Biomedical subjects

Yoshinao Wada

Publications and source records attributed to Yoshinao Wada.

30 records · Page 2Linked to original sources

PubMed2004

Immunohistochemical distribution of Toll-like receptor 4 in term and preterm human placentas from normal and complicated pregnancy including chorioamnionitis.

Recently discovered Toll-like receptors (TLRs) are essential for the induction of innate immune responses. One member of the TLR family, TLR4 mediates lipopolysaccharide (bacterial endotoxin)-induced inflammatory responses. Although the innate immune system appears to be important in the pathogenesis of infection-induced preterm delivery, the distribution of TLRs in human placenta is poorly understood. Here we investigated the expression of TLR4 protein in 43 human placentas obtained from normal and complicated pregnancies delivered in the second and third trimesters, using immunohistochemistry. TLR4 was localized to the extravillous trophoblasts, intermediate trophoblasts/X cells in the degenerative villi, and villous Hofbauer cells of all preterm and term placentas examined and to the inflammatory cells in placentas with chorioamnionitis (CAM). The villous Hofbauer cells of preterm CAM placentas demonstrated increased TLR4 immunoreactivity compared with those of preterm placentas without CAM or those of term placentas with or without CAM. These results suggest an important role of the villous Hofbauer cells in the activation of innate immune system in response to infectious pathogens in preterm placentas. Elucidation of biological functions of placental TLR4 under physiological conditions requires further investigation.

Chorioamnionitis↗

PubMed2004

Differential expression and localization of decorin in human choriodecidual membrane during preterm and term pregnancy.

PROBLEM: This study was aimed to isolate cDNAs for genes whose expression levels are dynamically regulated at the feto-maternal interface around parturition. METHOD: The suppression subtractive hybridization (SSH) method was used to compare the gene expression patterns of human choriodecidual membranes from term elective Caesarian delivery before labor (BL) and term vaginal delivery (VD). Northern blotting, quantitative real-time RT-PCR and in situ hybridization were performed to determine temporal and spatial expression of candidate clones. RESULT: After SSH procedure, we selected decorin from the cDNA library of BL for further investigation. The mean level of decorin transcription tended to be higher in BL samples than in VD or preterm samples. Decorin mRNA was mainly expressed in the decidual cells. Decorin gene expression was also upregulated in the term myometrium. CONCLUSION: These results suggest that dynamically increased expression of decorin in the choriodecidual membrane during parturition may be a part of the uterine preparation for labor.

Cloning, Molecular↗

PubMed2004

Technical aspects of gel-based proteomics designed for elucidating an aryl hydrocarbon receptor complex.

The identification of proteins by mass spectrometry has revolutionalized the basic method of identifying proteins constituting an intracellular unit or network for certain biological functions. The gel-based strategy following immunoprecipitation was applied to elucidating proteins associated with the aryl hydrocarbon receptor (AhR). Two hundred femtomoles of AhR was recovered from approximately 2 x 10(7) HepG2 cells by immunoprecipitation and was sufficient for identification by peptide mass fingerprinting. Possible candidates for the AhR-associated proteins were also identified. Improvements of the current strategy to increase the overall sensitivity tenfold are required to clarify the AhR complex in full detail. For example, a combination of trypsin and Achromobacter protease I for in-gel digestion allows the number of missed cleavage sites to be set at zero for database searching, thereby reducing random matches and facilitating identification. There is also room for improvement in each step of sample preparation prior to mass spectrometry.

Cell Line↗

PubMed2003

Molecular dissection of the alpha-dystroglycan- and integrin-binding sites within the globular domain of human laminin-10.

The adhesive interactions of cells with laminins are mediated by integrins and non-integrin-type receptors such as alpha-dystroglycan and syndecans. Laminins bind to these receptors at the C-terminal globular domain of their alpha chains, but the regions recognized by these receptors have not been mapped precisely. In this study, we sought to locate the binding sites of laminin-10 (alpha5beta1gamma1) for alpha(3)beta(1) and alpha(6)beta(1) integrins and alpha-dystroglycan through the production of a series of recombinant laminin-10 proteins with deletions of the LG (laminin G-like) modules within the globular domain. We found that deletion of the LG4-5 modules did not compromise the binding of laminin-10 to alpha(3)beta(1) and alpha(6)beta(1) integrins but completely abrogated its binding to alpha-dystroglycan. Further deletion up to the LG3 module resulted in loss of its binding to the integrins, underlining the importance of LG3 for integrin binding by laminin-10. When expressed individually as fusion proteins with glutathione S-transferase or the N-terminal 70-kDa region of fibronectin, only LG4 was capable of binding to alpha-dystroglycan, whereas neither LG3 nor any of the other LG modules retained the ability to bind to the integrins. Site-directed mutagenesis of the LG3 and LG4 modules indicated that Asp-3198 in the LG3 module is involved in the integrin binding by laminin-10, whereas multiple basic amino acid residues in the putative loop regions are involved synergistically in the alpha-dystroglycan binding by the LG4 module.

Amino Acid Sequence↗

PubMed2003

Decomposition of protein nitrosothiolsin matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry.

The S-nitrosylation of proteins is involved in the trafficking of nitric oxide (NO) in intra- and extracellular milieus. To establish a mass spectrometric method for identifying this post-translational modification of proteins, a synthetic peptide and transthyretin were S-nitrosylated in vitro and analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The intact molecular ion species of nitrosylated compounds was identified in the ESI mass spectrum without elimination of the NO group. However, the labile nature of the S-NO bond was evident when the in-source fragmentation efficiently generated [M + H - 30](+) ions. The decomposition was prominent for multiply charged transthyretin ions with high charge states under ordinary ESI conditions, indicating that the application of minimum nozzle potentials was essential for delineating the stoichiometry of nitrosylation in proteins. With MALDI, the S-NO bond cleavage occurred during the ionization process, and the subsequent reduction generated [M + H - 29](+) ions.

Humans↗

PubMed2003

Characterization of the ligand-binding specificities of integrin alpha3beta1 and alpha6beta1 using a panel of purified laminin isoforms containing distinct alpha chains.

Integrins alpha3beta1 and alpha6beta1 are two major laminin receptors expressed on the surface of mammalian cells. Interactions of cells with laminins through these integrins play important roles in cell adhesion, differentiation, motility, and matrix assembly. To determine the binding specificity and affinity of these integrins toward various types of laminins at the level of direct protein-protein interactions, we purified integrins alpha3beta1 and alpha6beta1 from human placenta, and examined their binding to a panel of laminin isoforms, each containing distinct alpha chains (i.e., laminin-1, laminin-2/4, laminin-5, laminin-8, and laminin-10/11). Integrin alpha3beta1 showed clear specificity for laminin-5 and laminin-10/11, with no significant binding to laminin-1, laminin-2/4, and laminin-8. In contrast, integrin alpha6beta1 showed a broad spectrum of specificity, with apparent binding affinity in the following order: laminin-10/11 > laminin-5 > laminin-1 > laminin-2/4 congruent with laminin-8. Integrin titration assays demonstrated that laminin-10/11 was the most preferred ligand among the five distinct laminin isoforms for both alpha3beta1 and alpha6beta1 integrins. Given that laminin-10/11 is the major basement membrane component of many adult tissues, the interaction of laminin-10/11 with these integrins should play a central role in the adhesive interactions of epithelial cells with underlying basement membranes.

Animals↗

PubMed2002

Advanced analytical methods for hemoglobin variants.

Hemoglobin variants are the protein mutations most often encountered in the clinical scene. They have been useful for developing methods to analyze mutant proteins because of their size and ease of collection in large amounts. Improvements in analytical methods have been directed toward higher resolution in electrophoresis and shorter elution times in chromatography. More importantly, in the last 20 years, hemoglobin variants have been used in the development of mass spectrometric strategies for analyzing protein mutations. This approach consists of a series of steps: measurement of the molecular mass of globins to detect or confirm the presence of mutations, peptide mass mapping or peptide mass fingerprinting of an enzymatic digest to identify mutated peptides, and tandem mass spectrometry to determine or confirm the site and type of mutation. The mass spectrometric strategy has enabled rapid analysis and demonstrated a superb ability to detect a number of hemoglobin variants, particularly those without a change in electrophoretic or chromatographic properties. Even with the recent advances in DNA analysis, protein analysis is still essential, because post-translational modifications following amino acid substitutions can occur including N-terminal acetylation, deamidation and oxidation-mediated processes.

Chromatography↗

PubMed2002

Tyrosine phosphorylation of clathrin heavy chain under oxidative stress.

In mouse pancreatic insulin-producing betaTC cells, oxidative stress due to H(2)O(2) causes tyrosine phosphorylation in various proteins. To identify proteins bearing phosphotyrosine under stress, the proteins were affinity purified using an anti-phosphotyrosine antibody-conjugated agarose column. A protein of 180kDa was identified as clathrin heavy chain (CHC) by electrophoresis and mass spectrometry. Immunoprecipitated CHC showed tyrosine phosphorylation upon H(2)O(2) treatment and the phosphorylation was suppressed by the Src kinase inhibitor, PP2. The phosphorylation status of CHC affected the intracellular localization of CHC and the clathrin-dependent endocytosis of transferrin under oxidative stress. In conclusion, CHC is a protein that is phosphorylated at tyrosine by H(2)O(2) and this phosphorylation status is implicated in the intracellular localization and functions of CHC under oxidative stress. The present study demonstrates that oxidative stress affects intracellular vesicular trafficking via the alteration of clathrin-dependent vesicular trafficking.

Animals↗

PubMed2002

Paranodal junction formation and spermatogenesis require sulfoglycolipids.

Mammalian sulfoglycolipids comprise two major members, sulfatide (HSO3-3-galactosylceramide) and seminolipid (HSO3-3-monogalactosylalkylacylglycerol). Sulfatide is a major lipid component of the myelin sheath and serves as the epitope for the well known oligodendrocyte-marker antibody O4. Seminolipid is synthesized in spermatocytes and maintained in the subsequent germ cell stages. Both sulfoglycolipids can be synthesized in vitro by using the isolated cerebroside sulfotransferase. To investigate the physiological role of sulfoglycolipids and to determine whether sulfatide and seminolipid are biosynthesized in vivo by a single sulfotransferase, Cst-null mice were generated by gene targeting. Cst(-/-) mice lacked sulfatide in brain and seminolipid in testis, proving that a single gene copy is responsible for their biosynthesis. Cst(-/-) mice were born healthy, but began to display hindlimb weakness by 6 weeks of age and subsequently showed a pronounced tremor and progressive ataxia. Although compact myelin was preserved, Cst(-/-) mice displayed abnormalities in paranodal junctions. On the other hand, Cst(-/-) males were sterile because of a block in spermatogenesis before the first meiotic division, whereas females were able to breed. These data show a critical role for sulfoglycolipids in myelin function and spermatogenesis.

Animals↗

PubMed2002

Detection of cytomegalovirus DNA in human placenta.

Although human cytomegalovirus (CMV) is one of the most common causes of viral intrauterine and perinatal infection, its distribution in the placenta is poorly understood. The purpose of this study was to determine the frequency of CMV DNA positivity in placentas, to demonstrate the localization of the viral genome, and to identify the clinical features related to placental CMV. A total of 254 placentas from 231 mothers were investigated, and the maternal serum CMV immunoglobulin antibodies were measured. Specimens from both the placental parenchyma and the placental membrane close to the ruptured site in each placenta were examined for the presence of CMV DNA using dot blot hybridization after PCR amplification. None of 57 placentas from seronegative mothers was positive for CMV DNA. Of 197 placentas from seropositive mothers, 60 (30.5%) had CMV DNA in either the parenchyma or the membrane by dot blot analysis. In situ hybridization was carried out on these 60 placentas, and the localization of the viral genome was established in 19; CMV DNA was localized mostly to the villi, including the mesenchyme and trophoblasts, extravillous trophoblasts, and decidual cells. The mean gestational age at delivery was significantly later in the CMV DNA-positive placentas than in the negative placentas (36.9 +/- 5.1 vs 34.7 +/- 6.2 weeks, P = 0.0059). CMV DNA was detected in only 6 of 33 placentas delivered in the second trimester, and all six were associated with either severe maternal nephritis or severe chorioamnionitis. These results suggest that the CMV genome is common in placentas at later gestational ages and in those of earlier gestational ages with certain maternal complications.

Antibodies, Viral↗

PubMed2002

Expression of vascular endothelial growth factor, placental growth factor, and their receptors Flt-1 and KDR in human placenta under pathologic conditions.

The vascular endothelial growth factor (VEGF) family and its receptors have multifunctional activities besides angiogenesis, and some of these molecules are induced by hypoxia/ischemia. They are known to be expressed in human placenta, but little is known about their involvement in pathologic conditions. We have investigated the expression patterns of VEGF, placental growth factor (PlGF), and their receptors fms-like tyrosine kinase (Flt-1) and kinase insert domain-containing region (KDR) in placentas with histopathological changes. Forty-two placentas from normal and complicated pregnancies delivered in the second and third trimesters were fixed with paraformaldehyde and embedded in paraffin. In situ hybridization and immunohistochemistry were performed on serial sections. In the villi with characteristic hypoxic/ischemic changes (HIC), including increased syncytial knots, infarction, or hypercapillarization, intense immunostaining for VEGF was detected in the media of blood vessels, and increased staining for KDR was demonstrated in the endothelial cells. Strong PlGF immunoreactivity was localized to the degenerative trophoblasts around the infarctions. Marked Flt-1 mRNA expression in the syncytiotrophoblast layers of HIC villi was identified, but some samples did not show ligand expression in these regions. Positive immunostaining for VEGF, PlGF, and Flt-1 was observed in infiltrated neutrophils and macrophages in the placentas with chorioamnionitis (CAM). These findings suggested that in the hypoxic/ischemic regions, VEGF and KDR expression is increased within the villous vessels by paracrine regulation, whereas the expression of PlGF and Flt-1 is enhanced in villous trophoblasts by autocrine regulation. The Flt-1 gene may also be up-regulated directly by hypoxia/ischemia independently of ligand mediation. Furthermore, the results indicated that VEGF and PlGF stimulate inflammatory cell migration by autocrine regulation via the Flt-1 receptor in the CAM placenta. Thus, various functions of VEGF family members participate in the development of pathologic changes in the placenta.

Adult↗

PubMed2002

Placental features in preterm infants with periventricular leukomalacia.

OBJECTIVE: Evaluation of the placenta provides some important insights into pathophysiologic changes that take place during the prenatal and intrapartum process. We investigated the relationship between placental findings and periventricular leukomalacia (PVL) to obtain a better understanding of its cause. METHODS: Thirty-two preterm infants with PVL delivered before 34 weeks' gestation, between 1990 and 1999, were classified into 4 groups according to the onset of brain injury assumed from ultrasonographic presentation and clinical course: 2 Antenatal, 22 Peripartum, 5 Postnatal, and 3 in an unknown time of onset group. We evaluated the gross and histopathologic features of the placentas of each group and compared them with those of a control group matched by birth weight and gestational age in terms of the frequency of major placental findings. Potential confounding factors were controlled in logistic regression analyses. RESULTS: Gross lesions with disturbance of uteroplacental circulation, including massive retroplacental hematoma, extensive infarction or thrombosis, and marked basal or perivillous fibrin deposition, were observed more frequently in the Antenatal + Peripartum combined subgroup than in the controls (41.7% vs 13.7%). Placentas from the Antenatal + Peripartum subgroup also demonstrated a significantly higher frequency of ischemic changes in villi, based on histopathologic examination, as compared with the control group (54.2% vs 13.7%). These associations remained after adjustment for confounding factors in logistic regression analyses (odds ratio: 4.04, 95% confidence interval: 1.40-11.67; and odds ratio: 7.28, 95% confidence interval: 2.50-21.20; respectively). Frequencies of chorioamnionitis and twin placentation tended to be higher in PVL cases than in the controls, although the differences were not statistically significant (46.9% vs 37.9%, 37.5% vs 20.0%, respectively). CONCLUSIONS: These results suggest that disturbed placental circulation underlies the development of PVL in the majority of cases with prenatal and peripartum brain injury. In chorioamnionitis cases, certain additional factors were suggested in the genesis of PVL. Thus, placental examination is essential for elucidating the pathophysiologic changes leading to PVL in the perinatal process.

Chorioamnionitis↗