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Biomedical subjects

H Yokota

Publications and source records attributed to H Yokota.

At least 55 records · Page 3Linked to original sources
PubMed2001

Early relapse after high-dose chemotherapy rescued by tumor-free autologous peripheral blood stem cells in acute lymphoblastic leukemia: importance of monitoring for WT1-mRNA quantitatively.

A 24-year-old woman who suffered from ALL with MLL gene rearrangement received high-dose chemotherapy followed by autologous PBSC transplantation during complete remission (CR). Reverse transcriptase-polymerase chain reaction (RT-PCR) used to detect MLL/LTG4 chimeric mRNA showed no minimal residual disease (MRD) in the graft or bone marrow at the transplantation. However, the leukemia relapsed four months after transplantation. Retrospective analysis of quantitative measurement of Wilms tumor gene (WT-1) mRNA showed an increased level in the bone marrow although it was within the normal range. These observations suggest that careful monitoring of MRD by quantitative measurement of WT-1 mRNA in addition to disease-specific chimeric mRNA is required to predict relapse.

Adult↗
PubMed2001

Bisphenol a glucuronide, a major metabolite in rat bile after liver perfusion.

The environmental estrogen bisphenol A, orally introduced into the body, passes through the liver and modulates the endocrine system to elicit irreversible changes in the functioning of reproduction. To elucidate the actual and dynamic metabolism of bisphenol A in the liver before its arrival at target organs, this study evaluated the metabolism and disposition of the compound within the passage through the liver in Sprague-Dawley rats. On perfusion of 7.5 micromol of bisphenol A into the liver via the portal vein, approximately 91% of the infused bisphenol A was absorbed by the liver tissue, and about 65% of the absorbed bisphenol A was glucuronidated within 60 min. Roughly 65% of the bisphenol A glucuronide that formed in the liver was excreted into the bile and about 35% into the hepatic vein. On perfusion of 0.01, 0.05, and 0.1 mM bisphenol A solution into the liver, free bisphenol A was excreted only into the vein at 5.6, 9.3, and 14.6%, respectively, of the total bisphenol A. These results suggest that most bisphenol A absorbed by the intestine is probably glucuronidated exclusively in the liver and the conjugate is excreted mainly into the bile.

Animals↗
PubMed2001

Microleakage of self-etching primers after thermal and flexural load cycling.

PURPOSE: To evaluate the adhesive properties of a one-bottle self-etching primer system. MATERIALS AND METHODS: 150 wedge-shaped cervical cavities on bovine teeth were restored with Clearfil Liner Bond 2 (LB), Clearfil Liner Bond 2V (LV) or Clearfil SE Bond (SE), according to the manufacturer's instructions. Twenty specimens of each adhesive system were finished 15 mins after light curing and 30 specimens were finished after a 24-hr storage period in water. From each group, 10 restorations were immediately immersed in 0.5% basic fuchsin solution and kept there for 24 hrs to examine microleakage. The other 10 restorations were thermocycled (5-60 degrees C, 15 s dwell time, 5,000 cycles), and then immersed in the dye solution. The remaining 10 specimens of the 24-hr storage group were subjected to flexural load cycling (0.5 mm labio-lingual displacement at the incisal edge, 10,000 cycles, 1 cps) prior to immersion in the dye solution. The data was analyzed using the Mann-Whitney U-test and the Kruskal-Wallis test. The tensile bond strengths of the adhesive systems to bovine enamel and dentin were also determined. The data was analyzed using the Student's t-test and ANOVA. In addition, SEM examinations were made to evaluate the effects of self-etching primers on enamel and dentin surfaces. RESULTS: LV and SE showed significantly better marginal sealing than LB (P<0.05). The marginal integrity of SE did not deteriorate even after immediate finishing, thermal stresses or flexural loads. There were no significant differences in bond strengths among the adhesive systems tested. All adhesive systems showed similar bond strengths to enamel and dentin. The enamel etching patterns using LV and SE systems were obscure and difficult to assess. Although the smear plugs were not fully removed, no smear layer was observed on the treated dentin surfaces, regardless of the self-etching primer system used. CLINICAL SIGNIFICANCE: Clearfil SE bond could possibly improve the clinical performance of cervical cavities.

Acid Etching, Dental↗
PubMed2000

Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-A resolution.

The intracellular protease from Pyrococcus horikoshii (PH1704) and PfpI from Pyrococcus furiosus are members of a class of intracellular proteases that have no sequence homology to any other known protease family. We report the crystal structure of PH1704 at 2.0-A resolution. The protease is tentatively identified as a cysteine protease based on the presence of cysteine (residue 100) in a nucleophile elbow motif. In the crystal, PH1704 forms a hexameric ring structure, and the active sites are formed at the interfaces between three pairs of monomers.

Amino Acid Sequence↗
PubMed2000

Reversed phase HPLC of Met58 oxidized rhIL-11: oxidation enhanced by plastic tubes.

The hydrophobicity of human recombinant interleukin 11 (rhIL-11) with an oxidized Met58 residue is nearly identical to the hydrophobicity of native rhIL-11. Consequently, separation of these species using standard gradient elution or isocratic elution is very difficult. Using an optimized, shallow gradient RP-HPLC method. Met58 oxidized rhIL-11 could be separated sufficiently from native rhIL-11. The identity of the oxidized form detected with this method was confirmed by peptide mapping with trypsin and endoproteinase Asp-N, N-terminal sequencing and mass spectrometric analysis. This method was employed to determine the effect of disposable laboratory plastic tubes for the oxidation. The amounts of Met58 oxidized rhIL-11 were increased when rhIL-11 samples were stored in plastic tubes at 37 degrees C in the dark. Samples stored in polypropylene tubes were oxidized much more than samples stored in polystyrene tubes. Additionally, the oxidation was greatly enhanced when samples were stored in polypropylene tubes exposed to light before rhIL-11 sample storage. The extent of the oxidation was also affected by the sources of polypropylene tubes. A maximum increase in Met58 oxidized rhIL-11 was more than 30% when samples were stored at 37 degrees C for 14 days in polypropylene tubes exposed to a daylight fluorescent lamp for 25 days. Consequently, these results indicate that attention should be paid for selection of suitable plastic tubes used for storage of protein samples, and for protection of the plastic tubes themselves from extended exposure to light while in storage.

Amino Acid Sequence↗
PubMed2000

Electrostatic interactions of androgens and progesterone derivatives with rainbow trout estrogen receptor.

In primary cultures of immature male rainbow trout (rt) hepatocytes, vitellogenin (Vg) gene expression is regulated by E(2) via the estrogen receptor (ER). However, steroids other than estrogens can also stimulate Vg gene expression. These steroids are hardly converted into E(2) during incubation and their stimulatory activity is completely inhibited by tamoxifen implying rtER involvement. These steroids have no or a slightly positive charge on the Connolly surface. In contrast, steroids that failed to stimulate Vg gene expression had a strong positive or negative charge around rings C and D due to polarization. The amino acid sequences of the ligand binding domains (LBD) of rtER and human ER alpha have 57.7% homology; only one amino acid differs in the presumed steroid binding site. We modeled the three-dimensional structure of the LBD of rtER using X-ray crystallographic data for hER alpha in order to investigate the fit (structural and electrostatic) between steroid and rtER. Two factors are essential for binding to rtER: (i) hydroxyl or carbonyl groups near C3 and C17 of the steroids (hydrophilic regions) that can form hydrogen bonds with His(489), Arg(359), and Glu(318), (ii) a hydrophobic steroid nucleus that interacts with a hydrophobic region of the rtER LBD through van der Waals forces. If polar functional groups are present, the hydrophobic interaction between steroid and the rtER LBD is considerably weakened.

Amino Acid Sequence↗
PubMed2000

Differential expression of neurexin mRNA in CA1 and CA3 hippocampal neurons in response to ischemic insult.

A short period of cerebral ischemia will trigger a cascade of events leading to neuronal death. In an effort to elucidate molecular mechanisms underlying differential vulnerability of CA1 and CA3 hippocampal neurons to neurodegeneration, we performed a transcriptional analysis of rat hippocampal neurons following transient global ischemia. In response to 15-min ischemia, the mRNA level of neurexins II alpha and III alpha was elevated in CA1 neurons and CA3 neurons, respectively. Interestingly, the up-regulated neurexin III alpha mRNA in postischemic CA3 consisted of the insert corresponding to the fourth splicing site, while the transcripts in postischemic CA1 neurons and control CA3 neurons lacked the insert. The observed tissue specific expression and the splicing pattern suggest functional importance of neurexins in postischemic degeneration of hippocampal neurons.

Alternative Splicing↗
PubMed2000

Crystal structure of archaeal RNase HII: a homologue of human major RNase H.

BACKGROUND: RNases H are present in all organisms and cleave RNAs in RNA/DNA hybrids. There are two major types of RNases H that have little similarity in sequence, size and specificity. The structure of RNase HI, the smaller enzyme and most abundant in bacteria, has been extensively studied. However, no structural information is available for the larger RNase H, which is most abundant in eukaryotes and archaea. Mammalian RNase H participates in DNA replication, removal of the Okazaki fragments and possibly DNA repair. RESULTS: The crystal structure of RNase HII from the hypothermophile Methanococcus jannaschii, which is homologous to mammalian RNase H, was solved using a multiwavelength anomalous dispersion (MAD) phasing method at 2 A resolution. The structure contains two compact domains. Despite the absence of sequence similarity, the large N-terminal domain shares a similar fold with the RNase HI of bacteria. The active site of RNase HII contains three aspartates: Asp7, Asp112 and Asp149. The nucleotide-binding site is located in the cleft between the N-terminal and C-terminal domains. CONCLUSIONS: Despite a lack of any detectable similarity in primary structure, RNase HII shares a similar structural domain with RNase HI, suggesting that the two classes of RNases H have a common catalytic mechanism and possibly a common evolutionary origin. The involvement of the unique C-terminal domain in substrate recognition explains the different reaction specificity observed between the two classes of RNase H.

Amino Acid Sequence↗
PubMed2000

ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs) is transcriptionally induced in beta-amyloid treated rat astrocytes.

Beta-amyloid (Abeta) deposition is considered to be of the most crucial events in the onset of Alzheimer's disease (AD). To identify factors involved in the exacerbation of AD, we investigated transcriptionally Abeta-induced genes using a cDNA subtraction technique in rat astrocytes as previously reported. One subtracted gene that showed the Abeta-induced expression was rat ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs). In this report, we present the deduced sequence for the mature form of rat ADAMTS-4 and demonstrate the induction of its mRNA by treatment of cells with Abeta. Our results suggest a degradation of the extracellular matrix occurring in the brain of AD patients and a possibly significant role of this enzyme in the progression of AD.

ADAM Proteins↗
PubMed2000

Atomic force microscopy-based detection of binding and cleavage site of matrix metalloproteinase on individual type II collagen helices.

Type II tropocollagen molecules were reacted with matrix metalloproteinase 8 (MMP-8) and the binding sites as well as the cleavage site of MMP-8 were detected on individual molecules using atomic force microscopy (AFM). Approximately 300-nm-long coiled-coil tropocollagen molecules were straightened and immobilized on an atomically flat surface for detection by AFM. The direct visualization of individual collagen molecules revealed heterogeneous characteristics of MMP-8:collagen complexes. We observed that there existed multiple MMP-8 nonspecific binding sites on the collagen molecules, but cleavage always took place at a unique site. When collagen molecules, straightened and immobilized on the surface, were reacted with MMP-8, a site of cleavage appeared as a gap in stretched molecules. This is the first report to visually show direct collagenase:collagen interactions using AFM. The described AFM-based analysis has potential as a protein analysis tool for understanding a complex mechanism of enzyme:substrate interactions.

Animals↗
PubMed2000

MutS-mediated detection of DNA mismatches using atomic force microscopy.

We have developed an atomic force microscopy-based method for detecting DNA base-pair mismatches using MutS protein isolated from E. coli. MutS is a biological sensor and a locator of DNA base-pair mismatches. It binds specifically to a mismatched DNA base pair and initiates a process of DNA repair. To test the possibility of visually detecting mismatched base pairs by atomic force microscopy, we prepared DNA templates approximately 500 bp in length consisting of a single or multiple base-pair mismatches. We demonstrate that MutS binding sites on individual DNA molecules were readily detectable by atomic force microscopy and that the observed positions were in good agreement with the predicted sites of base-pair mismatches at a few-nanometer resolution. The technique described here is rapid and sensitive and is expected to be useful in screening mutations and DNA polymorphisms.

Adenosine Triphosphatases↗
PubMed2000

Expression of prostaglandin E synthase mRNA is induced in beta-amyloid treated rat astrocytes.

Deposition of beta-amyloid (A beta) in the brain is considered to be one of the most critical events in the onset of Alzheimer's disease (AD). In order to identify factors involved in the exacerbation of AD, we investigated transcriptionally A beta-induced genes using a cDNA subtraction technique in rat astrocytes. One gene obtained was rat prostaglandin (PG) E synthase. In this report, we present the deduced sequence for rat PGE synthase for the first time and demonstrate the induction of PGE synthase mRNA by treatment of cells with A beta. Our results suggest a possibly significant role of this enzyme in the progression of AD.

Alzheimer Disease↗
PubMed2000

Genome-wide detection of unknown subtle mutations in bacteria by combination of MutS and RDA.

We propose a procedure for detecting unknown, subtle DNA changes throughout the entire bacterial genome by a combination of MutS and RDA. Current techniques detect subtle mutations after PCR amplification of the target regions, so the mutation detection is done between amplified PCR fragments. In this paper, genome-wide subtle mutation scanning in bacteria was performed by combining the MutS and RDA techniques. Our strategy for cloning a small mutation region is composed of two steps: an enrichment of fragments containing subtle mutations using MutS, followed by an RDA subtraction procedure for further enrichment. We successfully identified small mutations such as a four-base insertion, a two-base insertion, and transition mutations in bacteria.

Adenosine Triphosphatases↗
PubMed2000

Morphological assessment of the effect of growth hormone on preantral follicles from 11-day-old mice in an in vitro culture system.

The present study was carried out to verify that the cells attached to the outside of the basement membrane of mechanically isolated follicles are theca cells and to evaluate the effect of growth hormone (GH) on these cells. Preantral follicles, 100-140 micrometer in diameter, were mechanically isolated from 11-day-old BDF1 hybrid immature mice, divided randomly into two groups, and cultured in vitro. One group was treated with 0.1% collagenase immediately after mechanical isolation in an attempt to remove theca cells attached to the outside of the basement membrane. The other group was untreated. Morphological examination revealed that 86.1% of mechanically isolated follicles before collagenase treatment had at least one theca cell around the basement membrane on the single section. However, after collagenase treatment no theca cells remained on the basement membrane of the follicles. Androstenedione secretion as a result of stimulation by 100 ng/ml hCG was significantly higher in the culture medium of the follicles with theca cells than in those of collagenase-pretreated follicles (p < 0.0001), indicating that the cells attached to the outside of the basement membrane were actually functional theca cells, not interstitial cells. To elucidate the effect of GH on theca cells, preantral follicles cultured in the presence of 1.0 mIU/ml GH were morphologically examined. Preantral follicles mechanically isolated from immature mice showed significant proliferation of not only granulosa cells but also theca cells in the presence of GH. In particular, theca cells, which remained dotted on the basement membrane in a small number just after isolation, proliferated and finally formed complete layers after the culture with GH. This is the first report that GH induced the proliferation of theca cells to form morphologically complete layers around the preantral follicle from 11-day-old mice.

Androstenedione↗
PubMed2000

Size-dependent positioning of human chromosomes in interphase nuclei.

By using a fluorescence in situ hybridization technique we revealed that for nine different q-arm telomere markers the positioning of chromosomes in human G(1) interphase nuclei was chromosome size-dependent. The q-arm telomeres of large chromosomes are more peripherally located than telomeres on small chromosomes. This highly organized arrangement of chromatin within the human nucleus was discovered by determining the x and y coordinates of the hybridization sites and calculating the root-mean-square radial distance to the nuclear centers in human fibroblasts. We demonstrate here that global organization within the G(1) interphase nucleus is affected by one of the most fundamental physical quantities-chromosome size or mass-and propose two biophysical models, a volume exclusion model and a mitotic preset model, to explain our finding.

Cell Nucleus↗
PubMed2000

Isolation and structures of grammistins, peptide toxins from the skin secretion of the soapfish Grammistes sexlineatus.

Two peptide toxins (named grammistins Gs 1 and Gs 2) with hemolytic and ichthyotoxic activities were isolated from the skin secretion of the soapfish Grammistes sexlineatus. Grammistin Gs 2 showed 6-11 x higher hemolytic activity and 10x higher ichthyotoxicity than grammistin Gs 1. The complete amino acid sequences of Gs 1 comprising 25 residues and Gs 2 comprising 24 residues were determined. Although a search by the database failed to find any homologous toxins from other sources, the grammistins were similar in secondary structures as well as biological activities to the two classes of peptide toxins, melittin from the bee venom and pardaxins from the skin secretion of two species of soles. CD experiments and helical wheel projections showed that the grammistins were randomly coiled in distilled water but formed amphiphilic alpha-helices in the presence of SDS micelles. In addition, they were found to be surface seeking peptides by the Eisenberg plot and assumed to exist as aggregates of 3-4 molecules. Interestingly, grammistin Gs 2 is much more abundant in amphiphilic alpha-helices and much higher in biological activities than melittin and pardaxins as well as grammistin Cs 1.

Amino Acid Sequence↗