Biomedical subjects

M Hatakeyama

Publications and source records attributed to M Hatakeyama.

At least 19 recordsLinked to original sources

PubMed2005

Preservation of a transgenic strain of the sawfly, Athalia rosae (Hymenoptera) by artificial fertilization using cryopreserved sperm.

Germline transformation using a piggyBac-derived vector is feasible in the sawfly, Athalia rosae. A previously generated transgenic line carrying green fluorescence protein (GFP) genes as reporters was successfully maintained and preserved without consecutive rearing. Sperm taken from males that were frozen directly in liquid nitrogen and stored at -80 degrees C for a year were microinjected into mature unfertilized eggs dissected from female ovaries. A fraction of the sperm-injected eggs was fertilized and developed into diploid females, and all of them expressed GFP. Haploid male progeny from these females segregated into GFP-positive and GFP-negative individuals in a ratio of 1:1 indicating heterozygosity of the parental females. The GFP genes were stably inherited staying at the location where they were originally integrated.

Animals↗

PubMed2004

Transformation potency of ErbB heterodimer signaling is determined by B-Raf kinase.

Cellular transformation occurs only in cells that express both ErbB1 and ErbB4 receptors, but not in cells expressing only one or the other of these receptors. However, when both receptors are coexpressed and ligand-stimulated, they interact with virtually the same adaptor/effector proteins as when expressed singly. To reveal the underlying regulatory mechanism of the kinase/phosphatase network in ErbB homo- and heterodimer receptor signaling, extracellular signal-regulated kinase (ERK) and Akt activities were evaluated in the presence of several enzyme inhibitors in ligand-induced cells expressing ErbB1 (E1), ErbB4 (E4), and ErbB1/ErbB4 (E1/4) receptor. The PP2A inhibitor okadaic acid showed receptor-specific inhibitory profiles for ERK and Akt activities. Moreover, B-Raf isolated only from E1/4 cells could induce in vitro phosphorylation for MEK; this B-Raf kinase activity was abolished by pretreatment of the cells with okadaic acid. Our study further showed that the E1/4 cell-specific B-Raf activity was stimulated by PLC gamma and subsequent Rap1 activation. The present study suggests that B-Raf kinase, which was specifically activated in the cells coexpressing ErbB1 and ErbB4 receptors, elevates total ERK activity within the cell and, therefore, can induce cellular transformation.

Animals↗

PubMed2003

Germline transformation of the sawfly, Athalia rosae (Hymenoptera: Symphyta), mediated by a piggyBac-derived vector.

A piggyBac construct carrying two green fluorescent protein (GFP)-coding sequences one driven by Bombyx mori actin gene promoter and the other by Drosophila melanogaster heat-shock protein 70 (hsp70) promoter were injected together with a nonautonomous helper plasmid containing an active piggyBac transposase gene into the posterior end of mature unfertilized eggs dissected from the ovaries of Athalia rosae (Hymenoptera: Symphyta). These injected eggs, which developed as haploid male embryos upon artificial activation, were cultured to adulthood. Of 278 injected eggs, 61 grew to G(0) haploid adult males. These G(0) haploid males were individually mated to diploid females. The progeny embryos (G(1) generation) were examined for GFP expression. Four GFP-positive embryos (from three independent G(0) matings) were obtained. Two eclosed as diploid adult G(1) females. Mature unfertilized eggs dissected from the GFP-positive G(1) diploid females were activated artificially, and the resultant embryos were examined for GFP expression, separated and cultured to adulthood (G(2) generation). The G(2) haploid embryos segregated to GFP-positive and -negative individuals. By mating the G(2) adult haploid males individually to diploid females, stocks were established in which the piggyBac construct was stably integrated into the genome, as evidenced by GFP expression and Southern blot hybridization. The piggyBac transposition occurred at its canonical target TTAA sequence. These results, which demonstrate the first successful stable transposon-mediated germline transformation in Hymenoptera, will expand the usefulness of the piggyBac vector.

Animals↗

PubMed2003

The effects of cure of Helicobacter pylori infection on the signal transduction of gastric epithelial cells.

BACKGROUND: The CagA protein of Helicobacter pylori is directly injected from the bacteria into cells via the bacterial type IV secretion system and undergoes tyrosine phosphorylation in the gastric epithelial cells. Translocated CagA forms a physical complex with the SRC homology 2 domain (SH2)-containing tyrosine phosphatase SHP-2, which plays an important role in mitogenic signal transduction in the host cells. AIM: We examined the effect of eradication therapy on the signal transduction pathway of gastric epithelial cells induced by the CagA protein of H. pylori. METHODS: Gastric biopsy samples were obtained from 20 H. pylori-positive atrophic gastritis patients before, and 3 months after, H. pylori infection eradication therapy, and subjected to immunoblot analysis to detect tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2. RESULTS: Tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2 were detected in the gastric mucosa from H. pylori-positive atrophic gastritis patients. All H. pylori strains from these patients were cagA-positive type I strains. After curing H. pylori infection, the tyrosine phosphorylated CagA protein and CagA co-immunoprecipitated endogenous SHP-2 disappeared from the gastric mucosa. CONCLUSION: The cure of infection reduces the stimulated signal transduction of gastric epithelial cells by the translocated CagA protein of H. pylori, and may confer a beneficial effect on the reduction of cancer risk.

Bacterial Proteins↗

PubMed2002

Hypovitaminosis D and hyperparathyroidism in physically inactive elderly Japanese living in nursing homes: relationship with age, sunlight exposure and activities of daily living.

BACKGROUND AND AIMS: The purposes of this study were to investigate the prevalences of hypovitaminosis D and hyperparathyroidism in physically inactive elderly people living in Japanese nursing homes and determine any correlations with demographic, environmental and/or life-style factors. METHODS: We targeted 220 elderly people living in nursing homes for the physically disabled in Japan. Of these, 133 (women, 113; men, 20) subjects who agreed and met our inclusion criteria were studied. The average age of the subjects was 84.6 years (SD 8.2). Serum 25-hydroxyvitamin D3 (25[OH]D3), intact parathyroid hormone (intact PTH), creatinine, and albumin concentrations were measured in each subject. Total hours of sunshine during the previous 5 weeks, activities of daily living (ADL) levels, and dietary vitamin D intake were also measured. ADL levels were evaluated using the Physical Self-Maintenance Scale (5-point scale). The examinations were performed between October and December, 1999. RESULTS: The average concentrations of serum 25(OH)D3 and intact PTH were 29.9 nmol/L (SD 13.1) and 5.04 pmol/L (SD 2.27), respectively. The prevalences of hypovitaminosis D (25[OH]D3<30 nmol/L) and hyperparathyroidism (intact PTH>6.90 pmol/L) were 77/133 (57.9%) and 21/133 (15.8%), respectively. Serum 25(OH)D3 concentrations were positively associated with hours of sunshine (R2=0.371, p<0.001) and serum albumin concentrations (R2=0.086, p<0.001), but not with age (p=0.075) or total ADL score (p=0.527). A negative association between serum intact PTH and 25(OH)D3 levels was found in subjects who were 80 years or older (p<0.001) but not in subjects under the age of 80 years. CONCLUSIONS: Hypovitaminosis D and hyperparathyroidism are commonly seen in elderly Japanese with low ADL levels. Hypovitaminosis D seems to adversely affect PTH levels, especially in the very elderly.

Activities of Daily Living↗

PubMed2002

[Modified bentall operation using composite graft without resection of aortic valve].

A 27-year-old male whose diagnoses were aortic dissection (Stanford A), aortic regurgitation, annulo-aortic ectasia, and Marfan syndrome underwent modified Bentall operation using composite graft and total arch replacement. Modification of proximal suture without resection of aortic valve like intravalvular implantation might have resulted in good recovery without blood transfusion. Another 63-year-old male who suffered from spontaneous aortic rupture with aortic regurgitation also underwent modified Bentall operation in the similar manner as the first case with good result. Spontaneous aortic rupture reported here is a rare case, and operation is thought to be prerequisite for the patient who suffers from this disease to survive.

Adult↗

PubMed2001

Involvement of pRB-related p107 protein in the inhibition of S phase progression in response to genotoxic stress.

pRB family pocket proteins consisting of pRB, p107, and p130 are thought to act as a set of growth regulators that inhibit the cell cycle transition from G1 to S phases by virtue of their interaction with E2F transcription factors. When cells are committed to progressing through the cell cycle at the late G1 restriction point, they are hyperphosphorylated by G1 cyclin-cyclin-dependent kinase and are functionally inactivated. Consistent with such a G1 regulatory role, pRB and p130 are abundantly expressed in quiescent cells. In contrast, p107 is present at low levels in the hypophosphorylated form in quiescent cells. As cells progress toward late G1 to S phases, the levels of p107 increase, and the majority become hyperphosphorylated, suggesting a possible role of p107 in post-G1 cell cycle regulation. In this study, we have demonstrated that a nonphosphorylatable and thus constitutively active p107 has the potential to inhibit S phase progression. The levels of the phosphorylation-resistant p107 required for the S phase inhibition are significantly less than those of endogenous p107. We further show herein that the exposure of cells to the DNA-damaging agent, cisplatin, provokes S phase arrest, which is concomitantly associated with the accumulation of hypophosphorylated p107. Furthermore, the S phase inhibitory response to cisplatin is augmented by the ectopic expression of wild type p107, although it is diminished by the adenovirus E1A oncoprotein, which counteracts the pocket protein functions. Because p107 is a major pRB family protein expressed in S phase cells, our results indicate that p107 participates in an inhibition of cell cycle progression in response to DNA damage in S phase cells.

Amino Acid Substitution↗

PubMed2001

Collective inhibition of pRB family proteins by phosphorylation in cells with p16INK4a loss or cyclin E overexpression.

The activity of the retinoblastoma protein pRB is regulated by phosphorylation that is mediated by G(1) cyclin-associated cyclin-dependent kinases (CDKs). Since the pRB-related pocket proteins p107 and p130 share general structures and biological functions with pRB, their activity is also considered to be regulated by phosphorylation. In this work, we generated phosphorylation-resistant p107 and p130 molecules by replacing potential cyclin-CDK phosphorylation sites with non-phosphorylatable alanine residues. These phosphorylation-resistant mutants retained the ability to bind E2F and cyclin. Upon introduction into p16(INK4a)-deficient U2-OS osteosarcoma cells, in which cyclin D-CDK4/6 is dysregulated, the phosphorylation-resistant mutants, but not wild-type p107 or p130, were capable of inhibiting cell proliferation. Furthermore, when ectopically expressed in pRB-deficient SAOS-2 osteosarcoma cells, the wild-type as well as the phosphorylation-resistant pRB family proteins were capable of inducing large flat cells. The flat cell-inducing activity of the wild-type proteins, but not that of the phosphorylation-resistant mutants, was abolished by coexpressing cyclin E. Our results indicate that the elevated cyclin D- or cyclin E-associated kinase leads to systemic inactivation of the pRB family proteins and suggest that dysregulation of the pRB kinase provokes an aberrant cell cycle in a broader range of cell types than those induced by genetic inactivation of the RB gene.

Cyclin E↗

PubMed2001

Molecular evidence for two vitellogenin genes and processing of vitellogenins in the American cockroach, Periplaneta americana.

The American cockroach, Periplaneta americana has two vitellins (Vn1 and Vn2) and corresponding vitellogenins (Vg1 and Vg2). Vns/Vgs were separated on the SDS-PAGE as three major polypeptide bands [170, 100 (multisubunits), and 50 kD] and a minor polypeptide band (150 kD) both in the egg (mature terminal oocyte) extract and in the female hemolymph. We previously cloned one Vg (Vg1) cDNA and showed that the 170-kD polypeptide originated from the C-terminus of the Vg1. In the present study, we cloned the other Vg (Vg2) cDNA. It is 5,826 bp long encoding 1,876 amino acid residues (including 16 residues for putative signal peptide) in a single ORF. The deduced amino acid sequences of both Vgs (Vg1 and Vg2) of P. americana showed 30% identity. The GL/ICG motif is followed by eight cysteine residues at conserved locations near the C-terminal and the DGXR motif starts 18 residues upstream of the GL/ICG motif. The chemically determined N-terminal amino acid sequences of the 150-kD and of the 50-kD polypeptides matched exactly with each other and with the deduced N-terminal amino acid sequence of the Vg2 cDNA. The pattern of processing in P. americana Vns/Vgs is discussed.

Amino Acid Motifs↗

PubMed2001

Liver injury suppressing compounds from avocado (Persea americana).

To evaluate the protective activity of fruits against liver injury, 22 different fruits were fed to rats with liver damage caused by D-galactosamine, a powerful liver toxin. As measured by changes in the levels of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), avocado showed extraordinarily potent liver injury suppressing activity. Five active compounds were isolated and their structures determined. These were all fatty acid derivatives, of which three, namely, (2E,5E,12Z,15Z)-1-hydroxyheneicosa-2,5,12,15-tetraen-4-one, (2E,12Z,15Z)-1-hydroxyheneicosa-2,12,15-trien-4-one, and (5E,12Z)-2-hydroxy-4-oxoheneicosa-5,12-dien-1-yl acetate, were novel.

Alanine Transaminase↗

PubMed2001

O-ADP-Ribosylation in the NAD/NADase system: 2-alkanols as efficient substrates.

Several 2-alkanols (2-propanol, 2-butanol, 2-pentanol, etc.) were examined as substrates for ADP-ribosylation in the NAD/NADase enzymatic system. Even though these secondary alcohols have hydroxy groups that are subject to the steric influence of a methyl group, they were shown to be efficiently ADP-ribosylated. However, in the case of 3-alkanol (3-butanol), only slight ADP-ribosylation was observed. In this enzymatic reaction, 1,2-propanediol provided both 1-O- and 2-O-ADP-ribosylation products in the ratio 1:1 as determined by 1H-NMR spectrometry. On the other hand, an equimolar mixture system of 1- and 2-propanols provided major 1-O- and minor 2-O-ribosylation products in the ratio 4:1. This is the first report of O-ADP-ribosylation of terminal secondary alcohols with the NAD/NADase enzymatic system.

Adenosine Diphosphate Ribose↗

PubMed2001

[Off-pump coronary artery bypass grafting: comparison between standard CABG and off-pump CABG].

UNLABELLED: We have performed 321 cases of coronary artery bypass grafting (CABG), between October 15 1995 and November 20 2000. We have evaluated the operative results of 142 cases (44.2%) of conventional CABG and 179 cases (55.8%) of off-pump CABG performed during this period. The average numbers of bypassed grafts was 3.53 for conventional CABG, and 1.62 for off-pump CABG. The total number of 369 grafts were anastomosed to 501 coronary arteries for conventional CABG, and 283 grafts were anastomosed to 290 coronary arteries for off-pump CABG. RESULTS: Although two saphenous veins were occluded, the early postoperative patency rate was 100% for conventional CABG using RITA, LITA, GEA and RA. Three site of stenosis in 18 LITAs and 2 in 16 RITAs were recognized in off-pump CABG without the use of stabilizers. One site of stenosis in 130 LITAs and 3 string signs in 44 GEA were recognized in off-pump CABG with the use of stabilizers. Postoperative angiography in 52 off-pump CABG cases at one year later showed no new lesion. CONCLUSION: The use of stabilizers and LIMA suture enables adaptation of the MIDCAB procedure to a wider range of coronary artery bypass procedures, and a higher graft patency can be expected.

Adult↗

PubMed2001

[Minimally invasive direct coronary artery bypass performed via diaphragmatic approach].

From October 1996 until August 2000, coronary artery bypass grafting (CABG) for right coronary artery was performed via diaphragmatic approach. This procedure was done in 18 patients consist of 13 male and 5 female ranging in age from 47 to 81 years old. Ten of 18 cases were performed coronary bypass reoperation. Right gastroepiploic artery was used in 16 cases, and branched radial artery or saphenous vein graft from gastroduodenal artery was used in one case. Bypassed region in right coronary artery was segment 3 in 9 cases, right posterior descending artery in 8 and right atrio-ventricular branch in one. All grafts were patent in postoperative cardiac catheterization. This approach was desirable for the case, which bypass grafting to right coronary artery is necessary, on account of avoiding injury of patent bypassed graft by median sternotomy in re-do CABG. For the patient whose stomach had been resected, bypass grafting from gastroduodenal artery with radial artery or saphenous vein graft to right coronary artery is useful procedure.

Aged↗

PubMed2000

Identification and characterization of Elongin A2, a new member of the Elongin family of transcription elongation factors, specifically expressed in the testis.

The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites along the DNA template. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, the latter of which bind stably to each other to form a binary complex that interacts with Elongin A and strongly induces its transcriptional activity. To further understand the roles of Elongin in transcriptional regulation, we attempted to identify Elongin-related proteins. Here, we report on the cloning, expression, and characterization of human Elongin A2, a novel transcription elongation factor that exhibited 47% identity and 61% similarity to Elongin A. Biochemical studies have shown that Elongin A2 stimulates the rate of transcription elongation by RNA polymerase II and is capable of forming a stable complex with Elongin BC. However, in contrast to Elongin A, its transcriptional activity is not activated by Elongin BC. Northern blot analysis revealed that Elongin A2 mRNA was specifically expressed in the testis, suggesting that Elongin A2 may regulate the transcription of testis-specific genes.

Amino Acid Sequence↗

PubMed2000

Human p55(CDC)/Cdc20 associates with cyclin A and is phosphorylated by the cyclin A-Cdk2 complex.

The initiation of anaphase and exit from mitosis depend on the activation of the anaphase-promoting complex/cyclosome (APC/C), a multicomponent, ubiquitin-protein ligase. The WD-repeat protein called p55(CDC)(Cdc20) directly binds to and activates APC/C. By using yeast two-hybrid screening, we found that cyclin A, a critical cell cycle regulator in the S and G2/M phases, specifically interacts with p55(CDC). Ectopically expressed p55(CDC) and cyclin A form a stable protein complex in mammalian cells. The p55(CDC)-cyclin A interaction occurs through the region containing the WD repeats of p55(CDC) and the region between the destruction box and the cyclin box of cyclin A. In addition to the physical interaction, p55(CDC) is phosphorylated by cyclin A-associated kinase. These findings suggest that the function of p55(CDC) is mediated or regulated by its complex formation with cyclin A.

Animals↗

PubMed2000

Participation of testicular spermatids in development upon intracytoplasmic injection into eggs of the sawfly, Athalia rosae (Insecta, hymenoptera).

Fertilization by intracytoplasmic injection of mature sperm into mature eggs has previously been achieved in the sawfly, Athalia rosae (Insecta, Hymenoptera). In the present study, we examined the potential of spermatids, premature male gametes, for participating in development. When round spermatids and elongating spermatids from pupal testes were injected into the anterior end of mature eggs, about 5% of the total injected eggs developed into chimeric embryos (independent participation in development of the egg and spermatid nuclei). Some of them developed further, hatched, and pupated, with 1-2% of the total injected eggs becoming haploid chimeric male adults in which both the egg-derived and injected spermatid-derived nuclei contributed to the germline. No fertilized embryos were obtained by these injections. Elongated spermatids (immature sperm) from newly eclosed adult male testes upon injection did produce fertilized embryos that developed into normal diploid females (about 7% of the total injected). These results indicate that insect spermatids (round and elongating) have the potential to participate in development, but only independently of the egg nucleus. J. Exp. Zool. 286:181-192, 2000.

Animals↗

PubMed2000

beta3-endonexin as a novel inhibitor of cyclin A-associated kinase.

Cyclin A is indispensable for S phase cell cycle progression and is suggested to be a crucial target of cell adhesion signals. In this study, we demonstrate that beta3-endonexin, a molecule known to associate with the integrin beta3 cytoplasmic domain, specifically binds cyclin A. Deletion of the amino-terminal 52-amino-acid residues including the cyclin-binding RxL motif abolishes the ability of beta3-endonexin to interact with cyclin A. In an in vitro kinase assay, beta3-endonexin inhibits pRB kinase activity associated with cyclin A-Cdk2 while leaving its histone H1 kinase activity unaffected. Coexpression of beta3-endonexin in yeast cells overcomes growth suppression caused by an activation of cyclin A-associated kinase. Our results indicate that beta3-endonexin is a novel cyclin A-binding molecule that regulates cyclin A-associated pRB kinase activity.

Animals↗

PubMed2000

Artificial reproduction in a hymenopteran insect, Athalia rosae, using eggs matured with heterospecific yolk proteins and fertilized with cryopreserved sperm.

Previtellogenic ovaries of Athalia rosae (Hymenoptera, Symphyta, Tenthredinidae) were transplanted into the adult female abdominal hemocoel of Athalia infumata (Symphyta, Tenthredinidae), Arge nigrinodosa (Symphyta, Argidae), and Pimpla nipponica (Apocrita, Ichneumonidae). The donor oocytes accumulated heterospecific yolk proteins and matured in the A. infumata host. On average, six mature oocytes were obtained per transplanted ovary. In contrast, the donor oocytes accumulated a limited amount of yolk but did not mature in the Ar. nigrinodosa host and did not even accumulate yolk in the P. nipponica host. The eggs that matured in the A. infumata host were injected with cryopreserved A. rosae sperm that had been taken from adult male seminal vesicles and stored at -80 degrees C. Fertilization, as confirmed by the use of visible marker mutations, was achieved and a fraction of the injected eggs developed into fertile female adults.

Animals↗