Determining the internal morphology of root canals.
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Dental Pulp Cavity↗
Biomedical subjects
H Horiuchi
Publications and source records attributed to H Horiuchi.
Identification of cDNA clones coding for the style specific S11a-glycoprotein gene associated with gametophytic self-incompatibility in tomato (Lycopersicon peruvianum).
We isolated 18 cDNA candidates that were expected to encode parts of the self-incompatibility-associated RNase of Lycopersicon peruvianum PI 126441 (S11a-plant) from a style cDNA library. Polymerase chain reaction was used with oligonucleotides derived from conserved amino acid sequences of RNases from fungi and S-associated RNases in other species of Solanaceae. Two of these clones (both do not have full length sequences) gave 826-bp and 730-bp sequences, and they had the same open reading frame, named ORF-1. Comparison of the deduced amino acid sequence of ORF-1 with S-associated RNase of other Solanaceous plants showed a high degree of similarity. mRNA encoding this ORF-1 was about 1000 bases long and detected only in the style tissue by Northern analysis. Using one of the cDNA clones as a probe, we detected sequence variability among three different S-genotypes of randomly chosen wild-type tomatoes by Southern analysis. From these results, it was concluded that ORF-1 encodes the self-incompatibility associated S-glycoprotein of L. peruvianum PI 126441 (S11a-plant).
Amino Acid Sequence↗
Immunohistochemical localization of epidermal growth factor in human main and accessory lacrimal glands.
Immunohistochemical localization of epidermal growth factor (EGF) in human main and accessory lacrimal glands has been investigated using monoclonal and polyclonal antibodies. Positive stain of EGF was localized in the cytoplasm of the acinar cells and the epithelial cells of the intra- and interlobular ducts of the main lacrimal glands, including both the palpebral and orbital lobes, and of the accessory lacrimal glands (gland of Krause, gland of Wolfring) as well. Meibomian glands showed weak immunoreactivity. Positive stain in the conjunctival epithelium including goblet cells was not observed. These results suggest that the mature form of EGF is produced in both the main and accessory human lacrimal glands and secreted into the tear fluid as a constant and crucial component.
Aged↗
[New membrane filter (PORETEC) method for ocular diagnostic cytopathology].
We describe a new membrane filter (PORETEC) technique for processing cytopathologic fluid specimens. This procedure provides excellent cytologic preparations because there is no background staining and only a small amount of fluid specimen is necessary, as there is little cell loss. We compared the number of cells collected by the membrane filter technique with that collected by cytocentrifugation using conjunctival brush cytology specimens from 6 subjects. The number of cells obtained by the new method was significantly higher than that obtained by the cytocentrifugation technique. This method was very useful for ocular fluid specimens such as aqueous humor, vitreous specimens, and scrapings from the cornea and conjunctiva. We showed some examples of these specimens including immunocytochemical staining done by this method. We confirm that this is valuable for diagnostic cytopathologic study of various fluid specimens in ophthalmology.
Adult↗
[Intraoperative real time observation of tumor blood flow in hepatocellular carcinoma by using color Doppler imaging].
To evaluate the tumor blood flow in hepatocellular carcinoma, we have introduced color Doppler imaging during operation. 37 hepatocellular carcinoma patient with 54 nodules were included in this study. Following categories were analyzed, those were detectability of pulsatile blood flow and continuous blood flow signal in the tumor, pulsatile blood flow velocity depend on tumor size and pathological feature. Even the size of the tumor is less than 1 cm in diameter in 70% of those nodules we could detect and analyze the flow signal. This new modality could have a possibility to be a useful diagnostic device for small mass lesions in the liver.
Carcinoma, Hepatocellular↗
Clinical application of new membrane filter for cytopathological diagnosis in ophthalmology.
In order to evaluate the clinical usefulness of a new membrane filter method, PORETEC, for cytopathological diagnosis in ophthalmology, we performed a comparative study on conjunctival cell specimens using this method and a previously developed cytocentrifugation method. The subjects were 5 normal male volunteers (6 eyes) with an average age of 33 years. The conjunctival cells were obtained by brush cytology, and processed for both the new membrane filter and the cytocentrifugation methods. The total numbers of epithelial cells obtained by both methods were counted under a light microscope. The number of cells obtained by the cytocentrifugation method was 4933 +/- 2338 for each specimen, whereas that by the new membrane filter method was 12436 +/- 5281 (mean +/- SD). The difference was statistically significant (Wilcoxon signed-rank test, P < 0.05). The new membrane filter diagnostic method was also applied to specimens of aqueous humor from patients with uveitis or vitreous body from patients with proliferative diabetic retinopathy or endophthalmitis. The new method provided excellent cytopathological specimens which were also applicable for special staining and immunocytochemical study. We concluded that the new membrane filter is quite useful for cytopathological diagnosis of ocular fluid specimens.
Adult↗
A possible target protein for smg-25A/rab3A small GTP-binding protein.
The smg-25A/rab3A protein (smg p25A) is a small GTP-binding protein implicated in intracellular vesicle traffic, particularly in neurotransmitter release from the presynapse. In the present study, we attempted to identify a target protein in bovine brain crude membranes that might be interacted with the GTP-bound form of smg p25A. When the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of radioiodinated smg p25A and the crude membrane fraction of bovine brain were incubated with a cross-linker, disuccinimidyl suberate, and the sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography, one radioactive band with a M(r) of about 110,000 was detected. This radioactive band appeared to be composed of radioiodinated smg p25A and a molecule with a M(r) of about 86,000. This molecule, tentatively termed here smg p25A target, was extracted from the membranes by a detergent and highly purified by column chromatographies and sucrose density gradient ultracentrifugation. The purified smg p25A target was sensitive to heat boiling and tryptic digestion, indicating that smg p25A target is a protein molecule. The M(r) of the purified smg p25A target was estimated to be about 85,000-86,000 from SDS-PAGE and to be about 100,000 from the S value. The cross-linking of radioiodinated smg p25A with the purified smg p25A target was inhibited by the GTP gamma S-bound form of non-radioactive smg p25A with an IC50 of about 8 nM. The GDP-bound form of smg p25A was much less effective. Other small GTP-binding proteins, such as c-Ki-ras p21, rhoA p21, smg p21B, and rab11 p24 were ineffective. These results indicate that a protein with a M(r) of about 85,000-100,000 is a target for smg p25A.
Animals↗
Fragment formation studied with antisymmetrized version of molecular dynamics with two-nucleon collisions.
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Journal Article↗
Histochemical study on the maturation of human megakaryocytes using microfluorometry.
A microcytofluorometrical DNA measurement was basically studied and was applied to single megakaryocytes previously identified on a Wright-Giemsa stained smear. The smear was first photographed and the location of each megakaryocyte was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained with 4',6-diamidino-2-phenylindole (DAPI) reagent (pH 7.4) at 4 degrees C. Nuclear blue fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA with the coefficient of variation (CV) of 3.6% when stained for 30 min. After 30 min DAPI staining, the DNA measurement was microcytofluorometrically performed in single megakaryocytes which had been morphologically classified into 4 groups on the basis of cytoplasmic maturation, Bessis' classification, assessed on Wright-Giemsa-stained bone-marrow smears from normal human beings. The histograms of the cells did not show any difference in DNA ploidy distribution among the classes: that is, the DNA histograms disclosed ploidy distribution from 4 N to 64 N with the largest population of 16 N. These findings suggest that nuclear DNA synthesis is completed before platelet production starts. This method is useful for comparing the morphological features and DNA content of single megakaryocytes.
Bone Marrow Cells↗
Immunohistochemical study on the distribution of sarcoplasmic reticulum calcium ATPase in various human tissues using novel monoclonal antibodies.
Novel monoclonal antibodies were raised against sarcoplasmic reticulum calcium (Ca2+)-ATPase of human skeletal muscle. Immunohistochemical analysis demonstrated that these antibodies, designated 6F5 and 7F10, bind Ca(2+)-ATPase of non-muscle tissue of the adult including parathyroid, islets of Langerhans, anterior lobe of the pituitary gland and photoreceptor cells of the retina as well as skeletal muscle. A positive reaction was also found for fetal tissues including skeletal muscle, heart, chondrocytes and peripheral nerves. Our results for distribution suggest that Ca(2+)-ATPase is strongly expressed in the tissues and cells in which signal transduction is actively carried out by Ca2+ release from the cytoplasmic Ca2+ pool.
Antibodies, Monoclonal↗
Evidence that three histidine residues of a base non-specific and adenylic acid preferential ribonuclease from Rhizopus niveus are involved in the catalytic function.
In order to study the structure-function relationship of an RNase T2 family enzyme, RNase Rh, from Rhizopus niveus, we investigated the roles of three histidine residues by means of site-specific mutagenesis. One of the three histidine residues of RNase RNAP Rh produced in Saccharomyces cerevisiae by recombinant DNA technology was substituted to a phenylalanine or alanine residue. A Phe or Ala mutant enzyme at His46 or His109 showed less than 0.03%, but a mutant enzyme at His104 showed 0.54% of the enzymatic activity of the wild-type enzyme with RNA as a substrate. Similar results were obtained, when ApU was used as a substrate. The binding constant of a Phe mutant enzyme at His46 or His109 towards 2'-AMP decreased twofold, but that at His104 decreased more markedly. Therefore, we assumed that these three histidine residues are components of the active site of RNase Rh, that His104 contributes to some extent to the binding and less to the catalysis, and that the other two histidine residues and one carboxyl group not yet identified are probably involved in the catalysis. We assigned the C-2 proton resonances of His46, His104, and His109 by comparison of the 1H-NMR spectra of the three mutant enzymes containing Phe in place of His with that of the native enzyme, and also determined the individual pKa values for His46 and His104 to be 6.70 and 5.94. His109 was not titrated in a regular way, but the apparent pKa value was estimated to be around 6.3. The fact that addition of 2'-AMP caused a greater effect on the chemical shift of His104 in the 1NMR spectra as compared with those of the other histidine residues, may support the idea described above on the role of His104.
Adenosine↗
Evolution of reaction mechanisms in the light heavy-ion system.
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Journal Article↗
Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes.
Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.
Amino Acid Sequence↗
The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase.
Mammalian ras genes substitute for the yeast RAS gene, and their products activate adenylate cyclase in yeast cells, although the direct target protein of mammalian ras p21s remains to be identified. ras p21s undergo posttranslational processing, including prenylation, proteolysis, methylation, and palmitoylation, at their C-terminal regions. We have previously reported that the posttranslational processing of Ki-ras p21 is essential for its interaction with one of its GDP/GTP exchange proteins named smg GDS. In this investigation, we have studied whether the posttranslational processing of Ki- and Ha-ras p21s is critical for their stimulation of yeast adenylate cyclase in a cell-free system. We show that the posttranslationally fully processed Ki- and Ha-ras p21s activate yeast adenylate cyclase far more effectively than do the unprocessed proteins. The previous and present results suggest that the posttranslational processing of ras p21s is important for their interaction not only with smg GDS but also with the target protein.
Adenylyl Cyclases↗
Identification of fibronectin in chicken thrombocytes.
Cell attachment and spreading of chicken embryo fibroblasts and three mammalian cell lines were promoted by using thrombocyte secretion products (TSP) prepared from chicken thrombocytes which are analogous to mammalian platelets. The cell attachment activity following TSP treatment was interfered with by the addition of a synthetic peptide containing the RGD sequence from the cell attachment region of fibronectin (FN). Cell attachment on TSP-coated plates was inhibited by the addition of anti-chicken FN rabbit antiserum, but not by anti-chicken vitronectin (VN) rabbit antiserum. By immunoblotting, immunofluorescent test and sensitive enzyme linked immunosorbent assay using an anti-chicken FN antiserum, the presence of FN in thrombocytes and the release of FN from the cells were demonstrated. The release of FN from thrombocytes was partially induced by the in vitro cultivation of the cells and was further promoted by treatment of the cells with thrombin.
Amino Acid Sequence↗
Transformation of a Leu- mutant of Rhizopus niveus with the leuA gene of Mucor circinelloides.
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Blotting, Southern↗
1,25-Dihydroxyvitamin D3 inhibits thromboxane release from activated macrophages.
1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) in concentrations from 0.1 to 10 nM suppressed immunoreactive thromboxane B2 (iTXB2) release from Propionibacterium acnes (P. acnes)-elicited liver adherent cells stimulated with lipopolysaccharide (LPS, 1 microgram/ml). These suppressive effects of 1,25-(OH)2D3 were also observed in oyster glycogen-elicited peritoneal macrophages. On the contrary, it did not inhibit iTXB2 release from both resident Kupffer cells and peritoneal macrophages stimulated with the same concentration of LPS. Furthermore, 1,24(R)-dihydroxyvitamin D3 (1,24(R)-(OH)2D3), a vitamin D3 analogue, also inhibited iTXB2 release from liver adherent cells, but, another synthesized vitamin D3 analogue, 1 alpha-hydroxyvitamin D3 (1 alpha-OH-D3) tended to decrease iTXB2 release only at higher concentrations. These results suggest that active vitamin D3 analogues inhibit iTXB2 release from activated macrophages.
Animals↗
[The relationship between ploidy and morphological classification of human megakaryocytes].
The relation between polyploidy and morphological classification of human megakaryocytes was studied in bone marrow aspirates from five normal individuals. On a Wright-Giemsa stained smear, megakaryocytes were morphologically classified into four groups according to a modification of Feinendegen's classification which is considered to reflect megakaryocyte maturation. The DNA of the morphologically classified cell is measured by microcytofluorometry using DAPI (4',6-diamidino-2-phenylindole) staining after removing the Wright-Giemsa stain. Most of the normal megakaryocytes were classified into type III (mature megakaryocytes) and the maximum peak in population of the megakaryocyte ploidy was observed at 16N. In each individual, the ploidy showed a similar pattern regardless of the classification. These findings suggest that the development of ploidy depends on a factor different from the one that determined the megakaryocyte maturation of cytoplasm and the ploidy is determined at the level of a megakaryocyte precursor or the most juvenile megakaryocyte.
Adolescent↗