 6-sulfated sLe(x) hexasaccharide ceramide 1, 6'-sulfated sLe(x) hexasaccharide ceramide 2, and 6,6'-disulfated sLe(x) hexasaccharide ceramide 3 and examined their binding avidities for L-selectin. As a result, we found that the 6'-sulfated sLe(x) hexasaccharide ceramides 1-3 have similar binding avidities to L-selectin and their binding to L-selectin appeared somewhat stronger than that of sLe(x). For P-selectin, the sulfated sLe(x) derivatives 1-3 showed a similar avidity to sLe(x). On the other hand, 6-sulfated sLe(x) 2 was recognized to E-selectin and the binding avidity was apparently weak as compared to that of sLe(x) hexasaccharide ceramide. Surprisingly, 6'-sulfated and 6,6'-disulfated sLe(x)s 1 and 3 did not bind to E-selectin at all. We constructed the E-selectin-sLe(x) complex model and investigated the binding mode. Namely, the galactose 6'-position was directed toward the negatively charged residues, Glu80 and Asp100. Our results with E-selectin indicate that the replacement of 6'-OH position from anionic charged group to cationic charged one, e.g., amino groups, could have a marked affect on E-selectin recognition. These results could provide useful information for the drug design of selectin blockers.

Animals↗
PubMed1997

Identification of domains on the extrinsic 33-kDa protein possibly involved in electrostatic interaction with photosystem II complex by means of chemical modification.

The extrinsic 33-kDa protein of photosystem II (PSII) was modified with various reagents, and the resulting proteins were checked for the ability to rebind to PSII and to reactivate oxygen evolution. While modification of more than eight carboxyl groups of aspartyl and glutamyl residues with glycine methyl ester did not affect the rebinding and reactivating capabilities, modification of amino groups of lysyl residues with either N-succinimidyl propionate or 2, 4,6-trinitrobenzene sulfonic acid or modification of guanidino groups of arginyl residues with 2,3-butanedione resulted in a loss of rebinding and reactivating capabilities of the 33-kDa protein. Moreover, the number of lysyl and arginyl residues susceptible to modification was significantly decreased when the protein was bound to PSII as compared with when it was free in solution, whereas the number of carboxyl groups modified was little affected. These results suggested that positive charges are important for the electrostatic interaction between the extrinsic 33-kDa protein and PSII intrinsic proteins, whereas negative charges on the protein do not contribute to such interaction. By a combination of protease digestion and mass spectroscopic analysis, the domains of lysyl residues accessible to N-succinimidyl propionate or 2,4, 6-trinitrobenzene sulfonic acid modification only when the 33-kDa protein is free in solution were determined to be Lys4, Lys20, Lys66-Lys76, Lys101, Lys105, Lys130, Lys159, Lys186, and Lys230-Lys236. These domains include those previously reported accessible to N-hydroxysuccinimidobiotin only in solution (Frankel and Bricker (1995) Biochemistry 34, 7492-7497), and may be important for the interaction of the 33-kDa protein with PSII intrinsic proteins.

Amino Acid Sequence↗
PubMed1997

Activity of UDP-GlcNAc:GlcNAc beta 1-->6(GlcNAc beta 1-->2) Man alpha 1-->R[GlcNAc to Man] beta 1-->4N-acetylglucosaminyltransferase VI (GnT VI) from the ovaries of Oryzias latipes (Medaka fish).

UDP-GlcNAc:GlcNAc beta 1-->(GlcNAc beta 1-->2)Man alpha 1-R[GlcNAc to Man] beta 1-->4N-acetylglucosaminyltransferase VI (GnT VI) activity was shown to be present in crude homogenates of Medaka fish (Oryzias latipes) ovaries using UDP-[14C]GlcNAc and synthetic GlcNAc beta 1-->6 (GlcNAc beta 1-->2)Man alpha 1-->6Glc beta 1-->octyl as substrates. Characterization of this activity showed a pH optimum at about pH 7.0 and an absolute requirement for divalent cations. The optimum concentration of Mn2+ was at about 25 mM. This finding is the first report on GnT VI activity in fish; the enzyme has previously been described only in avian tissues.

Animals↗
PubMed1997

Altered intracellular localization of the glutamate receptor channel delta 2 subunit in weaver and reeler Purkinje cells.

The glutamate receptor (GluR) channel delta 2 subunit is expressed abundantly and specifically in cerebellar Purkinje cells. Our previous study demonstrated that the GluR is expressed as early as embryonic day 15 prior to Purkinje cell synaptogenesis, and its protein product accumulates in dendritic spines during normal Purkinje cell maturation. In this study, we examined expression and distribution of the GluR delta 2 in the weaver and reeler mutant cerebelli, which show abnormal cytoarchitecture and neural circuitry. In situ hybridization analysis showed that GluR delta 2 mRNA was expressed in entire Purkinje cells in both mutant mice. Immunohistochemical analysis revealed that intracellular localization of GluR delta 2 was altered in some region of mutant cerebelli. In the cortical surface where Purkinje cells from synapses with parallel fibers, GluR delta 2-immunoreactivity was restricted to dendritic spines of Purkinje cells as observed in normal mice. In contrast, in the subcortical region where granule cells and parallel fibers are absent, the immunoreactivity was found widely in Purkinje dendrites. Thus, the GluR delta 2 protein did not accumulate to the dendritic spines of Purkinje cells lacking synaptic contact with parallel fibers. These results suggest that the expression of both GluR delta 2 mRNA and protein is independent of abnormalities in the mutant cerebelli, but relocalization of the GluR delta 2 protein might depend on the formation of synapses between Purkinje cells and parallel fibers.

Animals↗
PubMed1997

One-point measurement of the peak-to-peak pulsatility index as an indicator for evaluation of infrainguinal bypass procedures.

While duplex scanning has been advocated as the most accurate modality for postoperative graft surveillance, it is time-consuming for evaluating the entire graft. The aim of the present study was to determine which parameter predicts graft failure most simply and precisely, by examining 62 men and 1 woman who collectively underwent 71 infrainguinal arterial bypasses. A total of 212 scannings were obtained using a duplex scanner, and the peak systolic velocity (PSV), PSV ratio, and peak-to-peak pulsatility index (PPI) were analyzed. This analysis revealed 7 occlusions, 9 stenoses, and 1 arteriovenous fistula. When a PSV < 45 cm/s and/or a PSV ratio > 2.0 was defined as graft failure the sensitivity was 84.0% and the specificity was 81.8%: however, a PPI < 7.0 at the midgraft, indicating graft failure, showed a sensitivity of 100% and a specificity of 83.3%. The PPI exhibited better sensitivity and specificity than the PSV, even though the PPI needs only to be measured at the midgraft whereas the PSV should be measured at at least two points. Thus, we believe that the PPI could be the most useful and simple parameter to assess infrainguinal bypass grafts.

Adult↗
PubMed1997

Flow characteristics of soft-tissue vascular anomalies evaluated by direct puncture scintigraphy.

Soft-tissue vascular anomalies such as haemangioma and vascular malformation are treated by surgical resection, arterial embolization or sclerotherapy. Because the effect of sclerotherapy, i.e. the percutaneous injection of sclerosing agents, depends on intralesional haemodynamics, estimation of flow characteristics of soft-tissue vascular anomalies is essential when determining appropriate patient management. However, lesions are at present divided into only two groups: high flow and low flow. We have developed a new method, direct puncture scintigraphy, to evaluate in detail the haemodynamics of vascular anomalies under conditions simulating sclerotherapy. Twenty-six soft-tissue vascular anomalies in 21 patients were studied. After 30 MBq of technetium-99m Sn colloid was injected percutaneously into the intravascular space of the lesion, dynamic imaging was performed for 5 min. A time-activity curve for the lesion was generated, with the infiltrated activity on injection subtracted. A monoexponential curve was fitted to the declining phase of the time-activity curve, and mean vascular transit time (MTT) was obtained. The lesions were classified into high-flow and low-flow lesions based on radionuclide angiography with intravenous injection of 99mTc-labelled red blood cells, and estimates of MTT in the two groups were compared. The imaging procedures were carried out with no major complications, and broad intralesional diffusion of 99mTc-Sn colloid was achieved in most lesions. The high-flow lesions (six lesions) had a short MTT, ranging from 1.6 to 3.4 s, while the low-flow lesions (20 lesions) had a longer MTT, with no overlap between the groups. MTT showed a wide range in low-flow lesions: it was less than 30 s in six lesions and more than 10 min in five other lesions. Direct puncture scintigraphy provides a quantitative indicator of the flow characteristics of soft-tissue vascular anomalies, and may aid in determining treatment strategies for patients with vascular anomalies.

Adolescent↗
PubMed1997

Postoperative delirium following gastrointestinal surgery in elderly patients.

Postoperative delirium is a common complication which can interfere with the surgical treatment and recovery of elderly patients, and is likely to prolong their hospitalization. Unfortunately, there is as yet no completely effective pre- and/or post operative technique of patient care to reduce or prevent postoperative delirium. In this study, 36 patients aged over 70 years undergoing gastrointestinal operations were assessed to examine the relationships between the preoperative cognitive state, the postoperative sleep cycle, and the occurrence of postoperative delirium. All patients were evaluated preoperatively using the revised version of Hasegawa's dementia scale (HDS-R). We correlated those test results and assessed the sleep-wakefulness disturbance postoperatively, to obtain a clinical DMS-III diagnosis of postoperative delirium. The incidence of postoperative delirium was 17% (6/36). The patients who developed postoperative delirium demonstrated preoperative cognitive impairment, and had a short sleep period during the night and a long sleep period during the day. Postoperatively, these results suggest that HDS-R is a useful method of evaluating preoperative cognition in elderly patients. Considering that sleep deficiency is likely to predispose elderly patients to postoperative delirium, techniques to prevent sleep deprivation may be of considerable value in minimizing the incidence of postoperative delirium.

Aged↗
PubMed1997

Pyruvate alleviates toxic effects of ethanol on cells in culture.

Cytotoxic effects of ethanol on cultured human hepatocytes and fibroblasts differed with the type of culture medium used. When cytotoxic effects of ethanol were compared between culture systems using either RPMI-1640 or Dulbecco's modified Eagle's medium (DMEM), the cytotoxicity was more prominent with the former medium than with the latter. A reduction of the cytotoxic effects appeared to be due to the pyruvate contained in DMEM. The protective effect of pyruvate against ethanol toxicity may be related to tricarboxylic acid (TCA) cycle activity because addition of malate to the medium also reduced the cytotoxic effects. Our results suggest that drug cytotoxicity testing in vitro must be done using various types of culture medium.

Cell Division↗
PubMed1997

Interaction between carbamazepine and bromperidol.

OBJECTIVE: The interaction between carbamazepine and bromperidol was studied in 13 schizophrenic inpatients. METHODS: Before carbamazepine addition, the subjects were taking bromperidol 12-24 mg.day-1 for 1-20 weeks. Carbamazepine 400 mg.day-1 was coadministered for 4 weeks, and blood samplings were performed before carbamazepine addition and at weekly intervals after the addition. Plasma concentrations of bromperidol and its reduced metabolite were measured by high-performance liquid chromatography. RESULTS: Carbamazepine significantly decreased plasma concentrations of both bromperidol and reduced bromperidol for all weeks. On average, the plasma concentrations of bromperidol and reduced bromperidol at 4 weeks were 37% and 23% of the corresponding precarbamazepine values. Despite these decreases in plasma concentration, the Clinical Global Impression scores decreased slightly but significantly after carbamazepine addition. CONCLUSION: The present study suggests that carbamazepine decreases plasma concentrations of bromperidol and its reduced metabolite by inducing the metabolism of these compounds. Nevertheless, adjunctive carbamazepine may be useful for schizophrenic patients treated with bromperidol.

Adult↗
PubMed1997

The urinary excretion of arsenic metabolites after a single oral administration of dimethylarsinic acid to rats.

The biotransformation following oral administration of dimethylarsinic acid (DMA), an organoarsenical herbicide and the main metabolite of inorganic arsenic in mammals, was studied in rats. Male F344/DuCrj rats were administered a single dose of DMA (50 mg/kg) orally. Urine was collected at 0, 2, 4, 8, 10, 24, and 48 h after administration by forced urination. Arsenic metabolites in urine were analyzed by ion chromatography with inductively coupled plasma mass spectrometry (IC-ICP-MS). The proportions of urinary elimination of DMA, trimethylarsine oxide (TMAO), methylarsonic acid (MMA), an unidentified arsenic metabolite, and arsenite were determined at various timepoints after administration. Unmetabolized DMA was the most common form excreted during the first 4 h. Thereafter, a gradual decrease in the proportion of DMA was observed, while progressive increases in those of TMAO, the unidentified metabolite, and arsenite occurred. The proportion of TMAO excreted amounted to over 50% of all arsenic in urine between 6 and 24 h. The proportion of the unidentified metabolite and arsenite were each approximately 10% at 10 and 24 h after administration. The findings indicate that DMA administered to rats was initially excreted as unchanged DMA, and later as the methylated metabolite, TMAO. Arsenite, a demethylated metabolite of DMA, also was excreted later than elimination of DMA and TMAO. The hypothesis of demethylation by intestinal microorganisms can be supported by comparing the metabolites following oral and intraperitoneal administration. The unidentified metabolite was readily decomposed by HCl but was left unchanged by NaOH; these findings suggest that it was present in a complexed form in urine.

Administration, Oral↗
PubMed1997

Efficient production of a functional mouse/human chimeric Fab' against human urokinase-type plasminogen activator by Bacillus brevis.

Expression/secretion vectors for the production of Fab' and single-chain (sc) Fab' by Bacillus brevis have been constructed. For the production of Fab', the cDNAs encoding the L chain and Fd' fragment (Fd with the hinge region) of a mouse-human chimeric Fab' against human urokinase-type plasminogen activator were fused directly with the translation-start and signal-peptide-encoding regions of the mwp gene, the gene for one of the major cell-wall proteins of Bacillus brevis. The two fused genes were placed tandemly downstream from the promoter of the cell-wall protein gene operon (cwp) of B. brevis. For the production of scFab', the two cDNAs were linked with a synthetic oligonucleotide encoding a flexible peptide linker of 17 or 24 amino acids, and fused with the translation start and signal-peptide-encoding regions of the mwp gene. Fab' was efficiently produced by B. brevis, being accumulated at a level of 100 mg/l in the culture medium in a simple shake-flask culture, which is the highest level obtained so far for a gram-positive bacterium. On the other hand, the scFab' remained at a level of a few milligrams per liter in the culture medium. The Fab' produced by B. brevis showed comparable antigen-binding activity to that of the parental antibody. The L chain and Fd' fragment, constituting the Fab', had the correct N-terminal amino acid sequences. These results indicate that B. brevis is a very promising host for the production of native Ig fragments.

Amino Acid Sequence↗