Biomedical subjects
K Kadomatsu
Publications and source records attributed to K Kadomatsu.
A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta.
Midkine is a 13-kDa heparin-binding growth factor with 45% sequence identity to pleiotrophin. Pleiotrophin has been demonstrated to bind to protein-tyrosine phosphatase zeta (PTPzeta) with high affinity. In this study, we examined the binding of midkine to PTPzeta by solid-phase binding assay. Midkine and pleiotrophin binding to PTPzeta were equally inhibited by soluble pleiotrophin and also by some specific glycosaminoglycans. For both bindings, Scatchard analysis revealed low (3.0 nM) and high (0.58 nM) affinity binding sites. These results suggested that PTPzeta is a common receptor for midkine and pleiotrophin. Midkine is structurally divided into the N- and C-terminal halves, and the latter exhibited full activity for PTPzeta binding and neuronal migration induction. The C-terminal half contains two heparin-binding sites consisting of clusters of basic amino acids, Clusters I and II. A mutation at Arg78 in Cluster I resulted in loss of the high affinity binding and reduced neuronal migration-inducing activity, while mutations at Lys83 and Lys84 in Cluster II showed almost no effect on either activity. Chondroitinase ABC-treated PTPzeta exhibited similar low affinity binding both to the native midkine and midkine mutants at Arg78. These results suggested that Arg78 in midkine plays an essential role in high affinity binding to PTPzeta by interacting with the chondroitin sulfate portion of this receptor.
Levels of expression of pleiotrophin and protein tyrosine phosphatase zeta are decreased in human colorectal cancers.
Pleiotrophin (PTN) and midkine (MK) form a distinct family of heparin binding growth factors. In a variety of human cancers, MK mRNA levels have been found to be increased as compared to adjacent non-cancerous tissues. We examined the expression of PTN, its putative receptor, namely protein tyrosine phosphatase zeta (PTPzeta, also known as RPTPbeta), and a related protein, receptor-type protein tyrosine phosphatase gamma (RPTPgamma), in human colorectal cancers and the adjacent normal mucosae. PTN and PTPzeta mRNA levels were generally decreased in colorectal cancers as compared to those in adjacent normal mucosae, while the RPTPzeta level was not significantly different between them.
Determination of the optimal time and dosage of all-trans retinoic acid for induction of murine exencephaly.
Murine exencephaly corresponds to human anencephaly, and provides a model for studying the mechanism of development of the central nervous system. A system which induces exencephaly at an extremely high rate is required in order to examine embryos, before the stage of neural tube closure, as samples of future exencephaly. Herein, we report on a system which is close enough to the best conditions for induction of this malformation, involving ICR mice and all-trans retinoic acid. The intraperitoneal administration of 30 mg/kg of all-trans retinoic acid at 03:00 hr on day 8 (copulatory plug, day 0) induced exencephaly in 81.6% of live embryos, as evaluated on day 10, with a 41.7% embryonic death rate. Earlier administration (more than 3 hr before) greatly increased the rate of embryonic death, whereas later administration resulted in a reduction in the rate of exencephaly. These findings suggest that a specific time during early development is crucial for neural tube closure, and provide a system which may facilitate the study of neural development and the pathophysiology of human anencephaly.
Syndecan-4 expression is associated with follicular atresia in mouse ovary.
Ovarian granulosa cells synthesize heparan sulfate proteoglycans (HSPGs), that have anticoagulant properties. Moreover, HSPGs greatly increase in the granulosa cells during follicular atresia. However, the species of ovarian HSPGs have not yet been identified. Syndecan-4 (ryudocan, amphiglycan) is a membrane-spanning HSPG and a member of the syndecan family. Herein, we demonstrate that syndecan-4 is expressed in the granulosa cells of type 4-5b follicles and, most intensely, in those of the atretic follicles in the mouse ovary, as revealed by in situ hybridization. There is no relationship between syndecan-4 expression and age or sexual cycle stage. Compared with syndecan-4 expression, syndecan-1 and -3 are expressed more abundantly in postovulatory follicles and the corpora lutea, but less in the type 4-5b follicles and much less in the atretic follicles. Immunohistochemistry also demonstrates syndecan-4 expression in atretic follicles with apoptosis. The present study has revealed the distinct modes of expression of the syndecan family members, and the association of syndecan-4 expression and apoptosis in ovarian atretic follicles.
Expression of midkine in the early stage of carcinogenesis in human colorectal cancer.
It has been suggested that a heparin-binding growth factor, midkine (MK), plays an important role in carcinogenesis because of its frequent overexpression in various malignant tumours. To clarify whether or not MK contributes to the early stage of carcinogenesis, we examined the status of MK mRNA in 20 adenomas with moderate- and severe-grade dysplasia, 28 carcinomas and 28 corresponding normal tissues, by means of Northern blotting. The MK expression level was significantly more elevated in adenomas than in normal tissues (P < 0.001, unpaired Student's t-test). A difference was also observed between carcinomas and the corresponding normal tissues (P < 0.04, paired Student's t-test). Moreover, MK immunostaining was positive in the adenomas with moderate- and severe-grade dysplasia and in the carcinomas, but not in mild-grade dysplasia or in normal tissues. These findings were in line with those on Western blotting. In three patients with both adenomas with moderate- or severe-grade dysplasia and carcinomas, elevated MK expression was observed in the neoplastic lesions. This is the first report of the association of elevated MK expression with the early stage of carcinogenesis in humans.
Histological characterization of defective spermatogenesis in mice lacking the basigin gene.
Basigin is a transmembrane protein belonging to the immunoglobulin superfamily. In the light of the fact that knockout mice lacking the basigin gene (Bsg) are azoospermic, the phenotype in the male reproductive system was extensively examined in this study. Spermatogenesis in Bsg (-/-) mice was found to be disrupted, and arrested at the metaphase of the first meiotic division. A few germ cells differentiated into young spermatids, but they were exfoliated. The lumens of the male reproductive system were filled with round degenerated cells. Using the TUNEL method and electron microscopy, some of the degenerated cells in the testis and epididymal head were shown to be apoptotic. Crystalloids of fine tubules and unusual ectoplasmic specializations were also observed in the Sertoli cells of Bsg (-/-) mice. These specializations displayed unusual 'circular' structures. Furthermore, unusual ectoplasmic specializations covering the spermatocytes rather than the mature spermatids were found. These structures were formed as a result of the lack of mature spermatids in the Bsg (-/-) testis. Results from analyses of azoospermia in the Bsg (-/-) mice suggest that basigin, through the interactions between germ cells and Sertoli cells, is an essential factor in the growth and/or survival of spermatids.
Overexpression of midkine in lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine in rats and its increase with progression.
The expression of midkine (MK) in lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats was examined. The animals were administered 2000 p.p.m. of BHP in their drinking water for 12 weeks, then maintained without further treatment until being killed 20-28 weeks after the beginning of the experiment. MK mRNA expression of adenocarcinomas and squamous cell carcinomas assessed by means of the reverse transcriptase-polymerase chain reaction and northern blot analysis was significantly higher than in rat embryonic tissues (positive controls) and contrasted strongly with the lack in normal lungs. MK protein was detected immunohistochemically in 58.3% of alveolar hyperplasias, 92.3% of adenomas and 100% of adenocarcinomas and squamous cell carcinomas. The extent of staining significantly increased along with malignant progression in adenomatous (pre-)neoplastic lesions and tended to become more pronounced with malignant progression in squamous lesions. The results suggest that MK may play some essential roles in the development and progression of lung tumors induced by BHP in rats.
Spatially and temporally regulated expression of N-acetylglucosamine-6-O-sulfotransferase during mouse embryogenesis.
GlcNAc-6-O-sulfotransferase is involved in formation of 6-sulfo-N -acetyllactosamine-containing structures such as 6-sulfo sialyl Lewis x. We investigated the mode of expression of GlcNAc-6-O-sulfotransferase during postimplantation embryogenesis in the mouse by in situ hybridization. Sulfotransferase mRNA was not detected on embryonic day (E) 6.5, while on E7.5 it was detected in the mesoderm, ectoderm, and ectoplacental cone. On E10.5, the sulfotransferase signals were mainly observed in the nervous tissue. On E12.5 and 13.5, various tissues in the process of differentiation expressed this mRNA. Several epithelial and mesenchymal tissues undergoing epithelial-mesenchymal interactions strongly expressed the mRNA. For example, in the developing tooth strong sulfotransferase mRNA expression was found only in the condensing mesenchyme on E13.5. On E13.5 and 15.5, the sites showing intense expression of the sulfotransferase again became restricted. In the brain, sulfotransferase mRNA was frequently found as discrete signals in narrow regions. These results suggest that 6-sulfo-N-acetyllactosamine structures have important roles in development. On E13.5 and 15.5, G152 (6-sulfo sialyl Lewis x antigen) was expressed in the neocortex, and AG223 (6-sulfo Lewis x antigen) in the thalamus and neocortex where the sulfotransferase signal was detected. However, in other organs, expression of these antigens did not correlate with the sulfotransferase mRNA, implicating complex nature of regulation of expression of the fucosyl 6-sulfo antigens.
Frequent expression of midkine gene in esophageal cancer suggests a potential usage of its promoter for suicide gene therapy.
We have examined the expression of midkine (MK), a neurotrophic factor with heparin-binding activity, in human esophageal cancer cells. Seven esophageal cell lines tested expressed the transcript and 8 out of 14 human esophageal tumor specimens were positively stained with anti-MK antibody, while surrounding normal esophageal tissues in these specimens were not stained. The 5'-flanking, 2.3 kb genomic region of the MK gene was shown to drive the transcription of a reporter gene in the esophageal cell lines in a cis acting manner. Forced expression in esophageal cancer cells of herpes simplex virus-thymidine kinase gene mediated by the flanking region of the MK gene conferred sensitivity to a prodrug, ganciclovir. The 5'-upstream region of the MK gene thus possesses putative promoter activity which can be used for suicide gene-based gene therapy for esophageal cancer.
Immunohistochemical analysis of midkine expression in human prostate carcinoma.
Midkine (MK) is a growth/differentiation factor frequently expressed at high levels in some types of human malignancies. To investigate whether MK is a useful marker in prostate carcinogenesis, immunohistochemical analysis was performed on samples of both latent and clinical prostate cancers of various stages, as well as on specimens of normal gland and prostatic intraepithelial neoplasia (PIN). Of the 80 clinical cancers examined, 69 specimens (86.3%) were immunoreactive for MK, with metastatic lesions generally showing higher expression than the corresponding primaries; normal prostate tissues were negative or showed only weak staining. Midkine was also detected in 12 of 15 latent cancers (80%) and in 12 of 16 cases of PIN (75%). In sections of whole prostate, MK showed variable expression through tumorous sections, probably in reflection of heterogeneous cell populations. The results demonstrate the possible value of MK as a marker for early and latent disease, as well as for more advanced clinical stages of prostate cancer.
Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase.
We isolated a cDNA clone encoding mouse N-acetylglucosamine-6-O-sulfotransferase based on sequence homology to the previously cloned mouse chondroitin 6-sulfotransferase. The cDNA clone contained an open reading frame that predicts a type II transmembrane protein composed of 483 amino acid residues. The expressed enzyme transferred sulfate to the 6 position of nonreducing GlcNAc in GlcNAcbeta1-3Galbeta1-4GlcNAc. Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc and various glycosaminoglycans did not serve as acceptors. Expression of the cDNA in COS-7 cells resulted in production of a cell-surface antigen, the epitope of which was NeuAcalpha2-3Galbeta1-4(SO4-6)GlcNAc; double transfection with fucosyltransferase IV yielded Galbeta1-4(Fucalpha1-3)(SO4-6)GlcNAc antigen. The sulfotransferase mRNA was strongly expressed in the cerebrum, cerebellum, eye, pancreas, and lung of adult mice. In situ hybridization revealed that the mRNA was localized in high endothelial venules of mesenteric lymph nodes. The sulfotransferase was concluded to be involved in biosynthesis of glycoconjugates bearing the 6-sulfo N-acetyllactosamine structure such as 6-sulfo sialyl Lewis X. The products of the sulfotransferase probably include glycoconjugates with intercellular recognition signals; one candidate of such a glycoconjugate is an L-selectin ligand.
Female sterility in mice lacking the basigin gene, which encodes a transmembrane glycoprotein belonging to the immunoglobulin superfamily.
Basigin (Bsg) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. Bsg knock-out mice exhibit infertility of both sexes. Based on limited results, defective implantation has been considered to be the cause of the female infertility. We demonstrate here that disruption of the Bsg gene produces the failure of female reproductive processes including not only implantation but also fertilization. Bsg mRNA expression in cumulus cells and basolateral localization of the Bsg protein in the endometrial epithelium further support the importance of Bsg in these processes.
A null mutation in basigin, an immunoglobulin superfamily member, indicates its important roles in peri-implantation development and spermatogenesis.
Basigin is a highly glycosylated transmembrane protein with two immunoglobulin-like domains. We generated mutant mice lacking the basigin gene (Bsg) by gene targeting. Bsg (-/-) embryos developed normally during preimplantation stages. However, the majority of Bsg (-/-) embryos died around the time of implantation. At this time, basigin mRNA was strongly expressed in the trophectoderm, embryo proper, and uterine endometrium of Bsg (+/+) mice. These results suggest that basigin is involved in intercellular recognition during implantation. Embryos which survived the critical period yielded Bsg (-/-) mutant mice. Half of the mutant mice died before 1 month after birth, due to interstitial pneumonia. The surviving adult mutant mice were small and sterile. Spermatogenesis was arrested in the mutant mice. Most of the spermatocytes in the Bsg (-/-) mouse were arrested and degenerated at the metaphase of the first meiosis, and only a small number differentiated to step 1 spermatids. In the female mutants, the ovaries and genital tract were morphologically normal, and the defect was probably in the capability of implantation of the uterus. In conclusion, basigin is an important cell-surface molecule involved in early embryogenesis and reproduction.
Functional modules and expression of mouse p40(phox) and p67(phox), SH3-domain-containing proteins involved in the phagocyte NADPH oxidase complex.
The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The formation of the active oxidase complex at the membrane requires translocation of the Rac GTPase and two specialized cytosolic proteins that harbor SH3 domains, p67phox and p47phox. Another SH3-domain-containing protein p40phox, which is constitutively associated with p67phox in phagocytes, also enters the complex upon cell stimulation. Here we describe how we cloned mouse cDNAs encoding p40phox and its partner in phagocytes, p67phox. Both p40phox and p67phox comprise several protein-binding modules that are structurally and functionally well conserved between mouse and human, indicating their nature as adaptor proteins. We have also systematically investigated expression of the gene for p40phox in comparison with those for p67phox and p47phox. Distributions of the mRNAs for the three proteins among tissues are similar, with the most abundant expression in the spleen. The messages are abundant not only in phagocytic cells, but also in B cell lineage. The p40phox gene, but not the other two, is expressed in some types of cells such as plasma cells and T lymphocytes. Furthermore, in situ hybridization analysis shows that the p40phox mRNA is distributed in neuronal cells of mouse brain, providing evidence that one of the genes for the specialized oxidase factors is expressed in neurons. These observations raise the possibility that the adaptor protein p40phox plays a heretofore unsuspected role via interacting with other proteins in the cells that do not express p67phox or p47phox.
Expression of basigin, a member of the immunoglobulin superfamily, in the mouse central nervous system.
Basigin (Bsg) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. Chicken Bsg (HT7/neurothelin/ 5A11) is expressed in neuroblasts, but disappears from neurons after a specific stage of cytodifferentiation, and becomes restrictedly expressed in the capillary endothelium in the adult brain. We show herein by means of in situ hybridization that Bsg mRNA was expressed in neuroblasts in 13.5 day old mouse embryos. In the adult mouse, Bsg was differentially expressed in subregions of the brain. Strong Bsg expression was detected in the limbic system, including the olfactory system, hippocampal formation, septal area, amygdala, thalamic anterior nuclei, hypothalamus, mesencephalic tegmentum, entorhinal cortex, and cingulate gyrus. Bsg was also intensely expressed in the retinal neuronal layers, the Vth layer of the cerebral neocortex, Purkinje cells of the cerebellum, several nuclei of the brain stem, and the gray matter of the spinal cord. Although in situ hybridization showed a weak signal in the brain capillary endothelium, protein expression of Bsg was strong enough to be detected by immunohistochemistry. Northern blot analysis confirmed the strong expression of Bsg in the central nervous system. Taking into account that Bsg knockout mice exhibit abnormalities in behavior, but a normal blood-brain barrier function, the present findings suggest that Bsg functions actively in neuronal interactions in the central nervous system.
Porcine alpha-1,3-galactosyltransferase: full length cDNA cloning, genomic organization, and analysis of splicing variants.
Full length cDNA and genomic DNA of porcine alpha-1,3-galactosyltransferase were isolated, and their structures were analysed. The coding region was encoded by six exons as in the mouse, and the length of each exon was conserved between the two species. The porcine exons were designated Exon 4-9, since in the mouse coding exons started from Exon 4. Introns tended to be longer in the porcine gene; the distance from Exon 4 to the 3'-end of Exon 9 was 24 kb, while this region was 18 kb in the mouse gene. The cDNA structure was extended from the previous data to the 3'-end and to the 5' side of the cDNA. In addition to a cDNA clone with all coding exons, clones lacking parts of these exons were isolated and their structures were determined. One variant lacked Exon 5; the second, Exons 5 and 6; and the third, Exons 5, 6 and 7. The last variant was not found in the mouse, and cDNA transfection of this variant yielded scarcely any enzymatic activity using asialo alpha1-acid glycoprotein as a substrate, and decreased activity using N-acetyllactosamine as a substrate.
Truncated midkine as a marker of diagnosis and detection of nodal metastases in gastrointestinal carcinomas.
Midkine (MK) is a growth factor identified as a product of a retinoic acid-responsive gene. A truncated form of MK mRNA, which lacks a sequence encoding the N-terminally located domain, was recently found in cancer cells. We investigated the expression of the truncated MK mRNA in specimens of 47 surgically removed human gastrointestinal organs using polymerase chain reaction. Truncated MK was not detected in all of the 46 corresponding non-cancerous regions. On the other hand, this short MK mRNA was expressed in the primary tumours in 12 of 16 gastric cancers, 8 of 13 colorectal carcinomas, five of nine hepatocellular carcinomas, two of two oesophageal carcinomas and one ampullary duodenal cancer. In addition, truncated MK was detectable in all of the 14 lymph node metastases but in none of three metastatic sites in the liver, suggesting that truncated MK mRNA could become a good marker of nodal metastases in gastrointestinal tract.