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Biomedical subjects

M Takagi

Publications and source records attributed to M Takagi.

At least 289 records · Page 16Linked to original sources
PubMed1998

Oocyte quality of small antral follicles coexisting with cystic follicles in the ovaries of the cow.

The present study was conducted with ovaries to evaluate the effect of the presence of a cystic follicle and its steroid hormone concentration on the oocyte recovery rates, oocyte morphology and in vitro maturation of the oocytes from coexisting small follicles. Ovaries, each bearing a follicular cyst (containing > 10 ml of follicular fluids, > 3 cm in diameter, and without a functional corpus luteum), were collected from each of the 26 Holstein cows from a local slaughterhouse. Small follicles (1-7 mm) from these ovaries were aspirated and their cumulus-oocyte complexes (COCs) classified into one of 5 groups (A to E), depending on oocyte and cumulus cell morphologies. Those oocytes with compact cumulus cells were cultured and their maturation rates determined. Concentrations of estradiol-17 beta (E2) and progesterone (P4) were measured in cystic follicular fluid using double antibody enzyme immunoassays (EIA). The morphology of the COCs and maturation rates of the oocytes were then evaluated using two comparisons. In first comparison, the left and right ovaries obtained from an individual cow were considered as a pair, with each pair being divided into two groups depending on the presence or absence of an E2 dominant or P4 dominant cystic follicle in one or another of the ovaries: E2 dominant cows; and P4 dominant cows. Oocytes collected from the ovaries of cyclic cows at follicular, luteal and post-ovulatory stages served as controls. The oocyte recovery rates, oocyte morphologies and oocyte maturation rates were independent of the presence or absence of a coexisting cystic follicle, or its steroid hormone classification or concentration. In the second comparison, each ovary was consider individually and divided into one of the two classes, depending on the presence or absence of a cystic follicle. Those ovaries with cystic follicles were then divided into three subclasses on the basis of E2 and P4 concentrations in the cystic follicular fluids: P4 dominant (P4/E2 ratio > 1); E2 dominant (P4/E2 ratio < 1); and both P4 and E2 dominant follicles present. The numbers of oocytes obtained from an ovary, their morphology and their maturation rates were not significantly different in the presence or absence of a coexisting cystic follicle. Moreover, the number of the oocytes aspirated from an ovary, their morphology and their maturation rates in small follicles coexisting with P4 dominant, E2 dominant and both P4 and E2 dominant cystic follicles were also not different. These results suggested that neither the presence of a cystic follicle in a cow's ovary nor the cyst's steroid hormone concentrations affected the oocyte recovery rate, oocyte morphology and maturation rates of the coexisting small follicles.

Animals↗
PubMed1998

Ectopic expression of lunatic Fringe leads to downregulation of Serrate-1 in the developing chick neural tube; analysis using in ovo electroporation transfection technique.

Lunatic Fringe (l-Fng) is one of the vertebrate homologues of Drosophila Fringe, which interacts with the Notch signal pathway and regulates activation of the Notch ligands, Delta and Serrate. To elucidate the roles of l-Fng in vertebrate neurogenesis, we transfected chick l-Fng (C-l-Fng) to chick neural tube using the in ovo electroporation technique and examined the subsequent changes in expression of Notch-related genes. We observed downregulation of C-Serrate-1 by ectopic C-l-Fng expression which implied that C-l-Fng acts on the vertebrate Notch pathway to regulate the expression of its ligand.

Animals↗
PubMed1998

Effects of follicular fluid on fertilization and embryonic development of bovine oocytes in vitro.

This study was conducted to evaluate the effect of bovine follicular fluid (BFF) on fertilizability and developmental capacity of bovine oocytes matured in vitro. Oocytes were collected from slaughterhouse ovaries, and matured in TCM199 supplemented with 5% superovulated cow serum (SCS), 2 mM pyruvate and 1 IU/mL PMSG. BFF was aspirated from small follicles (1 to 5 mm in diameter). In Experiment 1, BFF was added to the Brackett and Oliphant (BO) fertilization medium at concentrations of 0, 1, 5, 10 and 20%. After insemination with frozen-thawed and heparin-treated (10 micrograms/mL, 15 min) bull spermatozoa for 18 h, some of the oocytes were fixed and stained to evaluate the fertilization rate. The rest of the oocytes were co-cultured in serum-free embryo culture medium (ECM; TCM199 supplemented with 5% SCS, 2 mM pyruvate and 5 micrograms/mL insulin) with bovine oviductal epithelial cells (BOEC) at 38.5 degrees C under 5% CO2 in air, and the developmental capacity of embryos was examined at 2, 7 and 9 d. In Experiment 2, BFF was added to the serum-free ECM with BOEC at 0, 5, 10 and 20% concentrations, and embryos were cultured for 9 d. Fertilization rates and blastocyst rates in low (1 and 5%) BFF in fertilization medium were not significantly different from the control (without BFF). However, high concentrations of BFF (10 and 20%) in the fertilization medium suppressed both fertilization rates and development. Large vesicles with fast monolayer formation were observed at all concentrations of BFF added to ECM with BOEC. There were no significant differences in cleavage or development to blastocyst in different concentrations of BFF added to ECM. However, the rate of development to hatched blastocysts in 20% BFF was significantly lower than that of the control (P < 0.05). The results of the present study indicate that BFF addition to fertilization medium and ECM with BOEC does not improve fertilizability or developmental capacity and that high concentrations of BFF reduce the rate of both fertilization and development.

Animals↗
PubMed1998

Developmental capacity of bovine oocytes matured in two kinds of follicular fluid and fertilized in vitro.

This study was conducted to assess the ability of the follicular fluid derived from large and small follicles to support the in vitro oocyte maturation and subsequent fertilization and developmental capacity. Oocytes were cultured in bovine follicular fluid aspirated from small (SFF; 2-5 mm in diameter), large (LFF; 10 to 20 mm in diameter) follicles and TCM199 as a control under 5% CO2 in air. All maturation media were supplemented with 1 IU ml-1 pregnant mare serum gonadotropin. After 24 h culture, oocytes were fertilized in vitro with frozen-thawed and heparin-treated (10 micrograms ml-1, 15 min) bull spermatozoa and cultured in TCM199 with bovine oviductal epithelial cells (BOEC) for 7 days. Maturation of bovine oocytes cultured in LFF was inhibited and the low of male pronucleus formation was observed when compared with that of SFF (maturation rate: 69 vs. 78%; P < 0.05; male pronucleus formation rate: 58 vs. 80%; P < 0.05). Developmental capacity of bovine oocytes cultured in SFF was significantly (P < 0.05) higher than that of LFF (15 vs. 5%), but significantly (P < 0.05) lower than that of the control. There were no differences in the number of nuclei per blastocyst obtained after each treatment. These results indicate that the inhibitory action of follicular fluid on in vitro maturation, male pronucleus formation and developmental capacity of bovine oocytes is dependent on the developmental stage of the follicles from which fluid was obtained.

Animals↗
PubMed1998

Isozyme function of n-alkane-inducible cytochromes P450 in Candida maltosa revealed by sequential gene disruption.

An n-alkane-assimilating yeast Candida maltosa contains multiple n-alkane-inducible forms of cytochromes P450 (P450alk), which can be assumed to catalyze terminal hydroxylation of n-alkanes in the assimilation pathway. Eight structurally related P450alk genes have been identified. In the present study, the function of four major isoforms of P450alk (encoded by ALK1, ALK2, ALK3, and ALK5 genes) was investigated by sequential gene disruption. Auxotrophic markers used for the selection of disrupted strains were regenerated repeatedly through either mitotic recombination between heterozygous alleles of the diploid genome or directed deletion of the marker gene, to allow sequential gene disruptions within a single strain. The strain depleted of all four isoforms could not utilize n-alkanes for growth, providing direct evidence that P450alk is essential for n-alkane assimilation. Growth properties of a series of intermediate disrupted strains, plasmid-based complementation, and enzyme assays after heterologous expression of single isoforms revealed (i) that each of the four individual isoforms is alone sufficient to allow growth on long chain n-alkane; (ii) that the ALK1-encoding isoform is the most versatile and efficient P450alk form, considering both its enzymatic activity and its ability to confer growth on n-alkanes of different chain length; and (iii) that the ALK5-encoding isoform exhibits a rather narrow substrate specificity and thus cannot support the utilization of short chain n-alkanes.

Alkanes↗
PubMed1998

Cloning and characterization of an n-alkane-inducible cytochrome P450 gene essential for n-decane assimilation by Yarrowia lipolytica.

A gene encoding cytochrome P450 involved in n-alkane utilization was cloned from an n-alkane assimilating yeast, Yarrowia lipolytica CX161-1B. The RT-PCR was performed on the mRNA prepared from the cells grown on n-alkane as a template using degenerated PCR primers designed for the conserved amino acid sequences of the CYP52 family. The RT-PCR amplified fragment was then used as a probe to isolate genes coding for P450 of the CYP52 family from the genomic DNA library of the strain CX161-1B. The nucleotide sequence of one of the positive clones was determined. An open reading frame which had the same nucleotide sequence as the RT-PCR-amplified fragment was identified. It was of 523 amino acid residues, 60.2 kDa in molecular mass, and had 30-45% sequence identity with the other members of the CYP52 family of Candida species so far analysed. The expression of the P450 gene that was named as YlALK1 was induced by n-tetradecane and repressed by glycerol. A YlALK1 gene disruptant did not grow well on n-decane, but grew on longer-chain n-alkanes such as hexadecane as a sole carbon source. Introduction of YlALK1 on a plasmid to the disruptant restored the decane assimilation. These results suggest that the YlALK1 gene product is the major P450A1k to metabolize short-chain n-alkanes such as decane and dodecane in Y. lipolytica.

Alkanes↗
PubMed1998

Reconstruction of thoracic wall defects after tumor resection using a polytetrafluoroethylene soft tissue (Gore-Tex) patch.

BACKGROUND: Recently, there have been a few reports recommending use of a 2 mm thick polytetrafluoroethylene soft tissue (Gore-Tex) patch for repair of thoracic wall defects. The potential role of these Gore-Tex patches was examined. METHODS: Five patients underwent chest wall tumor resection with thoracic wall reconstruction using a Gore-Tex patch (2 mm). We present a review of the complications experienced by five patients with Gore-Tex patches, as well as a review of the literature. RESULTS: Functionally and cosmetically, satisfactory results were obtained for 5 patients with Gore-Tex patch. There were no cases of infectious complications. However, we experienced one case of a flail chest postoperatively, in which reconstruction with two Gore-Tex patches of 30 x 15 cm, and 3 days of mechanical ventilation and chest wall support was needed. CONCLUSION: Our experience with Gore-Tex patches has been positive, and we recommend patch closure for thoracic wall defects.

Adolescent↗
PubMed1998

[The usefulness of thoracoscopic surgery for pleuritis carcinomatosa].

We performed thoracoscopic surgery for pleuritis carcinomatosa, in which re-expansion of the collapsed lung was impossible, by employing tube thoracostomy. One of the treatments used for pleuritis carcinomatosa was drainage of the pleural effusion followed by re-expansion of the collapsed lung. The decortication and biopsy of the pleura and the pleural effusion drainage with the thoracoscopic procedure were performed, allowing control of the pleural effusion and re-expansion of the collapsed lung to be accomplished. We conclude that thoracoscopic surgery is useful for the thoracic treatment of pleuritis carcinomatosa.

Adenocarcinoma, Papillary↗
PubMed1998

Biocompatibility of silicon carbide in colony formation test in vitro. A promising new ceramic THR implant coating material.

We studied the possible use of silicon carbide (SiC) as a ceramic coating material of titanium-based total hip replacement (THR) implants. The idea is to prevent wear debris formation from the soft titanium surface. SiC is a hard and tightly bonding ceramic surface material, and because of these physical properties it is not easily degradable, as is the case with hydroxyapatite. Our previous in vivo and in vitro studies have indicated that SiC and hydroxyapatite are equally biocompatible regarding particle size for phagocytosis. The present cytotoxicity test using JCRB0603 cells showed that 5 microns SiC particles inhibited colony outgrowth by one-third (67% + 10% vs control), while SiC-coated pins did not cause any inhibition and acted similarly to uncoated titanium pins. The results support the hypothesis that SiC is a promising ceramic THR implant coating material.

Carbon Compounds, Inorganic↗
PubMed1998

Sequence analysis of glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 and comparison of the enzymatic characteristics of native and recombinant GDHs.

The gdhA gene encoding glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned and sequenced. Phylogenetic analysis was performed on an alignment of 25 GDH sequences including KOD1-GDH, and two protein families were distinguished, as previously reported. KOD1-GDH was classified as new member of the hexameric GDH Family II. The gdhA gene was expressed in Escherichia coli, and recombinant KOD1-GDH was purified. Its enzymatic characteristics were compared with those of the native KOD1-GDH. Both enzymes had a molecular mass of 47,300 Da and were shown to be functional in a hexameric form (284 kDa). The N-terminal amino acid sequences of native KOD1-GDH and the recombinant GDH were VEIDPFEMAV and MVEIDPFEMA, respectively, indicating that native KOD1-GDH does not retain the initial methionine at the N-terminus. The recombinant GDH displayed enzyme characteristics similar to those of the native GDH, except for a lower level of thermostability, with a half-life of 2 h at 100 degrees C, compared to 4 h for the native enzyme purified from KOD1. Kinetic studies suggested that the reaction is biased towards glutamate production. KOD1-GDH utilized both coenzymes NADH and NADPH, as do most eukaryal GDHs.

Amino Acid Sequence↗
PubMed1998

The O6-methylguanine-DNA methyltransferase from the hyperthermophilic archaeon Pyrococcus sp. KOD1: a thermostable repair enzyme.

The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is the most common form of cellular defense against the biological effects of O6-methylguanine (O6-MeG) in DNA. Based on PCR amplification using primers derived from conserved amino acid sequences of MGMTs from 11 species, we isolated the DNA region coding for MGMT from the hyperthermophilic archaeon Pyrococcus sp. KOD1. The MGMT gene from KOD1 (mgtk) comprises 522 nucleotides, encoding 174 amino acid residues; its product shows considerable similarity to the corresponding mammalian, yeast and bacterial enzymes, especially around putative methyl acceptor sites. Phylogenetic analysis of MGMTs showed that archaeal MGMTs were grouped with their bacterial counterparts. The location of the MGMT gene on the KOD1 chromosome was also determined. The cloned KOD1 MGMT gene was overexpressed using the T7 RNA polymerase expression system, and the recombinant protein was purified by ammonium sulfate fractionation, heat treatment, ion-exchange chromatography and gel filtration chromatography. The purified recombinant protein was assayed for its enzyme activity by monitoring transfer of [3H]methyl groups from the substrate DNA to the MGMT protein; the activity was found to be stable at 90 degrees C for at least 30 min. When the mgtk gene was placed under the control of the lac promoter and expressed in the methyltransferase-deficient Escherichia coli strain KT233 (delta ada, delta ogt) cells, a MGMT was produced. The enzyme was functional in vivo and complemented the mutant phenotype, making the cells resistant to the cytotoxic properties of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine.

Amino Acid Sequence↗
PubMed1998

Six-year survival after excision of cardiac malignant lymphoma.

We report a patient with a postoperative survival period of 6 years after the surgical excision of a cardiac malignant lymphoma. A 35-year-old woman underwent total excision of the tumor arising from the left ventricular outflow tract. After the operation, she was treated with chemotherapy for 6 months. She has been doing well thereafter without any medication. To date there is no evidence of recurrence.

Adult↗
PubMed1998

Characterization of the Candida rugosa lipase system and overexpression of the lip1 isoenzyme in a non-conventional yeast.

The fungus C. rugosa produces lipase isoenzymes (CRLs) homologous to the Geotrichum candidum and Yarrowia lipolytica lipases to which they share ca. 40 and 30% sequence identity, with a domain of sequence conservation at the N-terminal half of the protein. CRL proteins have high sequence homology but are not identical in their catalytic activity, therefore calling for the resolution of isoforms via heterologous expression. The non-conventional use of a serine codon in several Candida species frustrates overexpression in the currently available host systems. The LIP1 gene, coding for the major CRL form, was therefore expressed in C. maltosa, a related fungus with the same codon usage as C. rugosa. A recombinant lipase was produced and secreted in an active form in the culture medium upon engineering the 5' and 3' ends of the gene.

Candida↗
PubMed1998

Local distributions of oviductal estradiol, progesterone, prostaglandins, oxytocin and endothelin-1 in the cyclic cow.

The cyclic patterns of hormones which regulate the activity of the oviduct in the cow have not been adequately reported. We studied progesterone (P4), estradiol 17 beta (E2), prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), oxytocin (OT) and endothelin-1 (ET-1) concentrations in the cow oviduct. Reproductive tracts from cyclic Holstein cows in the follicular phase (n = 5), post ovulation phase (n = 5) and luteal phase (n = 5) were collected at a slaughterhouse. Oviducts were separated from the uterus, the lumen vas washed with physiological saline, and the enveloping connective tissues were removed. The fimbria was then separated at first and then the rest was divided into 2 parts of equal length (proximal and distal). After extraction, levels of different hormones in the tissues were measured using double antibody enzyme immunoassays (EIAs). There were no differences in any hormone concentration between the 3 parts of the oviduct at any stage of the estrous cycle. The highest concentration of oviductal P4 was observed during the luteal phase and in the oviduct ipsilateral to the functioning CL. Oviductal OT was unchanged throughout the cycle. The highest E2 concentration was observed during the follicular phase in the oviduct ipsilateral to the dominant follicle. The oviduct ipsilateral to the dominant follicle during the follicular phase and ipsilateral to the ovulation site post ovulation showed higher levels of PGE2, PGF2 alpha and ET-1 than those on the contralateral side or during the luteal phase. The highest PGE2 was observed in the oviduct ipsilateral to the ovulation site during the post ovulation phase. The results suggest that the ovarian products (P4, OT and E2) and the local oviductal products (PGE2, PGF2 alpha, and ET-1) may synergistically control oviductal contraction for optimal embryo transport during the periovulatory period, and provide further evidence for the local delivery of ovarian steroids to the adjacent reproductive tract.

Animals↗
PubMed1998

Genetic risk factor for abdominal aortic aneurysm: HLA-DR2(15), a Japanese study.

PURPOSE: Autoimmunity has been proposed to play a role in the pathogenesis of the abdominal aortic aneurysm (AAA). Several autoimmune diseases are associated with specific HLA DR alleles. These experiments were carried out to determine whether the same HLA DR types that have been reported to be associated with AAA in a mixed North American population are similarly associated with AAA in a more homogeneous group of patients in Japan. METHOD: HLA DR typing was performed by a serologic method on samples of peripheral blood of patients with nonspecific infrarenal AAA in Nagasaki University Hospital in Japan. The frequencies of HLA DR antigens were compared with those of volunteers approximately matched for age and sex from the same referral area. RESULTS: HLA DR haplotypes were determined in 46 Japanese patients with AAA and in 50 patients in a control group. The HLA-DR2(15) antigen was observed in 27 (58.7%) patients (29 alleles 31.5%) with AAA and in 14 (28%) subjects (16 alleles 26.0%) in the control group (p < 0.005). CONCLUSIONS: The data suggest that HLA-DR2(15) has an important role as a genetic risk factor for AAA in Japanese patients, as previously reported in a mixed North American population.

Aged↗
PubMed1998

New collagenolytic enzymes/cascade identified at the pannus-hard tissue junction in rheumatoid arthritis: destruction from above.

Our aim was to investigate the collagenolytic potential and localization of matrix metalloproteinase-2 (MMP-2) in relation to its regulatory proteins membrane type MT1-MMP and tissue inhibitor of metalloproteinases-2 (TIMP-2) in rheumatoid arthritis (RA). For this purpose, we have used purification of MMP-2, MMP-8, MMP-9 and interstitial type I, II and III collagens; SDS-PAGE/densitometric collagenase activity assay; zymography; Western blotting; reverse transcriptase polymerase chain reaction; in situ hybridization; and immunofluorescence, ABC, ABC-APAAP double immunostainings. MMP-2 degraded human type II collagen almost as effectively as MMP-8, whereas MMP-9 did not cleave type II collagen. In synovial tissue, MT1-MMP, TIMP-2 and MMP-2 were found in synovial lining in fibroblast- and macrophage-like cells, in stromal cells and in vascular endothelium. MT1-MMP, TIMP-2 and MMP-2 were strongly expressed in the pannocytes of the invasive pannus at the interface, but staining was weak and/or there were few positive cells both "above" and "below" the soft-to-hard tissue (cartilage and/or bone) interface. Rheumatoid synovial tissue extract contained proteolytically active 62/59 kDa MMP-2 and 43 kDa MT1-MMP, but no free TIMP-2. These results indicate that components of the ternary MT1-MMP/TIMP-2/MMP-2 complex are coexpressed in the normal synovial lining and in its pathological extension on the hyaline articular cartilage. MMP-2 may participate in the remodeling of the normal lining and also seems to be localized/focalized to pannocytes at a site critical for tissue destruction in arthritis.

Adult↗
PubMed1998

Archaeon Pyrococcus kodakaraensis KOD1: application and evolution.

Archaea is the third domain which is phylogenetically differentiated from the other two domains, bacteria and eucarya. Hyperthermophile within the archaea domain has evolved most slowly retaining many ancestral features of higher eukaryotes. Pyrococcus kodakaraensis KOD1, which grows at 95 degrees C optimally, is a newly isolated hyperthermophilc archaeon. The KOD1 strain possesses a circular genome, whose size is estimated to be approximately 2,036 kb. KOD1 enzymes involved in the genetic information processing system, such as DNA polymerase, Rec protein, aspartyl tRNA synthetase and molecular chaperonin, share features of eukaryotic enzymes. Rapid and accurate PCR method by KOD1 DNA polymerase and enzyme stabilization system by KOD1 chaperonin are also introduced in this article.

DNA, Archaeal↗