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Biomedical subjects

T Miyake

Publications and source records attributed to T Miyake.

At least 19 recordsLinked to original sources

Structure of the Drosophila melanogaster gene encoding cyclin A.

A P element (PE)-induced Drosophila melanogaster mutation, hari, affects the formation of the bristle mechanosensory organ in the adult fly. In this mutation, the site of PE insertion is in the first intron of the gene (CycA) encoding cyclin A (CycA). In order to analyze the hari mutant at the molecular level, we cloned and sequenced the cDNA and genomic DNA encoding CycA. CycA has seven exons and six introns, and its transcription unit spans 6 kb. All exon-intron junctions are compatible with the GT/AG consensus. Results of primer extension analysis and RNase protection assay indicate that CycA has major and minor transcription start points (tsp). To our knowledge, this is the first report on the CycA genomic sequence from a multicellular organism.

Amino Acid Sequence

Efficient amplification of Drosophila simulans copia directed by high-level reverse transcriptase activity associated with copia virus-like particles.

The number of retrotransposon copia per genome in Drosophila melanogaster cultured cells is two to three times higher than that in D. melanogaster embryo cells. Here, we have found that the genome of the related species, Drosophila simulans, contains in cultured cells more efficiently amplified copia DNA (approximately ten fold). Furthermore, we analyzed copia virus-like particles (VLPs) prepared from D. melanogaster and D. simulans cultured cells, which contain copia RNA and reverse transcriptase (RT) activity, and thus, play a major role in copia replication. The RT activity associated with the D. simulans VLPs was 25 times higher than that associated with the D. melanogaster VLPs. Taken together with the fact that copia is believed to transpose through an RNA intermediate, these results suggest that the amplification of copia DNA should relate to copia RNA-mediated transposition, and the higher RT activity associated with the D. simulans VLPs would lead to the efficient amplification of copia DNA. In a comparison between D. melanogaster and D. simulans copia nucleotide (nt) sequences, five nt substitutions, which cause the respective amino acid changes, were found in the copia RT-coding region. Polymerase chain reaction direct sequencing showed that these five substitutions are the vast majority in each Drosophila species. The substitutions, therefore, may be responsible for the high level of the RT activity associated with the D. simulans VLPs.

Amino Acid Sequence

Reactive proliferation of astrocytes studied by immunohistochemistry for proliferating cell nuclear antigen.

Astrocyte proliferation in the stab-wounded cerebral cortex of mice was studied using double immunohistochemistry for proliferating cell nuclear antigen (PCNA) and glial fibrillary acidic protein (GFAP). The number of GFAP-positive astrocytes increased markedly from day 0.5 to day 3 after stab wounding. Some GFAP-positive astrocytes in the immediate vicinity of the wound were found to be positive for PCNA. However, the maximum number of these double positive astrocytes was only 5-6% of the number of GFAP-positive astrocytes. This maximum value was observed on days 2.5 and 3. The present study revealed that astrocytes are able to reactively express PCNA, an intrinsic marker of DNA replication. On the other hand, it is suggested that the proliferation of astrocytes in the wounded cerebral cortex is limited, in contrast with their marked reactive up-regulation of GFAP.

Animals

Glutamine synthetase immunoreactivity in two types of mouse brain glial cells.

The localization and distribution of glutamine synthetase (GS) in the adult mouse brain were studied by immunohistochemistry. GS immunoreactivity was found in two morphologically distinct types of glial cells apart from Bergmann glia, one asteroid and the other ovoid. The light and electron microscopic features of the GS-positive asteroid and ovoid cells were well consistent with those of astrocytes and oligodendrocytes, respectively. The GS-positive asteroid cells were present in the hippocampus, cerebral cortex, neostriatum, and cerebellar granular layer, where many synapse receptors for excitatory amino acids such as glutamate are densely distributed. Weakly GS-positive asteroid cells were also scattered in the white matter. The GS-positive ovoid cells were present throughout the gray matter regions of the brain except for the hippocampus, and they were the predominant type of GS-positive cells in the thalamus and brainstem gray matter where excitatory amino acid receptors are relatively sparse. No GS-positive ovoid cells were found in the white matter. These results suggest that, in the mouse brain, GS is localized in oligodendrocytes of the gray matter and in astrocytes. These two types of GS-positive glial cells may play different roles in the metabolism of glutamate.

Animals

Production of a unique multi-lamella structure in the nuclei of yeast expressing Drosophila copia gag precursor.

Drosophila retrotransposon copia produces virus-like particles (VLPs) in the nuclei of cultured Drosophila cells. The VLPs contain copia RNA and reverse transcriptase activity, and thus, play a major role in copia replication. Here we have expressed the copia gag polyprotein precursor in yeast. The precursor, which includes copia protease itself, showed correct autoprocessing to produce a unique multi-lamella structure in the nuclei of the yeast cells. This expression system should be useful for the analysis of nuclear localization of the major copia VLP protein, and furthermore, would provide important information concerning the mechanism of copia VLPs formation.

Animals

Recognition of an antiparallel beta-sheet structure of human epidermal growth factor by its receptor. Site-directed mutagenesis studies of Ala-30 and Asn-32.

The Ala-30 and Asn-32 residues involved in the major antiparallel beta-sheet structure of human epidermal growth factor (hEGF) were substituted with various amino acid residues, and the receptor-binding affinities of the nine variant hEGFs were determined by the use of human KB cells. The Ala-30----Arg, Ala-30----His and Ala-30----Phe substitutions drastically reduced the binding affinity, suggesting that the side chain in position 30 of Ala-30 of hEGF is required to be small for the receptor binding. The Asn-32----Asp substitution significantly reduced the binding affinity, while the Asn-32----His variant could bind to the receptor as well as to the wild-type hEGF. Therefore, it seems to be important for receptor binding that the side chain in position 32 does not have a negative charge but does have an NH group. Thus, we propose that, in the ligand-receptor complex, the receptor recognizes, on one side of the antiparallel beta-sheet structure of hEGF, a wider contact area than previously suggested.

Alanine

A site-directed mutagenesis study on the role of isoleucine-23 of human epidermal growth factor in the receptor binding.

The isoleucine-23 residue of human epidermal growth factor (hEGF) was substituted by a variety of amino acid residues and the receptor-binding activities of variant hEGFs were determined by the use of human KB cell. Tight receptor binding was found of variants with hydrophobic amino acid residues in position 23. The size of the isoleucine residue was nearly optimum for the receptor binding as compared with other hydrophobic residues. The structure analysis by two-dimensional nuclear magnetic resonance spectroscopy showed that the substitution at position 23 only slightly affected the tertiary structure of hEGF. These indicate that the side chain of isoleucine residue in position 23, which is exposed on the protein surface, directly binds to a hydrophobic pocket of the receptor.

Amino Acid Sequence

Sensory mother cell division is specifically affected in a Cyclin-A mutant of Drosophila melanogaster.

Cyclin proteins are one of the important components of the mechanism regulating mitosis in eukaryotic cells. We isolated a Drosophila Cyclin-A mutant in which the progenitor cells of the peripheral nervous system (the sensory mother cells) do not divide properly, causing the loss and other abnormalities of mechanosensory organs in the adult fly. Sequence analysis of the mutant genome reveals that a P element is inserted into the first intron of the Cyclin-A gene. A 13 kb wild-type genomic DNA containing the Cyclin-A transcription units rescued the mutant phenotype when introduced into the mutant fly. The regulation of cell type specific expression of the Cyclin-A gene is discussed.

Animals

Edgeworth's legacy of cranial muscle development with an analysis of muscles in the ventral gill arch region of batoid fishes (Chondrichthyes: Batoidea).

A series of studies by Edgeworth demonstrated that cranial muscles of gnathostome fishes are embryologically of somitic origin, originating from the mandibular, hyoid, branchial, epibranchial, and hypobranchial muscle plates. Recent experimental studies using quail-chick chimeras support Edgeworth's view on the developmental origin of cranial muscles. One of his findings, the existence of the premyogenic condensation constrictor dorsalis in teleost fishes, has also been confirmed by molecular developmental studies. Therefore, developmental mechanisms for patterning of cranial muscles, as described and implicated by Edgeworth, may serve as structural entities or regulatory phenomena responsible for developmental and evolutionary changes. With Edgeworth's and other studies as background, muscles in the ventral gill arch region of batoid fishes are analyzed and compared with those of other gnathostome fishes. The spiracularis is regarded as homologous at least within batoid fishes, but its status within elasmobranchs remains unclear; developmental modifications of the spiracularis proper are evident in some batoid fishes and in several shark groups. The peculiar ventral extension of the spiracularis in electric rays and some stingrays may represent convergence, probably facilitating ventilation and/or feeding in both groups. The evolutionary origin of the "internus" and "externus" remains uncertain, despite the fact that a variety of forms of the constrictor superficiales ventrales in batoid fishes indicates an actual medio-ventral extension of the "externus." The intermandibularis is probably present only in electric rays. The "X" muscle occurs only in electric rays and is considered to be Edgeworth's intermandibularis profundus. Its association with the adductor mandibular complex in narkinidid and narcinidid electric rays may relate to its functional role in lower jaw movement. Contrary to common belief, in most batoid fishes as well as some sharks, muscles that originate from the branchial muscle plate and extend medially in the ventral gill arches do exist: the medial extension of the interbranchiales in most batoid fishes and some sharks and the "Y" muscle in the pelagic stingrays Myliobatos and Rhinoptera. The latter is another example of the medial extension of the "internus." Whether the interbranchiales and "Y" muscle are homologous within elasmobranchs and whether homologous with the obliques ventrales and/or transversi ventrales of osteichthyan fishes await further research. Four hypobranchial muscles are recognized in batoid fishes: the coracomandibularis, coracohyoideus, coracoarcualis, and coracohyomandibularis. The coracohyoideus is discrete from the coracoarcualis; its complete structural separation from the latter occurs in several groups of batoid fishes.(ABSTRACT TRUNCATED AT 400 WORDS)

Anatomy, Comparative

The membranous skeleton: the role of cell condensations in vertebrate skeletogenesis.

Elements of the vertebrate skeleton are initiated as cell condensations, collectively termed the 'membranous skeleton' whether cartilages or bones by Grüneberg (1963). Condensations, which were identified as the basic cellular units in a recent model of morphological change in development and evolution (Atchley and Hall 1991) are reviewed in this paper. Condensations are initiated either by increased mitotic activity or by aggregation of cells towards a centre. Prechondrogenic (limb bud) and preosteogenic (scleral ossicle) condensations are discussed and contrasted. Both types of skeletogenic condensations arise following epithelial-mesenchymal interactions; condensations are identified as the first cellular product of such tissue interactions. Molecular characteristics of condensations are discussed, including peanut agglutinin lectin, which is used to visualize prechondrogenic condensations, and hyaluronan, hyaladherins, heparan sulphate proteoglycan, chondroitin sulphate proteoglycan, versican, tenascin, syndecan, N-CAM, alkaline phosphatase, retinoic acid and homeo-box-containing genes. The importance for the initiation of chondrogenesis or osteogenesis of upper and lower limits to condensation size and the numbers of cells in a condensation are discussed, as illustrated by in vitro studies and by mutant embryos, including Talpid3 in the chick and Brachypod, Congenital hydrocephalus and Phocomelia in the mouse. Evidence that genes specific to the skeletal type are selectively activated at condensation is discussed, as is a recent model involving TGF-beta and fibronectin in condensation formation. Condensations emerge as a pivotal stage in initiation of the vertebrate skeleton in embryonic development and in the modification of skeletal morphology during evolution.

Animals

Blood-aqueous barrier in eyes with retinal vein occlusion.

PURPOSE: The purpose of this study is to examine the changes of blood-aqueous barrier function in cases of central or branch retinal vein occlusion. METHODS: In addition to fluorophotometry of the aqueous humor and posterior vitreous, the authors used a method that quantifies protein in the aqueous (aqueous flare) by measuring the intensity of scattered laser light. RESULTS: Aqueous and posterior vitreous fluorescein concentrations and aqueous flare were significantly higher than in unaffected fellow eyes or eyes of visually normal age-matched controls both in central and branch retinal vein occlusions. They also were significantly higher in eyes with central vein occlusion than in those with branch vein occlusion. Among those eyes with central vein occlusion, aqueous and posterior vitreous fluorescein concentrations and aqueous flare were significantly higher in the hemorrhagic than in the venous stasis type of disease. Among eyes with branch vein occlusion, cases at the acute stage showed significantly higher amounts of aqueous and posterior vitreous fluorescein concentrations and aqueous flare than did those with chronic occlusion. CONCLUSION: The authors believe that increased amounts mainly reflect blood-ocular barrier disruption. Not only the blood-retinal barrier but also the blood-aqueous barrier disruption is a common sequela in the disorder.

Aged

High survival of rabbit morulae after vitrification in an ethylene glycol-based solution by a simple method.

Rabbit morulae were exposed to a vitrification solution-modified PBS [PB1] medium containing 40% ethylene glycol + 18% Ficoll + 0.3 M sucrose (EFS) for 2, 5, or 10 min at 20 degrees C and were vitrified in liquid nitrogen. When morulae were rapidly warmed, 96% had an intact zona pellucida. When embryos were cultured after removal of the mucin coat, high proportions of them formed blastocoel (79-100%), but the percentage of embryos developed to fully expanded blastocysts decreased with increased exposure time 87%, 40%, and 17%). The survival rate of morulae vitrified after removal of the mucin coat was lower than that of mucin-intact embryos. To assess the development potential in vivo, 131 embryos were vitrified after 2 min of exposure to EFS solution; all the embryos were recovered and 120 were transferred to recipients without removal of the mucin coat, resulting in 78 (65%) full-term fetuses or young. This simple method, which yields high survival both in vitro and in vivo, will be of practical use for vitrifying rabbit embryos.

Animals

Longitudinal study on occlusal force distribution in lower distal-extension removable partial dentures with circumferential clasps.

In this study, longitudinal changes of the occlusal force distribution ratio were examined in lower distal extension removable partial dentures with cast circumferential clasps. Occlusal force applied to the denture base and forces transmitted to the retainers were measured on several separate occasions from the insertion of new dentures to about 4 months after. Two rates of loading were chosen. One was simulated mastication (fast loading rate) and the other was 10 Ns-1 (slow loading rate). Location of the loading points were first premolar (P1), second premolar (P2) and first molar (P3) of the denture. The occlusal force distribution ratio to the retainers was calculated when a load of 20N was applied to the loading point. The results are summarized as follows: (1) The occlusal force distribution ratio at fast loading rate on P1 and P2 was changed until 1 or 1 1/2 months after the insertion of the new dentures, and then became constant. This constant value was 30% on P1, 20% on P2 and 10% on P3. (2) Slow loading rate produced a greater ratio than the fast loading rate on P2 and P3 while there were no remarkable differences in the ratio between both loading rates on P1.

Aged

[Rapid diagnosis of influenza infection by PCR method--detection of influenza virus HA gene in throat swab].

We studied the detection of the HA gene of human influenza viruses in throat swabs obtained from the outbreaks of influenza in school children utilizing the polymerase chain reaction (PCR) method. Sensitivity and specificity of the PCR method was compared to conventional virus isolation using MDCK cells. Three pairs of primers for PCR in detecting the HA genes of AH1, AH3, and B influenza viruses showed both subtype and type specificity. The dilution experiments showed that influenza viruses, as few as 1.1-3.5 plaque-forming units per 50 microliters, were sufficient for the detection of HA genes by PCR method and the detection rate by PCR method was 2-3 fold higher than that by conventional method. Our results showed that the PCR method was a fast, sensitive and reliable method for the diagnosis of influenza infections.

Base Sequence

Insertional mutagenesis in Drosophila. II. P element mediated transformation of Drosophila yakuba.

Drosophila yakuba, a member of melanogaster subgroup being free of P element, acquired resistance to an antibiotic neomycin by the transformation utilizing P element. In this species, the transformation frequency was comparable to that of D. melanogaster. Further, the occurrence of 8 base pairs duplication upon the insertion of the element was confirmed. These facts suggest that the P element could be inserted into the genome in the same manner, even in D. yakuba. Any consensus for preferential insertion could not be found on the nucleotide sequence as in D. melanogaster. However, it is noticeable that a series of the short palindromic stretches was common around the insertion sites in both species. It suggests that a structural feature of DNA plays a role as a landmark for P element insertion.

Animals

Incomplete vascular casting for a scanning electron microscope study of the microcirculatory patterns in the rat pancreas.

Scanning electron microscopy of resin casts prepared by incomplete arterial injections showed that in the rat pancreas, the casting medium fills blood capillaries in the endocrine islets more promptly than those in the exocrine lobules and secretory ducts. Furthermore, the exocrine lobules containing endocrine islets allowed a more rapid resin flow through the insulo-acinar portal route than those lobules lacking an islet. The secretory ducts were the last portions to be filled with resin. Since the resin medium used in this study was as viscous as blood and injected under a physiological pressure, the microcirculatory modes demonstrated by the present method suggest the physiological flow of blood in the rat pancreas.

Animals

[Effect of HLA matching in renal transplantation].

Evaluation of HLA-matching is useful in determining graft function and survival in a recipient in cadaveric renal transplantation. The effects of HLA on graft function, which serum creatinine indicative of, and graft survival were examined in both living and cadaveric transplantation in 280 Japanese renal transplants performed at Osaka University and Osaka Prefectural Hospital between October, 1982 and December, 1991 (cadaver 68, living 212). HLA-DR mismatch had a strong effect in living transplants, whereas no disparity was found in cadaver transplants with HLA-DR mismatch. HLA-B, DR mismatch had more effect than HLA-DR in cadaver transplants, and the same effect as HLA-DR in living transplants. HLA-AB affected graft function. It can be used to predict the effect of long-term graft survival. The HLA-haplotype match had the strongest association with graft survival and function in both cases of living and cadaver transplants. HLA-haplotypes of a cadaveric donor and a recipient were presumed by means of the linkage disequilibrium. These findings showed that HLA-matching, especially of haplotype, is an essential factor in relatively low risk renal transplant survival and function. 1-haplotype-matched transplantation, determined by the family study or presumed by the linkage disequilibrium is recommended if not 2-haplotype matched.

Adult