Biomedical subjects

M Hatakeyama

Publications and source records attributed to M Hatakeyama.

At least 37 records · Page 2Linked to original sources

PubMed2000

Cloning of vitellogenin cDNA of the American cockroach, Periplaneta americana (Dictyoptera), and its structural and expression analyses.

A cDNA expression library constructed from poly (A)(+) RNA prepared from vitellogenic female fat body cells of the American cockroach, Periplaneta americana (Dictyoptera) was screened using a polyclonal antiserum against the 100-kD polypeptide(s) from the egg extract. A partial Vg cDNA clone was obtained and sequenced. The 5' end portion of the cDNA was then obtained by the RACE method, cloned, and sequenced. The combined complete Vg cDNA was 5,854 bp long and contained a single ORF encoding 1,896 amino acids. The entire deduced amino acid sequence was aligned confidently with those of the known insect Vgs. A GL/ICG motif, a number of cysteines at conserved locations following this motif, and a DGXR motif upstream of the GL/ICG motif were present near the C-terminal. The chemically determined N-terminal amino acid sequence of the 170-kD polypeptide from the egg extract completely matched the deduced sequence starting from just after one of the consensus (RXXR) cleavage sites, indicating the occurrence of post-translational cleavage in the fat body cells. The Vg gene begins to be expressed in the 2-day-old adult female fat body cells but is never expressed in ovaries or in male fat body cells. Hemolymph Vg was first detected by immunoblotting in 4-day-old adult females, 2 days after the beginning of gene expression. Western blot analysis of major yolk polypeptides in nine cockroach species belonging to the two superfamilies, Blattoidea and Blaberoidea, using the antisera against P. americana major yolk polypeptides showed that the similarities in Vn antigenicity are basically limited to within a superfamily.

Amino Acid Sequence↗

PubMed2000

Standard coronary artery bypass grafting and beating heart bypass. Indications and long-term results.

OBJECTIVES AND METHOD: We have performed 225 cases of coronary artery bypass grafting (CABG), between October 15 1995 and September 8 1999. We have evaluated the operative results of 121 cases (53.8%) of conventional CABG and 104 cases (46.2%) of minimally invasive coronary artery bypass grafting performed during this period. The average numbers of bypassed grafts was 3.45 for conventional CABG, and 1.41 for minimally invasive coronary artery bypass grafting. Sixty-seven right internal thoracic arteries, 145 left internal thoracic arteries, 71 gastroepiploic arteries, 38 radial arteries and 12 saphenous veins were used for conventional CABG, and 29 right internal thoracic arteries, 81 left internal thoracic arteries, 18 gastroepiploic arteries, 3 radial arteries, 10 saphenous veins and 2 inferior epigastric arteries were used for minimally invasive coronary artery bypass grafting. The total number of 303 grafts were anastomosed to 417 coronary arteries for conventional CABG, and 143 grafts were anastomosed to 147 coronary arteries for minimally invasive coronary artery bypass grafting. RESULTS: Although two saphenous veins were occluded, the early postoperative patency rate was 100% for conventional CABG using right internal thoracic arteries, left internal thoracic arteries, gastroepiploic arteries and radial arteries. Three site of stenosis in 18 left internal thoracic arteries and 2 in 16 right internal thoracic arteries were recognized in minimally invasive coronary artery bypass grafting without the use of stabilizers. One site of stenosis in 63 left internal thoracic arteries was recognized in minimally invasive coronary artery bypass grafting with the use of stabilizers. CONCLUSION: The use of stabilizers enables adaptation of the minimally invasive coronary artery bypass grafting procedure to a wider range of coronary artery bypass procedures, and a higher graft patency can be expected.

Coronary Artery Bypass↗

PubMed2000

Vitellogenin of the cicada Graptopsaltria nigrofuscata (Homoptera): analysis of its primary structure.

We cloned and sequenced the cDNA of vitellogenin (Vg) from the cicada Graptopsaltria nigrofuscata (Homoptera). The deduced amino acid sequence of 1987 residues (including 16 residues for a putative signal peptide) was obtained. The pro-Vg was cleaved into two subunits between residues 379 and 380 following a consensus RXXR cleavage site sequence, secreted as S-Vg (apparent molecular weight 43 kDa) and L-Vg (200 kDa), sequestered, and stored in the egg as two vitellins (Vns), S-Vn and L-Vn, with similar respective molecular weights. There was a single long serine-rich stretch closely following the cleavage site. The entire amino acid sequences of the Vgs from the eight insects so far reported could be aligned confidently. The presence of subdomains I-V (areas of relatively high amino acid conservation) and of 10 cysteines at conserved locations at the C-terminus, noted previously among insect Vgs, were confirmed. Antisera raised against G. nigrofuscata S- and L-Vn cross-reacted with the S- and L-Vg/Vn, respectively, of all three other cicada species examined. Another major egg protein (170 kDa) unrelated to Vg/Vn, was also detected in all species examined.

Amino Acid Sequence↗

PubMed2000

A simple and rapid method for cloning insect vitellogenin cDNAs.

We describe a simple and rapid method for cloning insect vitellogenin (Vg) cDNAs. The method relies on the facts that insect Vg amino acid sequences can be aligned confidently along their entire lengths and that a short, highly conserved GL/ICG motif and up to nine cysteine residues that follow at conserved locations are present near the C-termini. An adaptor-ligated double-strand cDNA library is constructed from poly(A)+ RNA prepared from vitellogenic female fat body tissues using a commercial kit, and subjected to PCR with each of the degenerate nucleotide sequences for the GL/ICG motif and the adaptor sequence as primers. The PCR products (0.7-0.9 kb, representing the 3' portion) are cloned, the nucleotide sequences are determined, and the deduced amino acid sequences are aligned with the known insect Vg sequences starting from the GL/ICG motif. Gene-specific primers corresponding to the sequences near the 5'-termini of the initial clones and the adaptor sequence are employed to obtain the remaining 5' portion of the Vg cDNAs. The method was successfully applied to the bean bug Plautia stali (Heteroptera), revealing three Vg genes.

Amino Acid Sequence↗

PubMed2000

High-performance affinity beads for identifying drug receptors.

We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-kappaB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-kappaB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.

Amino Acid Sequence↗

PubMed2000

Ras and signal transducer and activator of transcription (STAT) are essential and sufficient downstream components of Janus kinases in cell proliferation.

Cytokines exert their activities in cell growth and differentiation by binding specific cell membrane receptors. Janus kinases (JAKs) are cytoplasmic protein tyrosine kinases that physically interact with intracellular domains of the cytokine receptors and they play crucial roles in transducing signals triggered by the cytokine-receptor interaction. We have previously shown that conditional activation of JAK through membrane-proximal dimerization confers cytokine-independence on interleukin-3 (IL-3)-dependent Ba / F3 lymphoid cells and that the cytokine-independent proliferation is completely inhibited by dominant negative Ras. In this work, we demonstrate that ectopic expression of a dominant negative form of Stat5, a major signal transducer and activator of transcription (STAT) expressed in Ba / F3 cells, also inhibits JAK-triggered mitogenesis. In contrast, overexpression of constitutively active Ras or conditional activation of Stat5 by chemical dimerization fails to confer cytokine-independence. However, concomitant activation of ectopic Ras and Stat5 molecules in Ba / F3 cells suffices for cell proliferation in the absence of IL-3. Our results indicate that Ras and STAT are essential and sufficient components of JAK-triggered mitogenesis. Our findings further indicate that the cytokine signal bifurcates into Ras and STAT pathways following JAK activation.

Animals↗

PubMed2000

A linkage map of the turnip sawfly Athalia rosae (Hymenoptera: Symphyta) based on random amplified polymorphic DNAs.

A linkage map was constructed for the sawfly, Athalia rosae (Hymenoptera), based on the segregation of random amplified polymorphic DNA (RAPD) markers and a visible mutation, yellow fat body (yfb). Forty haploid male progeny (20 yfb and 20+) from a single diploid female parent (yfb/+) were examined. Sixty-one of the 180 arbitrary primers tested by polymerase chain reaction (PCR) produced one or more RAPD bands. A total of 79 RAPD markers were detected. Of these, seven showed significant deviation from the expected 1:1 ratio, and were therefore excluded from further analysis. The remaining 72 RAPD markers and the marker mutation, yfb, were subjected to linkage analysis. Sixty RAPD markers and the yfb marker were organized into 16 linkage groups, spanning a distance of 517.2 cM. Twelve RAPD markers showed no linkage relationship to any group. Thirteen gel-purified RAPD bands were cloned and sequenced to generate the sequence-tagged sites (STSs). A single locus was represented by two markers, with one of them having a short internal deletion.

Animals↗

PubMed1999

Granulocytic differentiation of myeloid progenitor cells by p130, the retinoblastoma tumor suppressor homologue.

The retinoblastoma protein (pRB) and the related pocket proteins, p107 and p130, play crucial roles in mammalian cell cycle control. Recent studies indicate that these pocket proteins are also involved in cellular differentiation processes. We demonstrate in this work that the pRB-related p130 selectively accumulates during the in vitro differentiation of the myeloid progenitor cell, 32Dcl3, into granulocyte in response to granulocyte-colony stimulating factor (G-CSF). This G-CSF-dependent granulocytic differentiation is blocked by the adenovirus E1A oncoprotein, which binds to and inactivates the pRB family of pocket proteins including p130. Furthermore, enforced overexpression of p130 but not pRB inhibits the myeloid cell proliferation that is concomitantly associated with granulocytic differentiation morphologically characterized by nuclear segmentation. However, simple G1-cell cycle arrest induced by cytokine deprivation or ectopic overexpression of the p27 cyclin-dependent kinase inhibitor, or inhibition of E2F activities by dominant negative DP-1 is not sufficient to trigger granulocytic differentiation. The differentiation-promoting activity of p130 in myeloid cells requires both the pocket domain and the spacer domain. Our results indicate that the pRB-related p130 plays a critical role in myeloid cell differentiation and suggest that coupling of cell cycle exit with the cellular differentiation program may be specifically achieved by p130.

Adenovirus E1A Proteins↗

PubMed1999

Physical interaction and functional antagonism between the RNA polymerase II elongation factor ELL and p53.

ELL was originally identified as a gene that undergoes translocation with the trithorax-like MLL gene in acute myeloid leukemia. Recent studies have shown that the gene product, ELL, functions as an RNA polymerase II elongation factor that increases the rate of transcription by RNA polymerase II by suppressing transient pausing. Using yeast two-hybrid screening with ELL as bait, we isolated the p53 tumor suppressor protein as a specific interactor of ELL. The interaction involves respectively the transcription elongation activation domain of ELL and the C-terminal tail of p53. Through this interaction, ELL inhibits both sequence-specific transactivation and sequence-independent transrepression by p53. Thus, ELL acts as a negative regulator of p53 in transcription. Conversely, p53 inhibits the transcription elongation activity of ELL, suggesting that p53 is capable of regulating general transcription by RNA polymerase II through controlling the ELL activity. Elevated levels of ELL in cells resulted in the inhibition of p53-dependent induction of endogenous p21 and substantially protected cells from p53-mediated apoptosis that is induced by genotoxic stress. Our observations indicate the existence of a mutually inhibitory interaction between p53 and a general transcription elongation factor ELL and raise the possibility that an aberrant interaction between p53 and ELL may play a role in the genesis of leukemias carrying MLL-ELL gene translocations.

Cell Compartmentation↗

PubMed1999

Correlation between the activation of Neu tyrosine kinase and promotion of foci formation induced by selected organochlorine compounds in the MCF-7 model system.

Several studies have shown that some organochlorine compounds act like estrogen in certain animals and in vitro cell culture systems, and therefore, there is a possibility that they could promote the process of tumorigenesis in breast cancer cells. In our previous study, two representative organochlorines, 1,1,1-trichloro 2-o-chlorophenyl-2'-p-chlorophenyl ethane (o,p'-DDT) and beta-1,2,3,4,5,6-hexachlorocyclohexane (beta HCH), were found to directly activate the protein tyrosine kinase of Neu (c-erbB-2 proto-oncogene product) immunoprecipitates isolated from MCF-7 breast cancer cells. In the current study, we also found that 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) at 1 nM and alpha-HCH isomers at 100 nM could also significantly activate protein tyrosine kinase of Neu immunoprecipitates in a cell-free system. We also found that organochlorines result in an increase of Neu protein tyrosine kinase after intact cell treatment in estrogen-depleted medium. This Neu kinase activation by beta-HCH (100 nM) was blocked when the cells were pretreated with Neu mRNA antisense oligonucleotide (p < 0.07, Student's t-test). Endogenously added alpha-, beta-, and gamma-HCH, o,p'-DDT, 2,2'-dichlorobiphenyl (2,2'-PCB), and 2,4,5-T at 100 nM were found to promote foci formation in postconfluent cultures of this cell line. This stimulatory effect caused by 17beta-estradiol, o,p'-DDT, and beta-HCH on foci formation was inhibited by coincubation with Neu monoclonal antibody (p < 0.05). Those two events induced by organochlorines (i.e., Neu kinase activation and foci formation) seemed causally correlated.

Antibodies, Monoclonal↗

PubMed1999

Structural organization and chromosome location of the mouse elongin A gene (Tceb3).

Elongin A is the transcriptionally active subunit of the Elongin complex, which strongly increases the rate of elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites within transcription units. In the present study, we obtained the cDNA sequence of the mouse Elongin A gene (Tceb3) and characterized its genomic structure. The deduced 773-amino acid sequence of mouse Elongin A shows 91% and 81% identity with rat and human Elongin A, respectively. The Elongin A gene was mapped to mouse chromosome 4D3 by fluorescence in situ hybridization.

Amino Acid Sequence↗

PubMed1999

Different absorption behaviors among steroid hormones due to possible interaction with P-glycoprotein in the rat small intestine.

The intestinal absorption of ten steroid hormones was evaluated in the rat small intestine, especially focusing on the interaction with intestinal P-glycoprotein (P-gp). Hydrocortisone, prednisolone, 6alpha-methylprednisolone, and dexamethasone (adrenocortical steroid hormones) all disappeared in a regional-dependent manner (duodenum>jejunum>ileum). The decreased rate of disappearance in the lower small intestine seemed to be due to the involvement of absorption barriers like P-gp. In contrast, all sex hormones including progesterone exhibited very high absorbability in the entire small intestine (duodenum=jejunum=ileum), possibly demonstrating the absence of restricted absorption by intestinal P-gp. Progesterone enhanced the rate of disappearance of vinblastine but did not affect 6alpha-methylprednisolone. In the presence of vinblastine and verapamil, on the other hand, the rate of disappearance of 6alpha-methylprednisolone increased significantly. It was demonstrated that there was a plural P-gp family, which had different substrate specificities, in the rat intestine and that steroid hormones interacted with them as substrates or inhibitors in a very complex manner.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

A novel human RasGAP-like gene that maps within the prostate cancer susceptibility locus at chromosome 1q25.

We report the molecular cloning of a human cDNA that encodes a molecule having striking homology with Ras-specific GTPase-activating proteins (RasGAPs). Among previously described RasGAPs, the cDNA product is most closely related to Caenorhabditis elegans GAP-2, including a predicted coiled-coil structure near the carboxyl terminus. Expression of the cDNA in Saccharomyces cerevisiae defective in one of two RasGAPs, Ira2, complemented loss of the Ira2 function, indicating that the cDNA product functions as a RasGAP. The RasGAP-like gene is located on the human chromosome 1q25, the locus that appears to contain a hereditary prostate cancer susceptible gene, HPC1.

Amino Acid Sequence↗

PubMed1998

Conditional activation of Janus kinase (JAK) confers factor independence upon interleukin-3-dependent cells. Essential role of Ras in JAK-triggered mitogenesis.

Cytokines play crucial roles in the growth and differentiation of hematopoietic cells. They bind to specific cell membrane receptors that usually do not possess intrinsic protein-tyrosine kinase activity. Janus kinases (JAKs) are cytoplasmic protein-tyrosine kinases that physically interact with intracellular domains of the cytokine receptors and have been implicated in playing important roles in signal transduction triggered by the cytokine-cytokine receptor interaction. However, it is still uncertain whether JAK activation alone suffices to induce cell proliferation. In this work, we modified Tyk2, a member of the JAK family, by adding a membrane localization sequence and a chemical dimerizer (coumermycin)-dependent dimerization sequence. The modified Tyk2 was activated in a coumermycin-dependent manner, and the activated Tyk2 conferred cytokine independence upon interleukin-3-dependent pro-B lymphoid cells. This cytokine-independent proliferation was completely inhibited by dominant-negative Ras. These results indicate that activation of JAK through membrane-proximal dimerization is sufficient to induce cell cycle progression and that Ras is essentially involved in JAK-triggered mitogenesis.

Aminocoumarins↗

PubMed1998

Highly controlled heterologous gene expression through combined utilization of the tetracycline-repressible transactivator and the lac repressor.

To achieve strictly on-off switching of a desired cDNA expression in a broader range of mammalian cell types, we introduced the Escherichia coli lac operator sequences into the tetracycline-repressible promoter and created a chimeric promoter (designated here as the TcIP promoter) whose activity was reciprocally controlled by tetracycline and isopropyl thiogalactopyranoside (IPTG). cDNAs were connected downstream of the TcIP promoter and stably transfected into interleukin (IL-2 or IL-3)-dependent lymphoid cells that ectopically coexpress the tetracycline-repressible transactivator and the lac repressor. Whereas the parental tetracycline-repressible promoter exhibited constitutive activities when stably introduced into the lymphoid cells, cDNA expression from the TcIP promoter was strongly inhibited by tetracycline and was potently induced by IPTG in stable transfectants. Hence, the TcIP promoter made it possible to achieve highly controlled cDNA expression in cells wherein the parental promoter did not function in an inducible manner. Potential application of this promoter was provided by expressing cyclin-dependent kinase inhibitor, p27(Kip1). Induced expression of p27(Kip1) by the TcIP promoter in the lymphoid cells strongly reduced cellular responsiveness to IL-2 or IL-3, consistent with the idea that p27(Kip1) is a critical target that must be inactivated by the interleukin-triggered mitogenic signals.

Animals↗

PubMed1998

Control of retinoblastoma protein-independent hematopoietic cell cycle by the pRB-related p130.

The retinoblastoma tumor suppressor protein (pRB) is a potent inhibitor of mammalian cell growth and the functional inactivation of pRB is widely presumed to be essential for progression of the cell cycle from G1 phase. In this work, the generality of pRB-based cell cycle control in mammalian cells was addressed by conditionally expressing pRB in cytokine-dependent hematopoietic cells. We show herein that these cells are able to progress through the cell cycle in response to cytokine despite the continued presence of supraphysiological amounts of wild-type pRB or phosphorylation-resistant pRB mutants. However, their growth was strongly blocked by ectopic expression of the pRB-related pocket protein, p130. This growth inhibition required the E2F-binding pocket domain but not the cyclin-binding domain of p130. Furthermore, increased amounts of the p130-controlled E2F, termed E2F-4, potentiated the mitogenic response of the cells to cytokine and the constitutive overexpression of E2F-4 rendered the cells cytokine-independent. Our results indicate the existence of a non-pRB-based cell cycle whose operation depends primarily on the interplay between p130 and E2F-4 in certain hematopoietic cells.

Animals↗

PubMed1998

Involvement of intestinal P-glycoprotein in the restricted absorption of methylprednisolone from rat small intestine.

The interaction between steroid hormones and intestinal P-glycoprotein was investigated by measuring intestinal absorption from rat small intestine in situ. Prednisolone and hydrocortisone were rapidly absorbed from the entire small intestine. In contrast, methylprednisolone absorption was significantly retarded in jejunum and ileum by an intestinal efflux system. In the presence of verapamil an quinidine, the retarded absorption of methylprednisolone was completely recovered, suggesting that P-glycoprotein is responsible for the unique features of methylprednisolone absorption. A requisite for the substrate of intestinal P-glycoprotein seemed to be 6 alpha-methyl group in the steroid structure. Substrate specificity of intestinal P-glycoprotein to steroid hormones was shown to be in part different from those in other tissues such as adrenal gland. Little of all three steroid hormones disappeared in the supernatant of mucosal homogenate from rat small intestine, indicating that intestinal metabolism of these steroid hormones was relatively small.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

K-252a-induced polyploidization and differentiation of a human megakaryocytic cell line, Meg-J: transient elevation and subsequent suppression of cyclin B1 and cdc2 expression in the process of polyploidization.

Megakaryocytes are unique haemopoietic cells which undergo DNA replication, giving rise to polyploid cells. However, little is known about the mechanism of megakaryocytic polyploidization. To address this issue, we used the human megakaryocytic cell line Meg-J. In the presence of K-252a (an indolocarbasole derivative), Meg-J cells stopped proliferation and exhibited additional megakaryocytic features, including morphological changes, polyploidization, and increases in the levels of surface expression of platelet glycoprotein (GP) IIb/IIa and GPIb. Thrombopoietin (TPO) promoted the K-2 52a-induced polyploidization and megakaryocytic differentiation. In the process of K-252a-induced polyploidization, levels of expression of both cdc2 and cyclin B1 were elevated transiently and subsequently decreased. This suggested that the polyploidization process in Meg-J cells was at least in part associated with a transient elevation and subsequent decrease in the expression of cdc2/cyclin B1 complex, a critical kinase involved in G2/M cell cycle transition.

CDC2 Protein Kinase↗