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Biomedical subjects

D Yamamoto

Publications and source records attributed to D Yamamoto.

At least 19 recordsLinked to original sources
PubMed2007

Quantitative analysis of messenger RNA expression of matrix metalloproteinases (MMP-2 and MMP-9), tissue inhibitor-2 of matrix metalloproteinases (TIMP-2), and steroidogenic enzymes in bovine placentomes during gestation and postpartum.

The relationship between the mRNA expression of proteolytic and steroidogenic enzymes in bovine placentomes was examined. Caruncle and cotyledon tissues were collected every 6 hr after spontaneous parturition until the fetal membranes were released. Based on the time of fetal membrane release after parturition, the specimens were classified as follows: (1) the early group, in which the fetal membranes were released within 6 hr after parturition; and (2) the late group, in which the fetal membranes were released 6-12 hr after parturition. The placentomes from a slaughterhouse were additionally collected as samples for the examination of enzymes during the gestation period. The mRNA expression of steroidogenic enzymes in the cotyledon was observed to be higher than that in caruncle tissues; however, the mRNA expression patterns of P450scc and StAR tended to be similar in both placental tissues. On the other hand, although the expression levels of TIMP-2 mRNA in both caruncle and cotyledon tissues were similar, during gestation and postpartum the expression levels of MMP-2 and MMP-9 mRNA were approximately 10 times higher in caruncle than in cotyledon tissue. Marked contrasting changes in mRNA expression patterns between pre- and postpartum periods were observed for MMP-2 and MMP-9 in caruncle tissues and for MMP-9 and TIMP-2 in cotyledon tissues. The present study provides the first evidence that MMP-2, MMP-9, and TIMP-2 mRNAs are expressed in bovine placentomes during the gestational and postpartum periods and suggests that these enzymes, in conjunction with steroidogenic enzymes, mediate fetal membrane detachment after parturition.

Animals↗
PubMed2006

Enucleated L929 mouse fibroblasts support invasion and multiplication of Shigella flexneri 5a.

Invasive bacteria can induce their own uptake and specify their intracellular localization; hence it is commonly assumed that proximate modulation of host cell transcription is not required for infection. However, bacteria can also modulate, directly or indirectly, the transcription of many host cell genes, whose role in the infection may be difficult to determine by global gene expression. Is the host cell nucleus proximately required for intracellular infection and, if so, for which pathogens and at what stages of infection? Enucleated cells were previously infected with Toxoplasma gondii, Chlamydia psittaci, C. trachomatis, or Rickettsia prowazekii. We enucleated L929 mouse fibroblasts by centrifugation in the presence of cytochalasin B, and compared the infection with Shigella flexneri M90T 5a of nucleated and enucleated cells. Percent infection and bacterial loads were estimated with a gentamicin suppression assay in cultures fixed and stained at different times after infection. Enucleation reduced by about half the percent of infected cells, a finding that may reflect the reduced endocytic ability of L929 cytoplasts. However, average numbers of bacteria and frequency distributions of bacterial numbers per cell at different times were similar in enucleated and nucleated cells. Bacteria with actin-rich tails were detected in both cytoplasts and nucleated cells. Lastly, cytoplasts were similarly infected 2 and 24 h after enucleation, suggesting that short-lived mRNAs were not involved in the infection. Productive S. flexneri infection could thus take place in cells unable to modulate gene transcription, RNA processing, or nucleus-dependent signaling cascades.

Animals↗
PubMed2006

Effect of fructose-rich high-fat diet on glucose sensitivity and atherosclerosis in nonhuman primate.

The present study examined a new model of impaired glucose tolerance (IGT) in a nonhuman primate fed with a high-fructose and high-fat (HFF) diet that contained 15% lard, 31% fructose, and 1% cholesterol. Female cynomolgus monkeys (age, 3-4 years) were divided into two groups: 1) those fed with normal control diet (N = 5) and 2) those fed with HFF diet (N = 5). In the HFF diet group, total cholesterol and low-density lipoprotein cholesterol in blood were significantly increased four- to fivefold when compared with the normal control diet group. No difference in the blood glucose, insulin, hemoglobin A1c, and triglyceride levels was detected between the two groups. Plasma levels of adiponectin, but not of resistin, were significantly higher in HFF diet at 20 weeks after HFF diet feeding. Oral glucose tolerance test was performed before HFF diet feeding and at 12 and 24 weeks after HFF diet feeding, but no significant changes in glucose sensitivity were observed even 24 weeks after HFF diet feeding. Twenty-four weeks after HFF diet feeding, accumulated foam cells and infiltrated macrophages were histologically detected in the thoracic aorta, in addition to a fatty liver. Interestingly, the pancreatic beta cells appeared normal in the HFF diet group. These results show that a chronic HFF diet does not induce IGT but can cause atherosclerotic lesions in conjunction with the generation of a fatty liver phenotype in cynomolgus monkey; however, the present results are very preliminary and they need to be validated in larger-scale studies in the future.

Adiponectin↗
PubMed2005

Blood flow: a key regulatory component of corpus luteum function in the cow.

Prostaglandin F2alpha (PGF2alpha) is the primary luteolysin in the cow. During the early luteal phase, the corpus luteum (CL) is resistant to the luteolytic effect of PGF2alpha. Once mature, the CL becomes responsive to PGF2alpha and undergoes luteal regression. These actions of PGF2alpha coincide with changes in luteal blood flow (BF): PGF2alpha has no effect on BF in the early CL, but acutely increases BF in the peripheral vasculature of the mature CL within 30 min of PGF2alpha injection. During spontaneous luteolysis, luteal BF increases on Days 17-18 of the estrous cycle, prior to any decrease in plasma progesterone (P). The increase in luteal BF is synchronous with an increase in plasma PGFM levels, suggesting that pulsatile release of PGF2alpha from uterus stimulates the increase in luteal BF. Serial biopsies of these CL showed that mRNA expression for endothelial nitric oxide synthase (eNOS) together with endothelin-1 (ET-1) and angiotensin converting enzyme (ACE) increases on Days 17-18 when the luteal BF is elevated. On Day 19 when plasma P level firstly decreases, eNOS mRNA returns to the basal level whereas ET-1 and ACE mRNA remains elevated. Cyclooxygenase-2 (COX-2) mRNA expression increases on Day 19. In support of these data, an in vivo microdialysis study revealed that luteal ET-1 and angiotensin II (Ang II) secretion increases and precedes PGF2alpha secretion during spontaneous luteolysis. In conclusion, we show for the first time that an acute increase of BF occurs in the peripheral vasculature of the mature CL together with increases in eNOS expression and ET-1 and Ang II secretion in the CL during the early stages of luteolysis in the cow. We propose that the increase in luteal BF may be induced by NO from large arterioles surrounding the CL, and simultaneously uterine or exogenous PGF2alpha directly increases ET-1 and Ang II secretion from endothelial cells of microcapillary vessels within the CL, thereby suppressing P secretion by luteal cells. Taken together, our results indicate that an acute increase in luteal BF occurs as a first step of luteolysis in response to PGF2alpha. Therefore, local BF plays a key role to initiate luteal regression in the cow.

Angiotensin II↗
PubMed2005

Structure and property of self-assemble valinyl bolaform amides having different chirality.

Bolaform amides were designed from N,N'-bis(carboethoxy-L-valinyl)-diaminoethane (1) by linking t-butyloxycarbonyl-valine through ethylenediamine (EDA) to enable spectroscopic and X-ray diffraction analyses. N,N'-Bis(Boc-L-valinyl)-diaminoethane (2) and N,N'-bis(Boc-D-valinyl)-diaminoethane (3) were composed of L-Val and D-Val, respectively. N-(Boc-L-valinyl)-N'-(Boc-D-valinyl)-diaminoethane (4) was composed of both L-Val and D-Val, and was achiral (meso-peptide). Peptide 5 was a 1:1 mixture of 2 and 3, and was also achiral (racemate). These peptides mediated gelation of corn oil at a concentration of approximately 1%. Within crystals, the peptides formed beta-sheet ribbons, but differences were observed in hydrogen-bonding patterns and side-chain arrangements. These differences were also deduced from temperature dependence of amide protons. Force-field calculations based on the crystal structures indicated that association of beta-sheet ribbons had energy benefits, and it was assumed that molecular aggregation progressed spontaneously. These structural studies indicated the chirality of amino acids affected for the properties of bolaform amides.

Amides↗
PubMed2003

HSpin1, a transmembrane protein interacting with Bcl-2/Bcl-xL, induces a caspase-independent autophagic cell death.

The Drosophila spinster (spin) gene product is required for programmed cell death in the nervous and reproductive systems. We have identified a human homologue of the Drosophila spin gene product (HSpin1). HSpin1 bound to Bcl-2 and apoptosis regulator Bcl-X (Bcl-xL), but not to proapoptotic members such as Bcl-2-associated X protein and Bcl-2 homologous antagonist killer, in cells treated with TNF-alpha. Exogenous expression of HSpin1 resulted in the cell death without inducing a release of cytochrome c from mitochondria. Overexpression of Bcl-xL inhibited the HSpin1-induced cell death. Interestingly, a necrosis inhibitor, pyrrolidine dithiocarbomate, but not the pancaspase inhibitors, carbobenzoxy-VAD-fluoromethyl ketone and p35, blocked the HSpin1-induced cell death. HSpin1-induced cell death increases autophagic vacuole and mature form of cathepsin D, suggesting a novel caspase-independent cell death, which is link to autophagy.

Autophagy↗
PubMed2001

Comparative analysis of the Band 4.1/ezrin-related protein tyrosine phosphatase Pez from two Drosophila species: implications for structure and function.

The FERM-PTPs are a group of proteins that have FERM (Band 4.1, ezrin, radixin, moesin homology) domains at or near their N-termini, and PTP (protein tyrosine phosphatase) domains at their C-termini. Their central regions contain either PSD-95, Dlg, ZO-1 homology domains or putative Src homology 3 domain binding sites. The known FERM-PTPs fall into three distinct classes, which we name BAS, MEG, and PEZ, after representative human PTPs. Here we analyze Pez, a novel gene encoding the single PEZ-class protein present in Drosophila. Pez cDNAs were sequenced from the distantly related flies Drosophila melanogaster and Drosophila silvestris, and found to be highly conserved except in the central region, which contains at least 21 insertions and deletions. Comparison of fly and human Pez reveals several short conserved motifs in the central region that are likely protein binding sites and/or phosphorylation sites. We also identified novel invertebrate members of the BAS and MEG classes using genome data, and generated an alignment of vertebrate and invertebrate FERM domains of each class. 'Specialized' residues were identified that are conserved only within a given class of PTPs. These residues highlight surface regions that may bind class-specific ligands; for PEZ, these residues cluster on and near FERM subdomain F1. Finally, the PTP domain of fly Pez was modeled based on known PTP tertiary structures, and we conclude that Pez is likely a functional phosphatase despite some unusual features of the active site cleft sequences. Biochemical confirmation of this hypothesis and genetic analysis of Pez are currently underway.

Amino Acid Sequence↗
PubMed2001

Inhibitory mechanism of daphnodorins for human chymase.

We investigated the inhibitory mechanisms of daphnodorins for human chymase using three-dimensional molecular modeling. In daphnodorin A-human chymase complex, daphnodorin A was fixed to the active site via hydrogen bonds with Ala177, Phe29, and Gly199 in human chymase, and it formed hydrogen bonds with Ser182 and Gly180, and this complex was formed stably. In daphnodorin B-human chymase complex, daphnodorin B formed hydrogen bonds with Lys28 and Phe29 in human chymase, but it could not form hydrogen bonds with Gly199, Ala177, and Lys179. The phenyl group of daphnodorin B shifted from the P1 hole in human chymase in comparison with that of daphnodorin A. For the inhibition of human chymase by daphnodorins, we indicated that it was significant whether daphnodorins formed hydrogen bonds with Ala177 located in the P1 hole, Ser182 located in the active site, Gly180 located in the anion hole, and with Gly199, Phe29, and Lys28 in human chymase.

Benzopyrans↗
PubMed2001

FTY720, a novel immunosuppressive agent, induces apoptosis in human glioma cells.

FTY720, a metabolite from Isaria sinclairii, has been developed to be a potent immunosuppressive drug with induction of apoptosis in T cells and several cell lines. We investigated whether FTY720 induces apoptosis in human glioma cell lines, since they are relatively resistant to multiple apoptotic stimuli. In human glioma cells including T98G, FTY720 induced apoptosiswith ED50 between 1 to 10 microg/ml, while etoposidedid not induce apoptosis at the same doses. Among the caspase family proteases, mainly caspase-6 was activated during the apoptosis by FTY720 but not etoposide. In addition, FTY720 caused tyrosine dephosphorylation of FAK and did not activate a FAK-PI3-kinase survival pathway. This was confirmed also by the observation that orthovanadate prevented FTY720-induced dephosphorylation of FAK and inhibited FTY720-induced cell death. We assumed that FTY720 induced FAK dephosphorylation and cut off the FAK-PI3-kinase pathway resulting in the induction of apoptosis via caspase-6 activation in these glioma cells.

Antineoplastic Agents, Phytogenic↗
PubMed2001

Statistical analysis of diagnostic failure of fine needle aspiration cytology (FNAC) in breast cancer.

Fine-needle aspiration cytology (FNAC) was performed on 300 patients. Among those, 57 cases failed in accurate diagnosis of malignancy and 243 were successful. Fourteen clinicopathological factors altogether were analyzed to elucidate any correlation with FNAC failure using uni- and multivariate analysis. The univariate analysis in each clinicopathlogical factor showed that these error cases were vaguely palpable cancers, estrogen receptor (ER) positive cancers, small-sized of tumors, scattered type of cancer cell distribution in tumor tissues, with low tumor grade, with low Nottingham prognostic index (NPI), with benign-like ultrasound findings and with low TNM stage. The multivariate analysis revealed tumor grade was the strongest factor for all, followed by cellular distribution type of cancer cells and benign-like ultrasound findings. From these results, we speculated that diagnostic failure of FNAC at first clinic visit seemed to be caused by mainly two histocytological factors: extrinsic factor (structural factors of tissue-like tumor cells' distribution pattern, etc.); and intrinsic one (cellular factors of low atypism such as benign-like ultrasound finding, low tumor grade, and so on).

Analysis of Variance↗
PubMed2001

Eosinophilic peroxidase deficiency: Identification of a point mutation (D648N) and prediction of structural changes.

ABSTRACT Hereditary eosinophil peroxidase (EPO; EC 1.11.1.7) deficiency is a rare abnormality without clinical symptoms characterized by decreased or absent peroxidase activity and decreased volume of the granule matrix in eosinophils. Nearly 100 cases have been reported, but a specific mutation has been reported in only one case. We report the genetic analysis of an EPO-deficient subject and his family. The case was found by automated blood analyzer. Sequencing of the entire coding region of the EPO gene disclosed a novel mutation, a 2060 G-A transition (g. 2060G>A) causing an amino acid change from aspartic acid to asparagine (D648N). Both the son and daughter of the propositus inherited the G-A transition, and in vitro expression experiments suggest this transition is responsible for the deficiency. We then analyzed the location of the affected amino acid within this molecule using a structural model of EPO based on myeloperoxidase (MPO). Asn648 is on the inside of the molecule; changing D to N would cause loss of the electrostatic interaction with Arg146 which is crucial for disulfide bonds of the light chain in the N terminus.

Amino Acid Substitution↗
PubMed2001

A utility of ductography and fiberoptic ductoscopy for patients with nipple discharge.

BACKGROUND: Breast carcinoma and precancer are thought to start in the lining of the milk duct or lobule. While ductography has been advocated as the main procedure in patients with nipple discharge, fiberoptic ductoscopy (FDS) is an emerging technique allowing direct visual access to the ductal system of the breast through nipple orifice exploration. METHODS: We applied ductography and FDS to 65 women who had nipple discharge, and compared their utility. RESULTS: Intraductal lesions occurred in the segmental duct and the first, second, third, fourth, fifth, and sixth branches in decreasing frequency with ductography and FDS screening. The detection rates of intraductal abnormal lesions by ductography and FDS and the their combination were 89.1 (37 patients), 97.4 (38 patients), and 97.5% (39 patients) respectively. Ductal washings performed during FDS were effective to obtain representative exfoliated ductal cells which could be evaluated (sensitivity 50%, specificity 94.3%, and diagnostic accuracy 89.7%). As a result, we diagnosed 35 cases of benign lesions and four cases of malignant lesions by cytological or/and histological examination. CONCLUSION: Ductography and fiberoptic ductoscopy are useful procedure in guiding subsequent breast surgery in the treatment of nipple discharge.

Adult↗
PubMed2001

Dual-tagging gene trap of novel genes in Drosophila melanogaster.

A gene-trap system is established for Drosophila. Unlike the conventional enhancer-trap system, the gene-trap system allows the recovery only of fly lines whose genes are inactivated by a P-element insertion, i.e., mutants. In the gene-trap system, the reporter gene expression reflects precisely the spatial and temporal expression pattern of the trapped gene. Flies in which gene trap occurred are identified by a two-step screening process using two independent markers, mini-w and Gal4, each indicating the integration of the vector downstream of the promoter of a gene (dual tagging). mini-w has its own promoter but lacks a polyadenylation signal. Therefore, mini-w mRNA is transcribed from its own promoter regardless of the vector integration site in the genome. However, the eyes of flies are not orange or red unless the vector is incorporated into a gene enabling mini-w to be spliced to a downstream exon of the host gene and polyadenylated at the 3' end. The promoter-less Gal4 reporter is expressed as a fusion mRNA only when it is integrated downstream of the promoter of a host gene. The exons of trapped genes can be readily cloned by vectorette RT-PCR, followed by RACE and PCR using cDNA libraries. Thus, the dual-tagging gene-trap system provides a means for (i) efficient mutagenesis, (ii) unequivocal identification of genes responsible for mutant phenotypes, (iii) precise detection of expression patterns of trapped genes, and (iv) rapid cloning of trapped genes.

Animals↗
PubMed2001

Mutations in the novel membrane protein spinster interfere with programmed cell death and cause neural degeneration in Drosophila melanogaster.

Mutations in the spin gene are characterized by an extraordinarily strong rejection behavior of female flies in response to male courtship. They are also accompanied by decreases in the viability, adult life span, and oviposition rate of the flies. In spin mutants, some oocytes and adult neural cells undergo degeneration, which is preceded by reductions in programmed cell death of nurse cells in ovaries and of neurons in the pupal nervous system, respectively. The central nervous system (CNS) of spin mutant flies accumulates autofluorescent lipopigments with characteristics similar to those of lipofuscin. The spin locus generates at least five different transcripts, with only two of these being able to rescue the spin behavioral phenotype; each encodes a protein with multiple membrane-spanning domains that are expressed in both the surface glial cells in the CNS and the follicle cells in the ovaries. Orthologs of the spin gene have also been identified in a number of species from nematodes to humans. Analysis of the spin mutant will give us new insights into neurodegenerative diseases and aging.

Amino Acid Sequence↗
PubMed2001

Trap a gene and find out its function: toward functional genomics in Drosophila.

Many declared aims of the genome projects have been achieved. The total genomic sequences of several relatively noncomplex/complex organisms (such as E. coli, yeast, Caenorhabditis, Drosophila) are being determined, and the nucleotide sequencing of the entire human genome will be complete in the near future. However, this achievement is not the end of the road but rather the first step toward the functional understanding of the genome of humans and other organisms. The determined linear nucleotide sequences remain only lists of A, C, G and T, unless they are given functional significance. The coding sequences of genes can be identified in a relatively reliable manner by computational methods, but the exact function of their protein products can rarely be determined without obtaining much additional information, e.g., by biochemical or cell biological methods. Thus, following sequencing, the next step must be to assign functions to the identified genes. The final goal of genome research today may look futuristic, but the knowledge of the function of every single gene and the interactions between them will finally allow us to understand the development and functioning of an organism as a whole. Gene-trapping methodology is a powerful strategy for cloning and identifying functional genes, as it marks a gene with a tag and simultaneously generates a corresponding genetic variation for that particular locus. Therefore, gene trapping is an extremely useful tool for functional genomics, establishing a correlation between the physical and genetic maps of the genome. The relative simplicity of its genome and the availability of huge bodies of genetic and molecular information make Drosophila melanogaster one of the most important model organisms. Its genome will serve as a "reference" for the in-depth analysis of the organization of more complex eukaryotic genomes. Multifaceted approaches to Drosophila functional genomics and the dual-tagging gene trap system newly developed for functional analysis of Drosophila genes are discussed in this review.

Animals↗
PubMed2000

Cycloprodigiosin hydrochloride, H(+)/CL(-) symporter, induces apoptosis and differentiation in HL-60 cells.

Cycloprodigiosin hydrochloride (cPrG * HCl), a novel H(+)/Cl(-) symporter, induces acidification of the cytosol and leads to apoptosis in rat and human liver cancer cells. In the present study, the effect of cPrG * HCl on a promyelocytic leukemia cell line (HL-60) was examined. cPrG * HCl lowered intracellular pH and induced apoptosis through up-regulation of Fas ligand, activation of stress-activated protein kinase (SAPK/JNK) and caspase. Apoptosis induced by cPrG * HCl was strongly suppressed when a cell-permeable weak base, imidazole, was present, indicating that cytosol acidification introduced by cPrG * HCl triggered caspase activation, leading to apoptosis. Concomitantly, cell differentiation into monocyte was also induced by cPrG * HCl both morphologically and functionally. However, the cPrG * HCl-induced differentiation was not suppressed by addition of imidazole, indicating that the differentiation process is unrelated to cytosol acidification. Further, the differentiation induced by cPrG * HCl was blocked by tyrosine kinase inhibitors (lavendustin A and HMA) but unaffected by the inhibitors of A-kinase (H-89) or C-kinase (H-7). Taken together, these findings suggest that cPrG * HCl, through apoptosis and differentiation induction, may be useful in leukemia treatment.

Antiporters↗
PubMed2000

Anti-apoptotic role of focal adhesion kinase (FAK). Induction of inhibitor-of-apoptosis proteins and apoptosis suppression by the overexpression of FAK in a human leukemic cell line, HL-60.

Focal adhesion kinase (FAK) has an anti-apoptotic role in anchorage-dependent cells via an unknown mechanism. To elucidate the role of FAK in anti-apoptosis, we have established several FAK cDNA-transfected HL-60 cell lines and examined whether FAK-transfected cells have resistance to apoptotic stimuli. FAK-transfected HL-60 (HL-60/FAK) cells were highly resistant to apoptosis induced with hydrogen peroxide (1 mm) and etoposide (50 microg/ml) compared with the parental HL-60 cells or the vector-transfected cells, when determined using viability assay, DNA fragmentation, and flow cytometry analysis. Because no proteolytic cleavage of pro-caspase 3 to mature caspase 3 fragment was observed in HL-60/FAK cells, FAK was presumed to inhibit an upstream signal pathway leading to the activation of caspase 3. HL-60/FAK activated the phosphatidylinositide 3'-OH-kinase-Akt survival pathway and exhibited significant activation of NF-kappaB with marked induction of inhibitor-of-apoptosis proteins (IAPs: cIAP-1, cIAP-2, XIAP), regardless of the hydrogen peroxide-treated or untreated conditions, whereas no significant IAPs were detected in the parental or vector-transfected HL-60 cells. Apoptotic agents induced higher NF-kappaB activation in HL-60/FAK cells than in HL-60/Vect cells, and it appeared that sustained NF-kappaB activation is critical to the anti-apoptotic states in HL-60/FAK cells. Mutagenesis of FAK cDNA revealed that Y397 and Y925, which are involved in the tyrosine-phosphorylation sites, were prerequisite for the anti-apoptotic activity as well as induction of IAPs, and that K454, which is involved in the kinase activity, was also required for the full anti-apoptotic activity of FAK. Taken together, we have demonstrated definitively that FAK-transfected HL-60 cells, otherwise sensitive to apoptosis, become resistant to the apoptotic stimuli. We conclude that FAK activates the phosphatidylinositide 3'-OH-kinase-Akt survival pathway with the concomitant activation of NF-kB and induction of IAPs, which ultimately inhibit apoptosis by inhibiting caspase-3 cascade.

Apoptosis↗