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Biomedical subjects

M Takagi

Publications and source records attributed to M Takagi.

At least 307 records · Page 17Linked to original sources
PubMed1998

Ultrastructural localization and biochemical characterization of vitronectin in developing rat bone.

This study has used light and electron microscope immunohistochemical and biochemical methods to localize and characterize vitronectin in early bone formation of developing rat mandible with rabbit antimurine vitronectin IgG. Developing jaws of foetuses were collected at embryonic day 15 (day 15) to day 18 from pregnant Wistar rats. After aldehyde fixation, specimens with and without osmium post-fixation were dehydrated and embedded in paraffin, Spurr's resin or LR gold resin for morphological and immunohistochemical examinations. At the light microscope level, in day 15 samples, positive vitronectin immunostaining was observed in small elongated areas of intercellular matrix and osteoblasts. Concomitant with initiation of matrix mineralization at day 16, vitronectin staining was similarly observed in small elongated areas containing intercellular matrix and osteoblasts but not clearly detected in fully mineralized bone matrix. The same staining profile was observed at days 17 and 18. At the ultrastructural level, immunogold particles were clearly detected over unmineralized matrix and cisterns of the rough-surfaced endoplasmic reticulum and the Golgi apparatus of osteoblasts as well as over demineralized bone matrix at day 16-18. In order to assess the presence of vitronectin in the mineral phase, mineral-binding bone proteins were extracted from fresh day 18 specimens using a three-step technique: 4M guanidine HCl (G1 extract), aqueous EDTA without guanidine HCl (E extract), followed by guanidine HCl. Subsequent Western blot analysis of sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis revealed that the antibodies produced only a single band at an M(r) of approximately 73000 in both G1 and E extracts, indicating the presence of vitronectin in the mineralized bone matrix. These results indicate that, at the onset of bone formation, osteoblasts synthesize and release vitronectin, which is subsequently incorporated into the bone matrix and becomes a specific component of bone tissues. The observation of vitronectin in these critical stages of bone formation suggests that it may be involved in the regulation of bone formation.

Animals↗
PubMed1998

Tissue reaction of hydroxyapatite sol to rat molar pulp.

Hydroxyapatite sol was prepared by dispersing unheated hydroxyapatite microcrystals into distilled water. A suspension of hydroxyapatite heated at 800 degrees C (HAp-800 degrees C) in distilled water was used as a comparative material. These were characterized by X-ray diffractometry, SEM and electroconductivity measurements. 0.001 ml of each material was injected into the upper first molar pulp of rats. After 7-14 days post-operatively, the group with hydroxyapatite sol showed slight inflammation in the pulp cavities and a highly calcified tubular dentine bridge was observed. Osteodentine had developed and adhered to the surface of the hydroxyapatite microcrystals. Matrix vesicles were observed in the extracellular matrix between collagen fibrils and electron-dense granules. After 28 days, the inflammation completely disappeared and tubular dentine was observed below the osteodentine. Acid phosphates (ACPase) activity was detected along all biosynthesizing pathways in odontoblast-like cells. Localized dehydrogenate (LDH) activity indicated phagocytosis of hydroxyapatite microcrystals by odontoblasts. In the group with HAp-800 degrees C after 7-14 days post-operatively, the inflammation was moderate. After 28 days, relatively fewer calcified dentine tubules were observed. ACPase activity was detected only along the membranes bounding cytoplasmatic vacuoles. It was concluded that hydroxyapatite sol is a safe biomaterial for use as dental pulp and induces early formation of dentine-bridge more so than HAp-800 degrees C.

Journal Article↗
PubMed1998

Coronary atherosclerosis with dual coronary artery fistulas.

Coronary artery fistula (CAF) is an uncommon congenital anomaly. Bilateral CAFs, arising from both right and left coronary arteries are rare. Myocardial infarction is by far a less frequent complication of CAF than angina pectoris. Ligation of CAF and coronary artery bypass graft were performed in the treatment of coronary artery stenosis with CAF.

Arterio-Arterial Fistula↗
PubMed1998

Comparisons of rice field mosquito (Diptera: Culicidae) abundance among areas with different agricultural practices in northern Thailand.

Adult mosquitoes were collected from 3 areas in northern Thailand with different availabilities of rice fields for larval habitats by using blacklight, truck, and pig-baited traps. Culex tritaeniorhynchus Giles and Culex gelidus Theobald were dominant in all samples. Significant study-area differences were found in light and truck trap collections for Cx. tritaeniorhynchus and Cx. gelidus, but not for Culex vishnui Theobald + Culex pseudovishnui Colless. In pig-baited sample area differences were significant only for Cx. gelidus. Adults of Culex fuscocephala Theobald were relatively rare, comprising only 0.6-7.9% of the total mosquitoes collected. When compared with the results from previous studies in the Chiangmai area, we conclude that Cx. fuscocephala has become a minor species and that changes in the mosquito fauna have occurred in northern Thailand.

Agriculture↗
PubMed1998

Cloning and sequencing of a 2,5-dichlorohydroquinone reductive dehalogenase gene whose product is involved in degradation of gamma-hexachlorocyclohexane by Sphingomonas paucimobilis.

Sphingomonas (formerly Pseudomonas) paucimobilis UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH), a halogenated organic insecticide, as a sole carbon and energy source. In a previous study, we showed that gamma-HCH is degraded to 2,5-dichlorohydroquinone (2,5-DCHQ) (Y. Nagata, R. Ohtomo, K. Miyauchi, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 176:3117-3125, 1994). In the present study, we cloned and characterized a gene, designated linD, directly involved in the degradation of 2,5-DCHQ. The linD gene encodes a peptide of 343 amino acids and has a low level of similarity to proteins which belong to the glutathione S-transferase family. When LinD was overproduced in Escherichia coli, a 40-kDa protein was found after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Northern blot analysis revealed that expression of the linD gene was induced by 2,5-DCHQ in S. paucimobilis UT26. Thin-layer chromatography and gas chromatography-mass spectrometry analyses with the LinD-overexpressing E. coli cells revealed that LinD converts 2,5-DCHQ rapidly to chlorohydroquinone (CHQ) and also converts CHQ slowly to hydroquinone. LinD activity in crude cell extracts was increased 3.7-fold by the addition of glutathione. All three of the Tn5-induced mutants of UT26, which lack 2,5-DCHQ dehalogenase activity, had rearrangements or a deletion in the linD region. These results indicate that LinD is a glutathione-dependent reductive dehalogenase involved in the degradation of gamma-HCH by S. paucimobilis UT26.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of EPD1, an essential gene for pseudohyphal growth of a dimorphic yeast, Candida maltosa.

Additional copies of the centromeric DNA (CEN) region induce pseudohyphal growth in a dimorphic yeast, Candida maltosa (T. Nakazawa, T. Motoyama, H. Horiuchi, A. Ohta, and M. Takagi, J. Bacteriol. 179:5030-5036, 1997). To understand the mechanism of this transition, we screened the gene library of C. maltosa for sequences which could suppress this morphological change. As a result, we isolated the 5' end of a new gene, EPD1 (for essential for pseudohyphal development), and then cloned the entire gene. The predicted amino acid sequence of Epdlp was highly homologous to those of Ggp1/Gas1/Cwh52p, a glycosylphosphatidylinositol-anchored protein of Saccharomyces cerevisiae, and Phr1p and Phr2p of Candida albicans. The expression of EPD1 was moderately regulated by environmental pH. A homozygous EPD1 null mutant showed some morphological defects and reduction in growth rate and reduced levels of both alkali-soluble and alkali-insoluble beta-glucans. Moreover, the mutant could not undergo the transition from yeast form to pseudohyphal form induced by additional copies of the CEN sequence at pH 4 or by n-hexadecane at pH 4 or pH 7, suggesting that EPD1 is not essential for yeast form growth but is essential for transition to the pseudohyphal form. Overexpression of the amino-terminal part of Epd1p under the control of the GAL promoter suppressed the pseudohyphal development induced by additional copies of the CEN sequence, whereas overexpression of the full-length EPD1 did not. This result and the initial isolation of the 5' end of EPD1 as a suppressor of the pseudohyphal growth induced by the CEN sequence suggest that the amino-terminal part of Epd1p may have a dominant-negative effect on the functions of Epd1p in the pseudohyphal growth induced by the CEN sequence.

Amino Acid Sequence↗
PubMed1998

Effects of lesions of the oculomotor vermis on eye movements in primate: saccades.

We studied the effects on saccades of ablation of the dorsal cerebellar vermis (lesions centered on lobules VI and VII) in three monkeys in which the deep cerebellar nuclei were spared. One animal, with a symmetrical lesion, showed bilateral hypometric horizontal saccades. Two animals, with asymmetrical lesions, showed hypometric ipsilateral saccades, and saccades to vertically positioned targets were misdirected, usually deviating away from the side to which horizontal saccades were hypometric. Postlesion, all animals showed an increase (2- to 5-fold) in trial-to-trial variability of saccade amplitude. They also showed a change in the ratio of the amplitudes of centripetal to centrifugal saccades (orbital-position effect); usually centrifugal saccades became smaller. In the two animals with asymmetrical lesions, for saccades in the hypometric direction, latencies were markedly increased (up to approximately 500 ms). There was also an absence of express and anticipatory saccades in the hypometric direction. When overall saccade latency was increased, centrifugal saccades became relatively more delayed than centripetal saccades. The dynamic characteristics of saccades were affected to some extent in all monkeys with changes in peak velocity, eye acceleration, and especially eye deceleration. There was relatively little effect of orbital position on saccade dynamics, however, with the exception of one animal that showed an orbital position effect for eye acceleration. In a double-step adaptation paradigm, animals showed an impaired ability to adaptively adjust saccade amplitude, though increased amplitude variability postlesion may have played a role in this deficit. During a single training session, however, the latency to corrective saccades-which had been increased postlesion-gradually decreased and so enabled the animal to reach the final position of the target more quickly. Overall, both in the early postlesion period and during recovery, changes in saccade amplitude and latency tended to vary together but not with changes in saccade dynamics or adaptive capability, both of which behaved relatively independently. These findings suggest that the cerebellum can adjust saccade amplitude and saccade dynamics independently. Our results implicate the cerebellar vermis directly in every aspect of the on-line control of saccades: initiation (latency), accuracy (amplitude and direction), and dynamics (velocity and acceleration) and also in the acquisition of adaptive ocular motor behavior.

Adaptation, Physiological↗
PubMed1998

Cloning and characterization of a chitinase-encoding gene (chiA) from Aspergillus nidulans, disruption of which decreases germination frequency and hyphal growth.

We cloned a chitinase-encoding gene from Aspergillus nidulans by polymerase chain reaction using degenerated oligonucleotide primers designed from the conserved amino acid sequences among chitinases from yeasts and Rhizopus spp. The cloned gene, named chiA, encoded a polypeptide consisting of 660 amino acids. Disruption of chiA had no effect on hyphal or conidiophore morphology, but germination frequency and hyphal growth rate decreased substantially. Expression of chiA was investigated using Escherichia coli beta-galactosidase as a reporter enzyme. The beta-galactosidase activity was present during hyphal growth and increased twice as the conidiophores developed. In situ staining of beta-galactosidase activity found high expression in metulae, phialides, and conidia during conidiophore development, indicating that the expression of chiA is developmentally regulated. This is the first report to isolate a chitinase gene from A. nidulans and investigate its functions using the gene disruption technique and gene fusion methods in filamentous fungi.

Amino Acid Sequence↗
PubMed1998

Analysis of tumor suppressor gene p53 in chicken lymphoblastoid tumor cell lines and field tumors.

To determine whether there is any abnormalities of the p53 gene in chicken lymphoblastoid tumor cell lines derived from Marek's disease (MD), lymphoid leukosis, reticuloendotheliosis, and field tumors, some portions of p53 cDNA corresponding to core and C-terminal domains (nucleotide positions 277-1104 in the p53 open reading frame (ORF)) were sequenced. Several mutations were identified in both cell lines and field tumors. However, none of these mutations is localized at the "hot spot", which has been reported as the site for transformation-activating mutations. Moreover, partial cDNA clones with a 122-bp deletion in the p53 ORF were identified in two cell lines, MSB1 and MTB1 derived from MD tumors. Southern blot analysis showed that no deletion occurred in the genome of p53 in MSB1, indicating that deletion occurred at the transcriptional level. This deletion could cause a frame shift of the encoding p53 protein, possibly resulting in the generation of a functionally different p53 protein. However, we confirmed that p53 mRNA without deletion is also present in each of these cell lines. These mutations of the p53 gene and deletion in the p53 transcript may be ones of molecular changes specific to the transformation induced by MD virus.

Amino Acid Sequence↗
PubMed1998

Increased interleukin-6 production in mouse osteoblastic MC3T3-E1 cells expressing activating mutant of the stimulatory G protein.

The McCune-Albright syndrome (MAS) is characterized by polyostotic fibrous dysplasia, café-au-lait spots, and multiple endocrine hyperfunction. An activating missense mutation of the alpha subunit of the Gs protein (Gs alpha) was found in several affected tissues, resulting in prolonged stimulation of adenylate cyclase. Our recent study has indicated that the cells derived from the fibrous bone dysplasia tissues in MAS patients produced increased levels of interleukin-6 (IL-6), which may be responsible for the increased bone resorption in this disease. In the present investigation, to analyze the molecular mechanism of the increased IL-6 production by activating mutant Gs alpha in bone cells, we established mouse osteoblastic MC3T3-E1 cells stably transfected with the activating mutant Gs alpha expression vector. These cells showed a significant increase of intracellular cAMP levels and produced a higher amount of IL-6 than the cells transfected with control vector or wild-type Gs alpha expression vector. Analysis of the IL-6 promoter revealed that any of the AP-1, nuclear factor (NF)-IL-6, and NF-kappa B binding elements are important for the activating mutant Gs alpha-induced gene expression. Electrophoretic mobility-shift assays using nuclear extracts of the mutant Gs alpha-expressing cells showed that phospho(Ser133)-cAMP-responsive element binding protein (CREB), AP-1, NF-IL6, and NF-kappa B were increased, compared with the control cells or the wild-type Gs alpha-expressing cells. These results indicate that activating mutant Gs alpha increases the transcriptional factors binding to CRE, AP-1, NF-IL6, and NF-kappa B elements to induce IL-6 gene expression in the osteoblastic cells.

3T3 Cells↗
PubMed1998

Immunocytochemical localization and biochemical characterization of large proteoglycans in developing rat bone.

This study used biochemical and light and electron microscopic immunohistochemical methods to localize and characterize large hyaluronate-binding proteoglycans in the developing mandible of fetal rats at embryonic day 15 (Day 15) to Day 18 using a monoclonal antibody (MAb) 5D5. This antibody is derived from bovine sclera and specifically recognizes the core protein of large proteoglycan such as versican, neurocan and brevican, but not that of aggrecan. At the light microscopic level, MAb 5D5 moderately stained the extracellular matrices among osteoblasts at the centers of ossification in Day 15 mandible specimens. Weaker staining was observed in osteoblasts, whereas Meckel's cartilage lacked staining. Ultrastructural immunocytochemistry showed the presence of immunogold particles over unmineralized matrices among osteoblasts and their intracellular organelles. In Day 16 to 18 specimens, bone nodules were recognized in LR gold sections before immunostaining, but, after immunostaining, consistently appeared devoid of mineral crystals and were seen as a demineralized structure that had an electron dense periphery within which fine filamentous and granular material were present. The appearance of these structures was created by the demineralization of thin sections on grids during immunostaining. Specific immunogold staining was clearly seen over the demineralized structures corresponding to bone nodules. The majority of immunogold particles tended to localize inside of the structures. Bone proteins were extracted from fresh, Day 18 specimens with a three-step technique: 4 M guanidine HCl (GdnCl,-extract), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis after chondroitinase ABC digestion, showed that G1-extract gave a 5D5 reactive band greater than 400 kDa, whereas E-extract produced two major reactive populations of small molecular size with core proteins approximately 63 and 74 kDa. These results indicate that the large proteoglycan having smaller molecular weight is preferentially localized to bone nodules and may correlate with bone matrix mineralization.

Animals↗
PubMed1998

Molecular cloning of the chick Nau gene and analysis of its expression patterns during neurogenesis.

We have previously identified a novel gene, Nau (Neuron-specific gene, which has a number of AU-rich RNA instability motifs), from a quail spinal cord cDNA library. Nau expression was observed mainly in post-mitotic neuroblasts, especially in the stage of axonogenesis. In this study, we cloned its chick orthologue (cNau, 5069 bp), which encodes 369 amino acid residues. cNau's open reading frame has three highly conserved regions with rat STOP (stable tubule-only polypeptide). cNau may be an orthologue of STOP gene considering its uniqueness in zoo blot analysis and its sequence similarity. Both the genetic structure and temporo-spatial expression patterns indicate that the cNau gene may be related to axonogenesis. In the hindbrain, cNau mRNA was accumulated in rhombomere boundary regions and in the rhombomere 2, 4 and 6. These observations suggest a specific role of cNau in the hindbrain or the difference in development between the hindbrain and spinal cord.

Amino Acid Sequence↗
PubMed1998

Mesenteric arterioportal shunt after hepatic artery interruption.

BACKGROUND: Massive hepatic necrosis from hepatic artery (HA) interruption is a complication after extended pancreatobiliary operation. The effectiveness of a mesenteric arterioportal shunt in preventing liver failure after massive hepatic necrosis was evaluated. METHODS: Of 98 patients who underwent pancreatic or hepatic resection for pancreatobiliary carcinoma between January 1989 and December 1995, six received a mesenteric arterioportal shunt. Clinical and hemodynamic analyses were done retrospectively. RESULTS: The six patients were classified into groups: A, postoperative hepatic arterial occlusion and, B, main HA excision without reconstruction. One patient in group A and three patients in group B had good arterioportal shunt patency and favorable clinical courses. However, fatal hepatic necrosis after ligation of the HA proper occurred in one patient in group A from small portal flow despite a presumed patent shunt. In another patient in group A angiogram revealed shunt occlusion. CONCLUSIONS: A mesenteric arterioportal shunt is beneficial when massive hepatic necrosis has occurred or is expected after main HA interruption under such conditions as postoperative hepatic arterial occlusion or HA excision without reconstruction. The procedure has the advantages of appropriate selection of artery size, a lower abdominal site apart from the primary operative field, and easy shunt closure by transarterial embolization.

Aged↗
PubMed1998

A new interpretation of salicylic acid transport across the lipid bilayer: implications of pH-dependent but not carrier-mediated absorption from the gastrointestinal tract.

Transport of several monocarboxylic acids across the lipid bilayer was examined in liposomes consisting of egg yolk phosphatidylcholine and cholesterol. In the presence of inward proton gradient, salicylic acid (SA) was taken up rapidly by liposomes showing overshoot, saturation and competitive inhibition phenomena. These carrier-mediated like profiles of SA uptake can be explained by assuming a very high permeability through the liposomal membrane of protonated SA. Protonated SA in the extraliposomal solution (pH 5.8) was taken up by liposomes rapidly, followed by a redissociation to anion according to the intraliposomal pH (pH 7.5). The concentration gradient of protonated SA across the liposomal membrane is maintained until the intraliposomal pH decreased to the extraliposomal level, which facilitates the uptake of SA into liposomes. The permeability of the lipid bilayer to several compounds was estimated from the inhibitory effects of those compounds on SA uptake by liposomes. Good linear relationships were observed between their inhibitory effects on the liposomal uptake of SA and the permeability of the intestinal membrane to them determined both in vivo and in vitro. These results clearly indicate that the carrier-independent transport mechanism of monocarboxylic acids observed in liposomes significantly contributes to their absorption from the intestinal tract under physiological conditions.

Animals↗
PubMed1998

The membrane-type-matrix metalloproteinase/matrix metalloproteinase-2/tissue inhibitor of metalloproteinase-2 system in periprosthetic connective-tissue remodeling in loose total-hip prostheses.

The aim of the present study was to investigate the proteolytic potential and localization of matrix metalloproteinase (MMP)-2 in relation to its regulatory protein, membrane-type-MMP (MT1-MMP), and tissue inhibitor of metalloproteinase-2 (TIMP-2), as well as to clarify an important step in the cascade of periprosthetic connective-tissue remodeling in loose total-hip prostheses. Immunohistochemical analysis revealed increased expression of MT1-MMP, MMP-2, and TIMP-2 in fibroblasts, synovial lining-like cells, and endothelial cells, as well as, to some extent, in monocyte/macrophage-like cells in both tissues from the bone-implant interface and reactive cellular tissues from regenerating capsules in loose hip joints, when compared with control fibrous tissues between bone and implants retrieved from unloosened hip joints. In loose hip joints, reverse transcription-PCR analysis showed the presence of MT1-MMP, MMP-2, and TIMP-2 mRNA in both the bone-implant interface and regenerating capsular tissues. Increased protein levels of MMP-2 and TIMP-2 were also demonstrated by an ELISA, and those of MT1-MMP were shown by immunoblot analysis. Gelatin-zymographic analysis confirmed the presence of both pro- and active forms of MMP-2, which suggested the in situ activation of MMP-2 by MT1-MMP in the loose hip joints. Collectively, these data suggest that the MT1-MMP/MMP-2/TIMP-2 system participates in the extracellular matrix degradation and periprosthetic connective-tissue remodeling in loose hip joints, and may thus contribute to the periprosthetic weakening, loosening, and osteolysis that can occur around implants.

Aged↗