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Biomedical subjects

S Enomoto

Publications and source records attributed to S Enomoto.

At least 109 records · Page 6Linked to original sources
PubMed1996

High-affinity calcium-binding site in the gama-carboxyglutamic acid domain of bovine factor VII.

The calcium-mediated interaction of factor VIIa with tissue factor is considered to be the primary trigger of blood coagulation. To determine the role of calcium ions in the action of factor VII, we prepared monoclonal antibodies whose binding to factor VII was calcium-dependent. A monoclonal antibody designated C6 strongly inhibited factor VII-induced clotting at a molar ratio of factor VII to antibody of 1:1. The half-maximal binding of factor VII to the C6 antibody was observed at a concentration of calcium ions of 80 microM. Proteolytic fragments of factor VII were assayed for their ability to inhibit competitively the binding of 125I-factor VII to immobilized C6 antibody. The binding was inhibited by increasing amounts of factor VII, by a fragment that contained the gamma-carboxyglutamic acid (Gla) domain linked to first epidermal growth factor-like domain, and by a Gla domain peptide (residues 1-41), over a range of concentration of 10(-9) to 10(-7) M. The antigenic site recognized by the monoclonal antibody C6, which was generated upon the high-affinity binding of calcium ions, was located in the Gla domain. The C6 antibody inhibited the activation of factor X and the amidolytic activity of factor VIIa in the presence of tissue factor. These results demonstrate that a high-affinity calcium-binding site(s) is located in the Gla domain of factor VII, which is concerned with the initiation of tissue factor-mediated blood coagulation by factor VIIa.

1-Carboxyglutamic Acid↗
PubMed1996

Identification of type I and type II serine/threonine kinase receptors for growth/differentiation factor-5.

Growth/differentiation factor-5 (GDF-5) is a member of the bone morphogenetic protein (BMP) family, which plays an important role in bone development in vivo. Mutations in the GDF-5 gene result in brachypodism in mice and Hunter-Thompson type chondrodysplasia in human. BMPs transduce their effects through binding to two different types of serine/threonine kinase receptors, type I and type II. However, binding abilities appear to be different among the members of the BMP family. BMP-4 binds to two different type I receptors, BMP receptors type IA (BMPR-IA) and type IB (BMPR-IB), and a type II receptor, BMP receptor type II (BMPR-II). In addition to these receptors, osteogenic protein-1 (OP-1, also known as BMP-7) binds to activin type I receptor (ActR-I) as well as activin type II receptors (ActR-II and ActR-IIB). Here we investigate the binding and signaling properties of GDF-5 through type I and type II receptors. GDF-5 induced alkaline phosphatase activity in a rat osteoprogenitor-like cell line, ROB-C26. 125I-GDF-5 bound to BMPR-IB and BMPR-II but not to BMPR-IA in ROB-C26 cells and other nontransfected cell lines. Analysis using COS-1 cells transfected with the receptor cDNAs revealed that GDF-5 bound to BMPR-IB but not to the other type I receptors when expressed alone. When COS-1 cells were transfected with type II receptor cDNAs, GDF-5 bound to ActR-II, ActR-IIB, and BMPR-II but not to transforming growth factor-beta type II receptor. In the presence of type II receptors, GDF-5 bound to different sets of type I receptors, but the binding was most efficient to BMPR-IB compared with the other type I receptors. Moreover, a transcriptional activation signal was efficiently transduced by BMPR-IB in the presence of BMPR-II or ActR-II after stimulation by GDF-5. These results suggest that BMPR-IB mediates certain signals for GDF-5 after forming the heteromeric complex with BMPR-II or ActR-II.

Alkaline Phosphatase↗
PubMed1996

Multitracer study on uptake and excretion of trace elements in rats.

The multitracer technique was first applied to the investigation of the uptake and excretion behaviour of trace elements in rats. A multitracer solution, prepared by irradiation of a gold target with a 14N-beam from the RIKEN Ring Cyclotron, was orally administered to male Wistar rats. The uptake and excretion rates of 23 elements, Be, Mn, Co, Zn, As, Rb, Sr, Y, Zr, Ce, Pm, Eu, Gd, Tb, Er, Tm, Yb, Lu, Hf, W, Re, Ir and Pt, were simultaneously determined under strictly identical experimental conditions. For some of the elements, the results obtained were consistent with previous reports on uptake and excretion of the elements in animals. For the other elements, unique behaviour was revealed for the first time as described in the present work. These results show that the multitracer technique has excellent reliability and versatility for a comparative study of the uptake and excretion of many different elements in animals.

Animals↗
PubMed1996

Bone uptake of vanadium in mice: simultaneous tracing of V, Se, Sr, Y, Zr, Ru and Rh using a radioactive multitracer.

The radioactive multitracer technique, which enables simultaneous tracing of a number of elements in a variety of bio-systems and an accurate comparison of their biobehavior, was employed in a study on uptake behavior of trace elements in various mice tissues, focusing on uptake of V by bone. We found that the bone uptake behavior of trace V closely resembles that of trace Sr and Y. It is suggested that V is a bone-seeking element which probably plays an important role in bone formation. The multitracer technique was thus shown to be useful for comparative studies of behavior of trace elements in living organisms.

Animals↗
PubMed1996

Rapid rewarming causes an increase in the cerebral metabolic rate for oxygen that is temporarily unmatched by cerebral blood flow. A study during cardiopulmonary bypass in rabbits.

BACKGROUND: Jugular venous hemoglobin desaturation during the rewarming phase of cardiopulmonary bypass is associated with adverse neuropsychologic outcome and may indicate a pathologic mismatch between cerebral blood flow (CBF) and cerebral metabolic rate for oxygen (CMRO2). In some studies, rapid rewarming from hypothermic cardiopulmonary bypass results in greater jugular venous hemoglobin desaturation. The authors wished to determine if rewarming rate influences the temperature dependence of CBF and CMRO2. METHODS: Anesthetized New Zealand white rabbits, cooled to 25 degrees C on cardiopulmonary bypass, were randomized to one of two rewarming groups. In the fast group (n = 9), aortic blood temperature was made normothermic within 4 min. In the slow group (n = 9), aortic blood temperature was made normothermic over 25 min. Cerebral blood flow (microspheres) and CMRO2 (Fick) were determined at baseline (25 degrees C), and at brain temperatures of 28 degrees, 31 degrees, 34 degrees, and 37 degrees C during rewarming. RESULTS: Systemic physiologic variables appeared similar between groups. At a brain temperature of 28 degrees C, CMRO2 was 47% greater in the fast rewarming group than in the slow group (2.2 +/- 0.5 vs. 1.5 +/- 0.2 ml O2.100 g-1. min-1, respectively; P = 0.01), whereas CBF did not differ (48 +/- 18 vs. 49 +/- 8 ml.100 g-1.min-1, respectively; P = 0.47). Throughout rewarming, CBF increased as a function of brain temperature but was indistinguishable between groups. Cerebral metabolic rate for oxygen differences between groups decreased as brain temperatures increased. CONCLUSIONS: Cerebral venous hemoglobin desaturation with rapid rewarming is caused by an increase in CMRO2 that is temporarily greater than the increase in CBF. This mismatch may indicate a transient abnormality in flow-metabolism coupling, or the effect of temperature gradients on oxygen transfer from hemoglobin to brain.

Animals↗
PubMed1996

Prediction of the human pharmacokinetics of troglitazone, a new and extensively metabolized antidiabetic agent, after oral administration, with an animal scale-up approach.

We have attempted to predict the human pharmacokinetics of troglitazone after oral administration based on animal data. Troglitazone is a new antidiabetic agent that exhibits a high-metabolic clearance and is metabolized mainly in the liver to sulfate and glucuronide conjugates. The prediction of the area under the plasma concentration-time curve (AUCp.o.) and bio-availability (F) in humans after oral administration was initially attempted by use of allometric equations involving the oral plasma clearance of total (CLp.o.) or unbound drug (CLp.o.,fu), or the hepatic intrinsic clearance of unbound drug (CLuint) and animal body weight. The exponents in the allometric equations between the clearances and body weights were 0.63 to 0.82 with high correlation coefficients (r > .98), and there was no marked difference in predictability by the three methods. Next, the prediction of the range of plasma profiles after oral administration to humans was attempted by the following series of steps: (1) calculation of the exponent and coefficients in the allometric relationships between body weight and parameters, such as total body plasma clearance (CLi.v.) and various distribution volumes (Vss, V beta and Vc) based on animal data; (2) estimation of the absorption rate constant (ka) from allometric relationship to body weight, and estimation of F value from the predicted AUCp.o. (3) description of the plasma concentration-time profiles after oral administration by an equation involving the allometric exponents and coefficients, ka, F and body weight. The observed and simulated plasma profiles were similar and the predicted AUCp.o. values were 60 to 120% of those observed. These methodologies will be useful for predicting the human pharmacokinetics after oral dosing from animal data.

Administration, Oral↗
PubMed1995

Dual brain stem projection from the cortical masticatory area in guinea-pig.

The cortical masticatory area (CMA) in the guinea pig is subdivided into the anterior and posterior parts (A-CMA and P-CMA), based on the pattern of the CMA-induced rhythmical digastric EMG burst and the cytoarchitecture. The anterograde tracing of horseradish peroxidase from the A-CMA and P-CMA revealed, in addition to a common projection to the region around the trigeminal motor nucleus and the parvicellular reticular formation bilaterally, a massive projection to the ipsilateral superior colliculus (SC) from the A-CMA but not from the P-CMA. The results suggest a dual brain stem projection system from the CMA: the direct pyramidal route from the P-CMA and the SC-mediated indirect route from the A-CMA.

Animals↗
PubMed1995

A class of single-stranded telomeric DNA-binding proteins required for Rap1p localization in yeast nuclei.

We have identified a class of proteins that bind single-stranded telomeric DNA and are required for the nuclear organization of telomeres and/or telomere-associated proteins. Rlf6p was identified by its sequence similarity to Gbp1p, a single-stranded telomeric DNA-binding protein from Chlamydomonas reinhardtii. Rlf6p and Gbp1p bind yeast single-stranded G-strand telomeric DNA. Both proteins include at least two RNA recognition motifs, which are found in many proteins that interact with single-stranded nucleic acids. Disruption of RLF6 alters the distribution of repressor/activator protein 1 (Rap1p), a telomere-associated protein. In wild-type yeast cells, Rap1p localizes to a small number of perinuclear spots, while in rlf6 cells Rap1p appears diffuse and nuclear. Interestingly, telomere position effect and telomere length control, which require RAP1, are unaffected by rlf6 mutations, demonstrating that Rap1p localization can be uncoupled from other Rap1p-dependent telomere functions. In addition, expression of Chlamydomonas GBP1 restores perinuclear, punctate Rap1p localization in rlf6 mutant cells. The functional complementation of a fungal gene by an algal gene suggests that Rlf6p and Gbp1p are members of a conserved class of single-stranded telomeric DNA-binding proteins that influence nuclear organization. Furthermore, it demonstrates that, despite their unusual codon bias, C. reinhardtii genes can be efficiently translated in Saccharomyces cerevisiae cells.

Amino Acid Sequence↗
PubMed1995

In vivo biological behavior of a water-miscible fullerene: 14C labeling, absorption, distribution, excretion and acute toxicity.

BACKGROUND: Water-soluble fullerenes have recently been shown to exhibit considerable in vitro biological activity including cytotoxicity, site-selective DNA cleavage and inhibition of HIV protease. To assess the potential of these compounds as drugs, studies on the in vivo behavior of fullerenes are needed. We therefore set out to synthesize a radiolabeled, water-soluble fullerene, in order to obtain data on the oral absorption, distribution and excretion of this class of compounds. RESULTS: We synthesized a 14C-labeled water-soluble [60]fullerene using dipolar trimethylenemethane, which undergoes cycloaddition to [60]fullerene. When administered orally to rats, this compound was not efficiently absorbed and was excreted primarily in the feces. When injected intravenously, however, it was distributed rapidly to various tissues, and most of the material was retained in the body after one week. The compound was also able to penetrate the blood-brain barrier. Acute toxicity of the water-miscible fullerene was found to be quite low. CONCLUSIONS: Although the water-soluble fullerenes (and possibly their simple metabolites) are not acutely toxic, they are retained in the body for long periods, raising concerns about chronic toxic effects. The fact that fullerenes distribute rapidly to many tissues suggests that they may eventually be useful to deliver highly polar drugs through membranes to a target tissue, however, and they may even have applications in the delivery of drugs to the brain. Recent advances in fullerene synthetic chemistry may also make it possible to control fullerene absorption/excretion profiles in the future.

Administration, Oral↗
PubMed1995

Production of functional human bone morphogenetic protein-2 using a baculovirus/Sf-9 insect cell system.

A clone of a human Bone Morphogenetic Protein-2 (hBMP-2) cDNA was obtained from a cDNA library established from human dental pulp cells. After subcloning hBMP-2 cDNA into Autographa californica nuclear polyhedrosis virus, the recombinant baculovirus was transfected to Sf-9 cells. Immuno-reactive recombinant hBMP-2 (rhBMP-2) was detected by a polyclonal antibody against Xenopus BMP-2 in the transfected insect cells but not in the culture media. Three days after treatment with the lysate of the transfected Sf-9 cells, increase in alkaline phosphatase activity of a murine stromal cell line, ST2, was detected. Subcutaneous implantation of rhBMP-2 produced in the insect cells induced formation of cartilage, bone and bone marrow in the rats. The present data indicated that the rhBMP-2 preparation produced in the insect Sf-9 cells had a comparable activity to that produced in mammalian cells.

Alkaline Phosphatase↗
PubMed1994

Expression on outer membranes of mannose residues, which are involved in osteoclast formation via cellular fusion events.

Osteoclast, the bone-resorbing cell, is formed from hematopoietic precursors via cell-cell fusion. To evaluate the possibility that under certain specific conditions mannose residues may be expressed on the mammalian cell surface, we examined the action of pradimicin derivatives, which bind specific sugars such as the mannose residue, on the formation of osteoclast induced in the coculture of mouse spleen cells with mouse stromal cells, a process in which cell-cell fusion is involved. Osteoclast formation was inhibited by treatment of this coculture system with pradimicin at the later stage (day 4-7), and this inhibition was specifically abrogated by mannose-rich yeast mannan. During the 8-day cocultivation, osteoclast formation was blocked by the pradimicin on days 6 and 7, when mononuclear preosteoclasts fused into multinucleated osteoclasts. With an interactive laser cytometer ACAS570, fluorescein isothiocyanate-labeled pradimicin was observed to bind osteoclast progenitors at the fusion stage and to have no binding affinity for osteoclast progenitors at the early stage (day 0-3) or for osteoclasts, which were formed after performing fusion between mononuclear preosteoclasts. These results suggest that mannose residues were expressed on outer membranes of monocytes under pathophysiological conditions and that they were involved in the osteoclast formation via cellular membrane fusion events.

Animals↗
PubMed1994

TEL+CEN antagonism on plasmids involves telomere repeat sequences tracts and gene products that interact with chromosomal telomeres.

In Saccharomyces cerevisiae, circular plasmids that include either a centromere (CEN-plasmids) or a telomere sequence (TEL-plasmids) segregate more efficiently than circular ARS-plasmids. In contrast, circular plasmids that include both telomere and centromere sequences were unstable, a property we term TEL+CEN antagonism. TEL+CEN antagonism required a telomere repeat tract longer than 49 bp although the distance and relative orientation of the centromere and telomere sequences was not critical. TEL+CEN antagonism was alleviated in strains carrying different rap1 alleles including rap1ts, rap1s, and rap1t alleles. Mutations SIR2, SIR3, SIR4, NAT1 and ARD1, genes that influence transcriptional silencing at telomeres and at the silent mating type loci, abolished TEL+CEN antagonism Mutation of SIR1 also partially alleviated TEL-CEN antagonism. In some sir mutant strains short yeast artificial chromosomes (YACs), which are normally unstable, became more stable, suggesting that the same mechanism that caused TEL+CEN antagonism on circular plasmids may contribute to the instability of short linear plasmids.

Alleles↗
PubMed1994

Effect of freeze-dried poly-L-lactic acid discs mixed with bone morphogenetic protein on the healing of rat skull defects.

Porous poly-L-lactic-acid discs were prepared by a freeze-drying method (FDPLLA). During preparation of the discs, semipurified bone morphogenetic protein (BMP) was mixed with the poly-L-lactic acid (500 micrograms BMP per disc). The discs were implanted in the skull defects of rats and retrieved after 2 or 4 weeks. With the FDPLLA/BMP discs, new bone formation was observed at 2 weeks after implantation. On the other hand, plain FDPLLA discs failed to produce new bone formation even at 4 weeks after implantation. Calcium content of the retrieved FDPLLA/BMP discs was statistically higher than that of plain FDPLLA discs. These results suggest that FDPLLA may be an effective delivery system for BMP and that the FDPLLA/BMP disc may potentially have usefulness as a bone graft substitute.

Animals↗
PubMed1994

Enhancement of telomere-plasmid segregation by the X-telomere associated sequence in Saccharomyces cerevisiae involves SIR2, SIR3, SIR4 and ABF1.

We have previously shown that circular replicating plasmids that carry yeast telomere repeat sequence (TG1-3) tracts segregate efficiently relative to analogous plasmids lacking the TG1-3 tract and this efficient segregation is dependent upon RAP1. While a long TG1-3 tract is sufficient to improve plasmid segregation, the segregation efficiency of telomere plasmids (TEL-plasmids) is enhanced when the X-Telomere Associated Sequence (X-TAS) is also included on the plasmids. We now demonstrate that the enhancement of TEL-plasmid segregation by the X-TAS depends on SIR2, SIR3, SIR4 and ABF1 in trans and requires the Abf1p-binding site within the X-TAS. Mutation of the Abf1p-binding site within the X-TAS results in TEL-plasmids that are no longer affected by mutations in SIR2, SIR3 or SIR4, despite the fact that other Abf1p-binding sites are present on the plasmid. Mutation of the ARS consensus sequence within the X-TAS converts the X-TAS from an enhancer element to a negative element that interferes with TEL-plasmid segregation in a SIR-dependent manner. Thus, telomere associated sequences interact with TG1-3 tracts on the plasmid, suggesting that the TASs have an active role in modulating telomere function.

Base Sequence↗