Biomedical subjects

T Fukuchi

Publications and source records attributed to T Fukuchi.

At least 55 records · Page 3Linked to original sources

PubMed1996

[Effect of duration of cerebral ischemia on pyruvate dehydrogenase activity (PDH) and metabolites in the gerbil brain].

Pyruvate dehydrogenase (PDH) is one of the mitochondrial enzymes which regulate the glucose metabolism. The purpose of this study is to determine the effect of the duration of cerebral ischemia on PDH activity and the metabolites. Cerebral ischemia was produced by bilateral common carotid artery occlusion in Mongolian Gerbils. 20-minute (1) and 60-minute ischemic groups (2) were made. PDH activity and energy metabolites (ATP, PCr, lactate) were measured in the caudate nucleus and cortex at each time period. 1) 20 min ischemic group: PDH activity significantly increased after 20-min ischemia in both the caudate nucleus and cortex, and decreased to levels less than that of the control after 20 min reperfusion. At 60 and 120 min reperfusion, PHD activity returned to the control levels. ATP and PCr concentrations were significantly depleted after the ischemic insult, returning to 60-80% of the control level after reperfusion. Lactate concentrations increased significantly after ischemia, and were reduced by reperfusion. 2) 60 min ischemic group: PDH activity significantly increased after 60 min ischemia, and decreased but remained higher than the control level after 20 min reperfusion. At 60 and 120 min reperfusion, PDH activity gradually decreased towards control levels. ATP and PCr concentrations were depleted after ischemia, and were gradually restored after 20 min reperfusion, recovering to 50% after 60 min reperfusion. Lactate concentrations increased after the ischemic insult, and became more elevated after reperfusion. These findings indicate that there is a significant difference in the PDH activity and metabolism depending on the duration of ischemia. The data suggest that impaired metabolism and persistent elevation of PDH activity may be caused by damage to the mitochondria allowing the influx of Ca2+ during prolonged ischemia.

Adenosine Triphosphate↗

PubMed1996

[Slow axonal transport in primate experimental glaucoma].

Axonal transport abnormality has been reported to be deeply related to glaucomatous optic nerve injury. These reports mainly investigate the axonal transport blockage using the model of acute elevation of intraocular pressure in normal primates. We studied such axonal transport abnormality especially of slow flow in laser-induced glaucomatous monkey eyes by tissue autoradiography. Abnormal silver grain accumulation suggesting axonal transport damage was seen in the areas of distorted lamina cribrosa in glaucomatous eyes. Accumulation of grains was also seen in the retro-laminar optic nerve in some areas. Normal control eyes showed almost homogenous grain distribution in the pre-lamina, lamina and post-lamina areas. Areas of slightly elevated, accumulation of grains were seen around the lamina cribrosa and disc margin suggesting physiological blockage of axonal transport.

Animals↗

PubMed1996

[Proteoglycan molecules in scleral tissue of enlarged eyeball].

We studied scleral specimens from experimentally induced enlarged eyeball with axial elongation by the transmission electron microscopy following cationic dye (cuplonic blue) staining. The animal model was prepared by the injection of alpha-chymotrypsin into the posterior chamber of young albino rabbits. Cuprolinic blue staining was applied to scleral specimens obtained from equatorial lesion and portions of the scleral tissues were subjected to enzyme digestion by chondroitinase ABC, AC and B before cuprolinic blue staining. In control eyes, dermatan and chondroitin type sulfated proteoglycan filaments were identified. Large, electron dense, and abnormal by shaped proteoglycan filaments were seen in the transmission electron microscopy. Such abnormal proteoglycan filaments were susceptible to enzyme chondroitinase ABC and AC digestion but resistant to chondroitinase B, suggesting that they are chondroitin sulfate dominant proteoglycans. Our morphological data corroborated a previous biochemical report of abnormally induced proteoglycan molecules in sclera with enlarged, axially elongated eyes.

Animals↗

PubMed1995

Splicing isoforms of rat Ash/Grb2. Isolation and characterization of the cDNA and genomic DNA clones and implications for the physiological roles of the isoforms.

We obtained three types of cDNA clones homologous to Ash/Grb2(Ash-l) cDNA from rats. One of these clones, Ash-psi, was an unusual transcribed gene having 93% identity in the nucleotide sequence to Ash-l. The other two clones, Ash-m and -s, had nucleotide sequences identical with Ash-l cDNA in the amino-terminal region. The coding sequence of Ash-m cDNA is 42 nucleotides shorter than that of Ash-l cDNA. The defective region of Ash-m cDNA encodes 14 amino acid residues (157 to 170 of Ash-l), which comprise the most conserved region of the second SH3 domain. On the other hand, the coding sequence of Ash-s terminated at the end of the first SH3 domain due to a stop codon at the boundary of the sequence, thereby differing from Ash-l cDNA. Cloning of the genomic DNA of the Ash-l-encoding gene, determination of the gene organization, and nucleotide sequencing revealed that the two isoforms, as well as Ash-l, are generated from a single gene by unusual alternative splicings. The gene spans more than 16 kilobases and contains 6 exons and 5 introns. Ash-m and Ash-s mRNAs were detected in various tissues by reverse-transcribed polymerase chain reaction. Ash-m physically associated with dynamin, but the association with Sos was less effective than that of Ash-l in rat pheochromocytoma PC12 cell lysates, irrespective of treatment with nerve growth factor. In contrast, Ash-s formed a complex with dynamin and Sos in cell lysates. Moreover, the newly formed carboxyl-terminal SH3 of Ash-m by splicing bound different proteins from those bound to the carboxyl-terminal SH3 domain of Ash-l, suggesting that Ash-m generates different signals. Microinjection of Ash-m or Ash-s into Balb/c 3T3 cells inhibited DNA synthesis induced by platelet-derived growth factor. These results show that these isoforms act as dominant negative regulators of mitogenic signals by Ash-l.

Adaptor Proteins, Signal Transducing↗

PubMed1995

[Seven cases of pseudoseizure unnecessarily treated with AEDs].

An increasing number of pseudoseizures and/or behavioral abnormalities are being unnecessarily treated with anti-epileptic drugs (AEDs) for long periods, often producing adverse effects and even aggravating abnormal behaviors. Seven of the 77 new referrals (43 men, 34 women) to our epilepsy clinic within a single year (1992-1993) were misdiagnosed with epilepsy and had pseudoseizures or behavioral abnormalities alone. The initial episodes for which AEDs had been given were febrile convulsions during childhood in two patients, nonfebrile convulsions of uncertain nature in four patients, and learning difficulty with EEG abnormality in one patient. The reasons AED treatment had continued for so long are probably the persistence of behavioral problems and/or EEG abnormalities and failure to reevaluate the patients to determine whether they had true epileptic symptomatology. Based on the results of this study, we concluded that: 1. AED treatment should be initiated only after a suspicious episode had been demonstrated to be truly epileptic in nature. 2. The diagnosis of pseudoseizures should be made on the basis of careful investigation of clinical symptomatology and ictal pattern as described in "Proposed International Classification of Clinical and Electroencephalographic Epileptic Seizures, 1981". 3. In doubtful cases, frequent reevaluation of the nature of the episodes and the necessity of AEDs is needed to avoid adverse effects.

Adolescent↗

PubMed1995

Loss of cell-matrix cohesiveness after phagocytosis by trabecular meshwork cells.

PURPOSE: To investigate the response of trabecular meshwork cells to phagocytic events. METHODS: Cultured bovine trabecular meshwork cells were established and exposed to latex microspheres for 40 to 44 hours. After phagocytosis, the cohesiveness of cells to their underlying matrix was measured by the susceptibility to trypsin, as indicated by the time needed to be liberated from culture plates. The amounts of two cell attachment proteins, fibronectin and laminin, in both the phagocytically challenged and the control cultures were measured at various postphagocytosis time points with an enzyme-linked immunosorbent assay. The fibronectin and laminin network was visualized with immunostaining. The mRNA levels were analyzed by Northern blot. Zymography using gelatin-containing gels was also performed to examine the gelatinase activities. RESULTS: Compared with controls, cells in phagocytically challenged cultures were more sensitive to trypsin. At the 4- and 8-hour postphagocytosis time points, the trypsinization time needed to suspend cells from tissue culture plates was significantly shorter for phagocytically challenged cells. Also, at these two time points, reduced amounts of fibronectin and laminin, as well as disruption of the fibronectin-laminin network, were observed in the phagocytically challenged trabecular meshwork cultures. The mRNA level for fibronectin was reduced, and a slightly increased gelatinase activity was noted. The fibronectin and laminin levels returned to normal by 24 hours. CONCLUSIONS: Results suggest that after phagocytosis, trabecular meshwork cells exhibit a short-term loss in cell-matrix cohesiveness. Such a loss may be related to diminished levels of cell attachment proteins.

Animals↗

PubMed1995

[Ultrastructural changes in the lamina cribrosa in experimental monkey glaucoma].

We examined the ultrastructural changes in the lamina cribrosa in monkeys with experimental chronic glaucoma. In normal monkey eyes, the extracellular matrix consists of tightly packed collagen fibers, elastic fibers and less ground substance in the beams, and basement membranes associated with vascular cells and astrocytes in the lamina cribrosa. In the experimental glaucomatous eyes there was a marked destruction of collagenous bundles. The empty spaces were expanded and filled with fine fibrillar materials. There were normal by appearing parts and clearly destroyed parts mixed in the same region. Elastic fibers looked isolated from the collagenous bundles around them. Basement membranes were generally thick, multi-laminated, and bent. In addition, basement membrane-like materials, separated from the cells, were often seen in the laminar beams. The lamina cribrosa in experimental chronic glaucoma showed evidence of both destruction and healing or remodeling. These changes might have a influence to the worse for the tissue characterization of the lamina cribrosa, such as resistence to intraocular pressure changes. In conclusion, this may be a factor related with the progression of glaucomatous optic nerve damage.

Animals↗

PubMed1995

A human monoclonal antibody to carbohydrate moiety of carcinoembryonic antigen.

Hybridoma BSRF-S-97, secreting a human monoclonal antibody of IgG1 subclass reactive to the carcinoembryonic antigen, was generated by fusing the regional lymph node lymphocytes from a cervical cancer patient with RF-S1 human-mouse heteromyeloma fusion line. This monoclonal antibody was found specifically reactive to carinoembryonic antigen-producing cell lines, including those of cervical cancer (SKG-II), mucinous type ovarian cancer (RMUG-L), stomach cancer (MKN-45), and lung cancer (PC-10). The monoclonal antibody reactivity with pepsin- and periodate-treated carcinoembryonic antigen demonstrated that this monoclonal antibody recognizes the carbohydrate moiety of carcinoembryonic antigen specifically. Possibilities of the monoclonal antibody reaction with mucin and blood-group antigens were excluded by the comparative studies with a placental mucin-containing protein which reacted with carcinoembryonic antigen-specific rabbit polyclonal antibody. The monoclonal antibody conjugated with Pseudomonas exotoxin showed potent regression effects on the growth of the MKN-45 cell line in both the dish culture and xenografted nude mice, indicating potential usefulness of this human monoclonal antibody as a promising tumor targeting vehicle.

Animals↗

PubMed1994

Recombinant rat nucleoside diphosphate kinase isoforms (alpha and beta): purification, properties and application to immunological detection of native isoforms in rat tissues.

We previously demonstrated that at least two isoforms of nucleoside diphosphate (NDP) kinase, the products of two different tandemly arrayed genes, are present in rat. To understand the physiological role of each isoform, some biochemical properties of recombinant rat NDP kinase alpha- and beta-isoforms, produced in large amount, were studied. cDNAs of the two isoforms were inserted in an expression vector pET3b and recombinant enzymes were overproduced in Escherichia coli. Their primary structures were different from the native enzymes in that the latter suffer from modification of the NH2-terminal end. The two recombinant isoforms were purified from the cell lysate to apparent homogeneity by ammonium sulfate fractionation followed by three successive column chromatographies. Despite their extreme similarity in the amino-acid sequences, the two showed somewhat different enzymic properties in terms of di- and triphosphate nucleotide substrate specificity. They showed similar mobilities on SDS-PAGE as expected from their calculated molecular weight (alpha-isoform, 17,283 versus beta-isoform, 17,192) but differed in isoelectric point (alpha-isoform, pI 6.7; beta-isoform, pI 7.8) and heat stability. Polyclonal antibody which reacted with both isoforms and alpha-isoform-specific monoclonal antibodies differentially recognized native enzymes from rat tissues after the tissue extracts were separated by isoelectric focusing gel electrophoresis under a denaturation condition. The results showed that the alpha-isoform, though its amount varied from one tissue to another, was the major form in rat tissues examined compared with the beta-isoform which was detectable in brain and testis. There was no preference in their subcellular localization when examined with myelin, synaptosomal supernatant and total homogenate fractions from the rat cerebrum and cerebellum.

Amino Acid Sequence↗

PubMed1994

Lysosomal enzyme activities in conjunctival tissues of patients with keratoconus.

OBJECTIVE: To examine the lysosomal enzyme activities in the conjunctival tissues of patients with keratoconus. METHODS: Tissues collected from 11 patients with keratoconus, eight patients with senile cataract, three patients with Fuchs' corneal dystrophy, and 11 normal control subjects were processed for histochemical staining for two lysosomal hydrolases, acid esterase and acid phosphatase. RESULTS: The epithelium of all conjunctival specimens stained positively for the two enzymes. The staining in the conjunctival tissues of patients with keratoconus was more prominent than that seen in specimens from either normal control subjects or patients with other diseases. CONCLUSION: Our results suggest that the conjunctival epithelium may be altered in keratoconus. Elevation of lysosomal enzyme levels has been demonstrated in the epithelium of corneas with keratoconus, implicating a role of this layer in the disease. The conjunctival abnormality seems to corroborate the corneal epithelial theory. It also adds one dimension to the pathogenesis of keratoconus.

Acetylesterase↗

PubMed1994

Sulfated proteoglycans in the lamina cribrosa of normal monkey eyes and monkey eyes with laser-induced glaucoma.

The distribution of the sulfated proteoglycans in the lamina cribrosa of normal monkey eyes and monkey eyes with laser-induced glaucoma were analysed by electron microscopy after cuprolinic blue dye binding. Three types of cuprolinic blue-positive filaments, CB-1, CB-2 and CB-3, were identified in the laminar beams of normal monkey eyes. CB-1 and CB-2 were both small filaments representing chondroitin and dermatan sulfate proteoglycan copolymers. The former lined up perpendicularly to the long axis of collagen fibrils, whereas the latter ran axially parallel to collagen fibrils. The large CB-3 filaments, representing chondrotin sulfate proteoglycans, were located around collagen bundles or in loose spaces within the beams. In addition, small CB-4 filaments or punctate structures representing heparan sulfate proteoglycans were found aligned on the basal laminae of blood vessels and glial cells. In the glaucomatous eyes, accumulation and enlargement of collagen-associated proteoglycan filaments were seen, accompanied by the destruction of collagenous beams. Accumulation of chondroitin sulfate proteoglycans was most evident. Prominent filamentous heparan sulfate/heparin proteoglycans were also noted in thickened astrocytic and vascular basal laminae. These may be alterations secondary to the destruction of collagen bundles. They may also represent cellular responses related to intraocular pressure elevation.

Animals↗

PubMed1994

Alpha 1-antichymotrypsin is present in and synthesized by the cornea.

The proteinase inhibitor alpha 1-antichymotrypsin is present in the epithelial, stromal and endothelial layers of the human cornea. This was determined by immunolocalization in corneal sections and by Western blot analysis of extracts from the three separated layers. The inhibitor was quantified in the extracts by immunodot blot analysis. The levels observed were 1.3 +/- 0.3 microgram/cornea for the epithelial layer, 22.8 +/- 3.8 micrograms/cornea for the stromal layer and an average of 0.17 micrograms/cornea for the endothelial layer. alpha 1-Antichymotrypsin is being synthesized by the cornea. Metabolically labeled inhibitor was immunoprecipitated from the three layers following organ culture of the intact cornea. Two major forms were detected. These were the native, mature 64 kDa form and a 50 kDa form which is either a degradation product or an incompletely glycosylated form. These results indicate that the cornea has the ability to locally control degradation through synthesis of this inhibitor. Local synthesis of this inhibitor releases the cornea from total dependance upon the vascular system for its supply of alpha 1-antichymotrypsin.

Adolescent↗

PubMed1994

The collagen fibrillar network in the human pial septa.

The three-dimensional organization of the collagen fibrils in the human pial septa was studied by transmission electron microscopy and by scanning electron microscopy after cell maceration with sodium hydroxide. The collagen fibrils along the nerve fibers were loosely distributed thin filaments with a diameter ranging from 25 to 40 nm (mean, 32 +/- 6 nm). Most fibrils were oriented perpendicularly to the long axis of the nerve fiber. In the deeper stroma of the pial septa, collagen fibrils were well packed and the diameter was 40 +/- 6 nm (range, 35 to 50 nm). The scanning electron microscopic study demonstrated a smooth, fabric-like structure formed by wavy and loosely interwoven thin collagen fibrils. Under high magnification, multiple collagenous sheets were observed in the pial septa by scanning electron microscopy. In addition to the structure of loosely arranged thin fibrils on the surface layer adjacent to the nerve fibers, two distinct patterns of collagen fibril network were demonstrated in the deeper stroma. One exhibited fibrils with random distribution, and the other showed well-packed, undirectionally oriented collagen. Small openings corresponding to elastic tissues were also observed. This collagen architecture may contribute to the flexibility and tensile strength of the pial septa required in the intraorbital portion of the optic nerve.

Adult↗

PubMed1994

Alpha 2-macroglobulin is present in and synthesized by the cornea.

PURPOSE: The purposes of this study were to determine whether the proteinase inhibitor alpha 2-macroglobulin is present in the cornea, and, if so, where it is located, and whether it is synthesized by the cornea, and, if so, where it is being synthesized. METHODS: alpha 2-Macroglobulin was immunolocalized using a double antibody technique and quantified by immunodot blot assays, and its identity was confirmed by Western blot analysis. Corneal synthesis of this inhibitor was determined by immunoprecipitation of extracts from corneas incubated in organ culture with 35S-methionine. mRNA was localized by in situ hybridization of 3H-labeled cDNA to the inhibitor. RESULTS: alpha 2-Macroglobulin was localized in the epithelial, endothelial, and stromal cells. It was also found in the stromal extracellular matrix. When extracts of the epithelium, stroma, and Descemet's membrane-endothelium were analyzed by Western blot, an immunoreactive band for this inhibitor was detected in all extracts. This band comigrated with the alpha 2-macroglobulin form isolated from plasma. Metabolically labeled inhibitor was immunoprecipitated from the stromal layer but not from the epithelial or endothelial layer. However, when examined by in situ hybridization, mRNA was localized to epithelial and endothelial cells in addition to stromal keratocytes. CONCLUSIONS: Because alpha 2-macroglobulin has the ability to inhibit a wide range of proteinases, it is probable that this inhibitor plays an important role in protecting the cornea from damage caused by proteinases. This includes proteinases synthesized by the cornea and those released from inflammatory cells and invading organisms.

Adolescent↗

PubMed1994

Corneal synthesis of alpha 1-proteinase inhibitor (alpha 1-antitrypsin).

PURPOSE: To determine if the cornea synthesizes alpha 1-proteinase inhibitor (alpha 1-antitrypsin). METHODS: Human corneas were placed in organ culture for 24 hours in the presence of 35S-methionine to radiolabel corneal proteins. Monoclonal antibodies were used to precipitate labeled alpha 1-proteinase inhibitor. The immunologically isolated inhibitor was electrophoresed on polyacrylamide gels and visualized by autoradiography or by staining for protein. Human corneas were also fixed with formalin and imbedded in paraffin. Sections were probed with 3H-labeled complementary DNA probes to the coding region of alpha 1-proteinase inhibitor. RESULTS: Metabolically labeled alpha 1-proteinase inhibitor was recovered from organ-cultured corneas and the cornea-conditioned medium. Specific messenger RNA was observed in the cornea by in situ hybridization most prominently in corneal epithelial cells. CONCLUSIONS: alpha 1-Proteinase inhibitor is synthesized and released by human corneal epithelial cells. These results indicate that the cornea has the ability to locally control degradation through synthesis of this inhibitor without total dependence on a supply of the inhibitor from the vascular system.

Adult↗

PubMed1994

[A case of malignant localized chest wall mesothelioma accompanying with abdominal aortic aneurysm].

The patient was a 78-year-old man with a history of large painful left lower lateral chest wall tumor that was fist sized, while he had large abdominal aortic aneurysm. Because of high age, two staged operation was done. Initially chest wall tumor was resected. The defect was 11 x 11 cm in sized. The reconstruction of the defect was made with Marlex mesh. The microscopical findings was biphasic malignant mesothelioma. Fourty one days later abdominal aortic aneurysm was excised and the aorta was reconstructed using a Y shaped artificial graft. Post-operative course was uneventful.

Aged↗

PubMed1993

Isolation, overexpression and disruption of a Saccharomyces cerevisiae YNK gene encoding nucleoside diphosphate kinase.

Nucleoside diphosphate kinase (NDPK) is the major enzyme responsible for the synthesis of all nucleoside triphosphates except ATP. A gene (YNK) encoding NDPK was isolated from the yeast Saccharomyces cerevisiae. The coding region consists of 459 bp encoding 153 amino acid (aa) residues. The M(r) of NDPK, calculated from the deduced aa sequence, is 17,166. Yeast NDPK was 59% and 58% identical to the rat NDPK alpha and beta, respectively. Overexpression of YNK in yeast showed high NDPK activity. The overproduced NDPK cross-reacted with anti-NDPK antibody raised against rat NDPK by Western blot analysis. Despite the fact that NDPK has features of a housekeeping enzyme, disruption of the YNK locus in a haploid strain was neither lethal nor significantly affected phenotypic behaviors such as growth rate, spore formation, mating ability and morphology. Yeast with a defective YNK still possessed NDPK activity to approximately 10% of the wild-type level. Possible sources of the remaining enzyme activity are discussed.

Amino Acid Sequence↗

PubMed1993

Nd:YAG laser trabeculopuncture (YLT) for glaucoma with traumatic angle recession.

Traumatic angle recession caused by blunt trauma often induces uncontrollable glaucoma despite the maximum medical therapy tolerated, such as argon laser trabeculoplasty (ALT), with no or little benefit. Therefore, instead of ALT, we tried Nd:YAG laser trabeculopuncture (YLT) on 11 patients with this type of glaucoma. The intraocular pressure of these patients was followed up for 15 +/- 7 months (average +/- SD). In 6 of 7 eyes treated initially with YLT, the IOP was significantly reduced, so medication was discontinued. Four other cases with uncontrollable IOP after failed ALT were treated with YLT. The IOP of 3 cases was successfully controlled by medication after YLT. These YLT results were then compared with those of ALT in 11 glaucoma patients with traumatic angle recession. Seven of 11 cases treated initially with ALT failed in less than 3 months, and surgical intervention or additional laser treatments were required. The probability of success from the time-series analysis at 12 months after each laser application was 0.909 in YLT, 0.273 in ALT. YLT offers significant advantages over ALT for the treatment of glaucoma with traumatic angle recession after blunt trauma and thus merits further study.

Adolescent↗