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Biomedical subjects

Y Fukui

Publications and source records attributed to Y Fukui.

At least 343 records · Page 19Linked to original sources
PubMed1991

Developmental ability of in vitro matured sheep oocytes collected during the nonbreeding season and fertilized in vitro with frozen ram semen.

During the nonbreeding season, oocytes recovered from ovaries of FSH-primed or nonprimed ewes were matured in the presence or absence of granulosa cells collected from ovaries of primed or nonprimed ewes prior to in vitro fertilization with either fresh or frozen-thawed sperm. Following fertilization, ova were cultured for 24 h in synthetic oviduct fluid medium (SOF) supplemented with 20% human serum at 39 degrees C under humidified 5% CO(2), 5% O(2), 90% N(2) and then assessed for cleavage. Overall, 52% of ova cleaved. Cleavage was not affected by the source of sperm. Significantly more oocytes from primed follicles cleaved after 24 hours than those from nonprimed follicles (P<0.001). Maturation of oocytes in the presence of granulosa cells from nonprimed ewes resulted in a lower cleavage rate (44%, P<0.05) than in the presence of granulosa from primed ewes (59%) or no granulosa cells (50%). Oocytes (n = 508) from primed ewes were matured in the presence of granulosa cells (also from primed ewes) and fertilized in vitro with frozen-thawed sperm. Following in vitro culture for 24 hours, 68 of the 270 (53%) cleaved embryos were transferred to 17 recipient ewes, 15 of which remained pregnant to term, producing 24 lambs. The remaining 202 cleaved embryos were cultured for a further 5 days, of which 73 appeared to reach the morula/blastocyst stage and 61 were transferred to 16 recipients. Two ewes remained pregnant to term producing two lambs. These results demonstrate that production of sheep embryos using in vitro maturation and fertilization techniques is possible in the nonbreeding season. However, the poor viability of embryos obtained following extended culture needs to be resolved before such techniques can be usefully applied.

Journal Article↗
PubMed1991

Effects of media and the presence of bovine oviduct epithelial cells during in vitro fertilization on fertilizability and developmental capacity of bovine oocytes.

The effect of the presence of bovine oviduct epithelial cells (BOEC; Experiment 1) as well as the effects of media (Tyrode fertilization medium: TFM vs synthetic oviduct fluid: SOF), fertilization containers (drops in petri dish vs 96-wells), and the number of oocytes per drop and well (5 vs 10) for in vitro fertilization (Experiment 2) on the fertilizability and in vitro development of bovine oocytes were investigated. Immature oocytes with cumulus cells were cultured in TCM199 supplemented with 10% ECS and 2.5x10(6) granulosa cells for 24 hours at 39 degrees C under 5% CO(2) in air. In vitro fertilization was performed with frozen-thawed, heparin-treated spermatozoa (100 mug/ml, 15 minutes) and with BOEC (Experiment 1). In Experiment 2, in vitro fertilization was performed with two different media (TFM and SOF) and various conditions (culture dish and different number of oocytes). Cleavage, development to the blastocyst stage were evaluated on Day 2 and Day 7 after the start of culture. Effect of the presence of BOEC on fertilizability and developmental capacity (Experiment 1) was not significantly different. In Experiment 2, alterations in media, containers and number of oocytes during in vitro fertilization had no affect. The SOF medium showed results similar to those of TFM (normal fertilization rate: 63.2 vs 64%; cleavage: 69.3 vs 73.9%; development to the blastocyst stage: 14 vs 15%; and mean number of nuclei per blastocyst: 80.5 vs 86.6). The results indicate that the presence of BOEC during in vitro fertilization did not improve fertilizability, and that SOF as well as TFM medium can be utilized as a simple fertilization medium.

Journal Article↗
PubMed1991

Effects of culture duration and time of gonadotropin addition on in vitro maturation and fertilization of red deer (Cervus elaphus) oocytes.

Immature red deer (Cervus elaphus) oocytes (n = 1208) were collected from 1 to 4 - mm diameter follicles on ovaries and then cultured for 16, 20, 24 or 28 h (Groups I to IV) in TCM 199 supplemented with 10% FCS, 1 x 10(6) granulosa cells/ml and 1 microg/ml estradiol at 39 degrees C under 5% CO(2) in air. Gonadotropins (10 microg/ml, FSH and LH) were added to the culture medium at the start of culture (0 h) or after 6 h. Approximately one-third of the oocytes were examined for maturation, and the remainder were fertilized in vitro with frozen-thawed semen collected from a stag by electroejaculation. In vitro fertilized oocytes (n = 309) from four of the maturation treatment (Groups II and III in both gonadotropin treatments) were cultured for 7 d and examined for cleavage. Oocytes cultured for 16 h (Group I) had lower (P < 0.001) maturation rates (4.7%) than those in the longer culture durations (Groups II to IV: 68.9%). Culture for 20 (Group II) and 24 h (Group III) resulted in higher (P <0.001) fertilization rates than culture for 16 (Group I) and 28 h (Group IV) (18.3, 20.5, 7.1, 7.8%, respectively). The time of gonadotropin addition did not affect maturation or fertilization rates, but its addition at 6 h increased (P < 0.05) the percentage of oocytes cleaving (5.7 vs 12.5%). Oocytes cultured for 20 h (Group II) and with the delayed addition of gonadotropins cleaved most readily (18.2%). No embryos developed beyond eight-cell stage.

Journal Article↗
PubMed1991

Direct amygdaloid projections to the superior salivatory nucleus: a light and electron microscopic study in the cat.

Amygdaloid projections to the superior salivatory nucleus (SSN) were investigated in the cat by using the anterograde and retrograde tracing techniques of horseradish peroxidase (HRP). After HRP injections were made into the lingual nerve, retrogradely labeled SSN neurons were located in the lateral tegmental field medial to the spinal trigeminal nucleus from the middle level of the superior olivary nucleus to the caudal level of the facial nucleus. These labeled neurons, triangular, oval or polygonal in shape, were small to medium-sized (12-29 microns) and formed loosely packed clusters. In further HRP studies, HRP injections were made into the amygdala and in the reticular formation containing the SSN neurons. The results suggested that the SSN receives direct afferents from the central nucleus of the amygdala with ipsilateral predominance. Final proof of such direct connections from amygdala to the SSN can be obtained only by electron microscopic study. Therefore, HRP injections were made into the lingual nerve and in the amygdala in the same animal and electron microscopic observations were carried out on the SSN. It appeared that anterogradely labeled amygdalo-tegmental fibers formed axosomatic and axodendritic synaptic contacts with retrogradely labeled SSN neurons.

Afferent Pathways↗
PubMed1991

Studies of the development of optical fiber sensors for biochemical analysis.

An optical fiber sensor utilizing Thymol blue and an ion-exchange resin complex in a cellulose acetate membrane was developed. By monitoring several different chromophores of Thymol blue, the sensor could measure the pH of the solution from 1.0 to 12.0 with good reproducibility. An optical fiber glucose sensor utilizing a cellulose acetate membrane containing glucose oxidase, 2,7-diaminofluorene dihydrochloride, and sodium N-(3-sulfopropyl)-3,3',5,5'-tetramethylbenzidine was developed. Reflectance changes at 580 nm were large enough to trace changes in glucose concentration in physiological saline solution.

Biosensing Techniques↗
PubMed1991

Deletions in the SH2 domain of p60v-src prevent association with the detergent-insoluble cellular matrix.

p60v-src has been shown to associate with a detergent-insoluble cellular matrix containing cytoskeletal proteins, but p60c-src does not bind to this matrix. We analyzed the association of mutant src proteins with the matrix and found that mutants which lack an amino-terminal portion (residues 149 to 169) of the SH2 domain cannot bind to the matrix. Neither the SH3 region nor other portions of the SH2 region were required for association. We also tested protein kinase-defective mutants and chimeras of p60v-src and p60c-src. We found a strong correlation between the kinase activity of p60src and its association with the detergent-insoluble matrix. Double infection of kinase-defective and kinase-active mutants did not result in matrix binding of the kinase-defective src proteins. We also found that Tyr-416, the major site of autophosphorylation in p60v-src, was not required for matrix association.

Amino Acid Sequence↗
PubMed1991

Requirement of phosphatidylinositol-3 kinase modification for its association with p60src.

When purified p60v-src was mixed with lysates of chicken embryo fibroblasts and immunoprecipitated with anti-Src antibody, phosphatidylinositol (PI)-3 kinase activity was found to be present in the Src protein immunoprecipitates. The level of bound PI-3 kinase activity was 5 to 10 times higher in lysates obtained from cells transformed by the src, fps, or yes oncogene than in lysates of uninfected cells. This increase in associated PI-3 kinase activity appears to be due to increased binding of this enzyme to p60v-src. This change most likely resulted from tyrosine phosphorylation of PI-3 kinase or an associated protein, since the PI-3 kinase activity that can bind to p60v-src was depleted by antiphosphotyrosine antibody. Binding of PI-3 kinase did not require either p60src protein kinase activity or autophosphorylation of p60v-src tyrosine residues. Furthermore, binding was markedly decreased by deletions in the N-terminal SH2 region but unchanged by deletion of the C-terminal half of p60v-src containing the catalytic domain. Taking these data together, it appears that PI-3 kinase or its associated protein is phosphorylated on tyrosine and that the phosphorylated form can bind to the N-terminal half of p60v-src, which contains the SH2 domain.

1-Phosphatidylinositol 4-Kinase↗
PubMed1991

Protein-secretory patterns of normal and abnormal human placentas with special reference to human chorionic gonadotropin.

[35S]Methionine-labeled protein-secretory patterns resolved by two-dimensional polyacrylamide gel electrophoresis in abnormal hydatidiform-mole placentas were compared with those in normal full-term placentas with special reference to human chorionic gonadotropin (hCG) by means of immunoblotting and immunoelectron-microscopic techniques. Although basic protein-secretory patterns of both placentas were similar to each other, four polypeptide spots appeared and one spot disappeared in the hydatidiform-mole samples. Among four newly synthesized and secreted spots, three were immunoreacted with anti-hCG serum by an immunoblotting experiment. Ultrastructural localization of hCG showed that the labeling intensity of anti-hCG serum in hydatidiform-mole placentas was much heavier than that in full-terms ones. Particularly, the Golgi apparatus, middle-sized granules and large bodies were highly immunoreactive. The present study reveals that hydatidiform-mole placentas have different protein-secretory functions especially in hCG synthesis and secretion from those of normal pregnancy.

Autoradiography↗
PubMed1991

Neoadjuvant therapy for locally invasive bladder cancer: results of randomized trials in 40 patients.

The present investigation was conducted to examine the effect of a neoadjuvant cyclophosphamide, doxorubicin and cisplatin (CAP) regimen with radiotherapy for locally invasive bladder cancer as a well-controlled randomized trial. Since 1986, a total of 40 patients with primary transitional cell carcinoma of the urinary bladder have been randomized into two groups: neoadjuvant CAP plus radiation-treated group and control group. Of 18 patients who received neoadjuvant chemotherapy, complete and partial responses were observed in 52.9% of 17 measurable and evaluable patients and downstaging was observed in 92.9% of 14 evaluable patients. The 3-year survival rates of the neoadjuvant-treated and control group were 93.8 and 83.6%, respectively. No statistical significance was achieved in the survival rates. These results indicated that neoadjuvant CAP would be useful in the management of invasive bladder cancer.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1991

Cell behavior and actomyosin organization in Dictyostelium during substrate exploration.

The behavior of individual Dictyostelium amebae was quantitatively analyzed with the computer-assisted "Dynamic Morphology System" (Soll, Voss, Varnum-Finney and Wessels, (1988) J. Cell. Biochem., 37: 177-192.). The same amoebae were then fixed and analyzed for filamentous (F-) actin and myosin (myosin-II, or "conventional" myosin) by fluorescence microscopy using the "agar-overlay method" (Yumura, and Fukui (1985) Nature, 314: 194-196.). This procedure provides a novel description of the behavior and morphometric changes preceding the static analysis of cytoskeletal organization in the same cell. It is demonstrated that when translocating cells make contact with an etched-smooth glass interface, 14% cross the interface, 20% either reverse direction or migrate along the interface, and the remaining 45% stay at the site. Cells contacting the interface from the smooth or etched side show equivalent behavioral responses. Upon contact with the interface, they project numerous lamellipodia and pseudopodia. While the lamellipodial projections exhibit cycles of spreading and retraction, the pseudopodia show lateral scanning motion, analogous to "substrate exploration" in fibroblasts (Albrecht-Buehler (1976) J. Cell Biol., 69: 275-286.). F-actin is localized in the lamellipodia and pseudopodia of amoebae contacting the interface. There is also discernable cortical F-actin, while conventional myosin appears to be excluded from the cortex and dispersed throughout the cytoplasm. The myosin displays a transient filamentous lattice at the base of newly forming lamellipodia. The ultrastructural study suggests that the new lamellipodia are formed on the dorsal surface and subsequently make contact with the substrate, indicating the dorsoventral sequence of polarity of the motile/sensory cellular organs. The present study demonstrates substrate exploration in Dictyostelium amoebae, and suggests its coupling to dynamic reorganization of the actomyosin cytoskeleton. The possible role of single-headed small myosin(s) (myosin-I, or mini-myosin) is discussed.

Actomyosin↗
PubMed1991

Factors affecting the in-vitro development to blastocysts of bovine oocytes matured and fertilized in vitro.

The effects of media (TCM199 vs. synthetic oviduct fluid, SOF), sera (foetal calf serum, FCS vs. human serum, HS), gas atmosphere (5% CO2 in air vs. 5% CO2, 5% O2 and 90% N2) and coculture with bovine oviduct epithelial cells (cells vs. no cells) on the in-vitro development of in-vitro matured and fertilized bovine oocytes were examined. Immature oocytes surrounded with compacted cumulus cells were cultured for 24 h in TCM199 supplemented with 10% FCS, 10 micrograms follicle-stimulating hormone (FSH)/ml and 10 micrograms luteinizing hormone (LH)/ml, 1 microgram oestradiol/ml, and 1 x 10(6) granulosa cells at 39 degrees C under 5% CO2 in air. In-vitro fertilization was performed with frozen-thawed, heparin-treated (100 micrograms/ml, 15 min) spermatozoa from 2 bulls. Oocytes were incubated with 2.5 x 10(6) spermatozoa/ml for 24 h and then cultured in one of 16 treatments for 7 days. Cleavage (2-8-cell) and development to blastocysts were recorded on Days 2 and 7, respectively, after the start of culture. SOF was superior to TCM199 for cleavage (P less than 0.01), development to blastocysts (P less than 0.001) and for proportion of cultured ova resulting in blastocysts with at least 60 or at least 100 nuclei (P less than 0.001). FCS was superior to HS for development to blastocysts (P less than 0.001) and 5% oxygen was superior to air for the proportion of ova reaching at least 60 cells (P less than 0.01). For cleavage and development to blastocysts, there was an interaction between serum and cells (P less than 0.01). In the presence of cells, ova preferred FCS, in their absence, serum had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Phosphatidylinositol-3 kinase is activated in v-src, v-yes, and v-fps transformed chicken embryo fibroblasts.

PI-3 kinase activity has been shown to associate with p60v-src. We found that immunoprecipitates of p60v-src exhibit an activity that catalyzes the formation of PI-3-P, PI-3,4-P2 and PIP3 from PI, PI-4-P, and PI-4,5-P2, respectively. Transformation of chicken embryo fibroblasts (CEF) by p60v-src of Rous sarcoma virus (RSV) caused elevation of PI-3-P, PI-3,4-P2, and PIP3, suggesting that the PI-3 kinase may be activated in these cells. Similar elevations were seen in cells transformed with the v-yes or v-fps oncogenes, but not with v-ros or v-ras. We have established also a system that allows the binding of PI-3 kinase to purified p60v-src in vitro, reproducing the binding seen in vivo. This assay indicated that more PI-3 kinase activity binds to purified p60v-src in cell lysates from CEF transformed with v-yes or v-fps, suggesting that some modification or over-expression of PI-3 kinase takes place in these cells.

1-Phosphatidylinositol 4-Kinase↗
PubMed1991

[A case of retroperitoneal pleomorphic lipoma].

A 71-year-old male patient was referred to our department for further examination for right retroperitoneal tumor. Exploration was done through a flank approach and the tumor with right adrenal gland was removed. A pleomorphic lipoma was diagnosed histopathologically. There have been 8 reported cases of pleomorphic lipoma including our present case in Japan and we discuss the pathogenesis and treatment of this rare disease.

Aged↗
PubMed1991

Development of a compact extracorporeal membrane oxygenation (ECMO) system.

In recent years, extracorporeal membrane oxygenation (ECMO) has been used for treatment of neonates with respiratory failure. A prototype of a compact ECMO system for neonates was developed. A single-lumen catheter, inserted into the right atrium via a jugular vein, was used for withdrawal and infusion of blood through the catheter. An extracapillary flow hollow-fiber membrane lung made of microporous polypropylene has a total surface area of 0.6 m2. To prevent the increase of plasma free hemoglobin, the ratio of withdrawal/infusion is controlled by a microcomputer. The system is compact in size with a low priming volume (less than 90 ml), which allows for ECMO with no additional blood transfusions. Its potential application as a respiratory support system is evaluated in animal experiments. The total intermittent veno-veno bypass flow was 15-30 ml/min/kg. The O2 transfer rate was 20 ml/min and the CO2 transfer rate was 33 ml/min at a blood flow rate of 300 ml/min. The O2 and CO2 exchange with the ECMO system was efficient enough to eliminate the respiratory failure induced by mechanical ventilation. The increase in plasma free hemoglobin was only 4 mg/dl after 6 h of ECMO. The system was considered applicable to respiratory aid for neonates.

Animals↗
PubMed1991

Quantitative study of the development of the optic nerve in rats reared in the dark during early postnatal life.

Male rats were placed in complete darkness from birth until 30 days of age, followed in some cases by a 35 days period of rehabilitation in control lighting conditions. Groups of control and experimental animals were killed at 30 and 65 days of age by perfusion with buffered 2.5% glutaraldehyde. The right optic nerve was dissected out from each animal and processed for embedding in Epon. Quantitative stereological procedures were used to estimate the total number of both myelinated and non-myelinated optic nerve fibres and their mean minimum diameters. There were no significant differences in the total number of optic nerve fibres between dark- and light-reared rats. However dark-reared rats had myelinated and non-myelinated fibres with significantly larger fibre diameters than those in age-matched light-reared rats. The proportion of optic nerve fibres which were myelinated increased with age in both groups of animals. However by 65 days of age the degree of myelination was slightly but significantly greater in the previously dark-reared rats than in the light-reared controls. These results indicate that rats reared in complete darkness for the first 30 days of postnatal life show morphological changes in the optic nerves. The possible significance of these changes is discussed.

Animals↗
PubMed1991

Quantitative study of the development of neurons and synapses in rats reared in the dark during early postnatal life. 1. Superior colliculus.

Rearing animals in dark conditions during early postnatal life has been shown to affect both the morphology and the normal functioning of the visual system. We have investigated the effects on the synapse-to-neuron ratios in the superior colliculi of rearing male rats in the dark from birth until 30 days of age, followed in some cases by a 35 day period of rehabilitation in control lighting conditions. Control lighting conditions consisted of a room on a 12 hour light/12 hour dark cycle. Synapse-to-neuron ratios were calculated from estimates of the numerical densities of neurons and synapses. These estimates were made using the 'disector' method at the light and electron microscopical levels. Neuronal nuclei were used as the counting unit for neurons and paramembranous densities for synapses. There were no significant differences in the numerical densities of neurons, synapses or synapse-to-neuron ratios between dark-reared and control rats at 30 days of age. Sixty five days old rats, previously raised in the dark, had a significantly smaller numerical density of neurons than light-reared controls. Two-way analysis of variance techniques showed significant effects of age on the estimates of the numerical densities of neurons and synapses. However, there were no significant main effects of the lighting conditions, nor any significant interaction for any of the measures.

Animals↗