Biomedical subjects

Masashi Mizuno

Publications and source records attributed to Masashi Mizuno.

At least 19 recordsLinked to original sources

PubMed2006

CD55 in rat male reproductive tissue: differential expression in testis and expression of a unique truncated isoform on spermatozoa.

CD55 is a key regulator of complement activation, expressed on most tissues and cells in man and other mammals. In the rat, alternative splicing in the gene encoding CD55 yields GPI-anchored (GPI-CD55) and transmembrane (TM-CD55) forms. Published Northern blot analysis indicated that while GPI-CD55 was broadly expressed, TM-CD55 was primarily expressed in the testis, although the precise site of expression was not identified. To clarify the distribution of CD55 isoforms in rat reproductive tissues, we first performed immunohistochemistry and Western blot analysis with an anti-rat CD55 mAb that recognized all reported CD55 isoforms, and a polyclonal immunoglobulin specific for TM-CD55. CD55 was absent in testis prior to puberty. Post-puberty, CD55 was expressed at high levels on all spermiogenic cells from step 6 spermatid onward, and on mature spermatozoa focussed on the acrosome, but was absent from support cells and early progenitors. Enzymatic digestion revealed that GPI-CD55 was predominant in testis and spermatozoa. Staining for TM-CD55 with specific immunoglobulin confirmed its absence from mature sperm and expression on spermatids only between steps 11 and 14 of development. GPI-CD55 on spermatozoa was of lower molecular weight than that in testis and other tissues; sequencing from spermatozoal mRNA identified a unique isoform of GPI-CD55 missing short consensus repeat 4. The predominant acrosome expression and presence of a unique, truncated isoform of CD55 on spermatozoa provides further support for the hypothesis that the acrosome is a highly specialized region in which closely regulated complement activation may contribute to reproductive function.

Acrosome↗

PubMed2006

Immunization with autologous CD46 generates a strong autoantibody response in rats that targets spermatozoa.

CD46, a membrane complement regulator, has been implicated as pathogen receptor, T cell activator and contributor to spermatozoa-egg interactions. In man, a role in the fertilization process was suggested by its localization on the acrosome. In rodents, CD46 is expressed only on the spermatozoal acrosome, suggesting an essential role at this site. This restricted expression led us to ask whether immunization with CD46 would generate anti-CD46 antibody responses that might target spermatozoa and influence fertility. We immunized male and female rats with rat CD46. Strong immune responses were generated in all rats and immune sera stained CD46 in testis extracts and in situ in testis and sperm. Incubation of spermatozoa with immune sera caused deposition of immunoglobulin and C3b in an acrosome pattern and reduced motility. We mated immune male rats with naïve females and female immune rats with naïve males. The incidence of pregnancy and number of fetuses were not different in matings involving immune male or female rats compared to controls. Testis sections from immune rats revealed no immunoglobulin deposition on CD46-positive sperm precursors, suggesting that acrosomal CD46 was inaccessible in this location. A minority of spermatozoa harvested from epididymis of immune rats had immunoglobulin and C3b bound to the acrosome, suggesting that anti-CD46, present in genital tract fluids, bound after acrosome reaction. These data demonstrate that the restricted expression of CD46 allows strong anti-CD46 responses in rats that target spermatozoa in vitro and in vivo. The anti-CD46 response did not influence fertility, perhaps reflecting the considerable redundancy for fertilization in rodents.

Acrosome↗

PubMed2006

Synthesis and evaluation of 4-substituted benzylamine derivatives as beta-tryptase inhibitors.

Since beta-tryptase is considered a critical mediator of asthma, potent tryptase inhibitors may be useful as new agents for the treatment of asthma. We investigated 4-substituted benzylamine derivatives and obtained M58539 (15h) as a potent inhibitor of beta-tryptase (IC50 = 5.0 nM) with high selectivity against other serine proteases, low molecular weight, clog P value less than 5, lack of amidino and guanidino groups, and independence of Zn2+ ion.

Benzylamines↗

PubMed2006

Complement and complement regulators in the male reproductive system.

Spermatozoa are almost unique among cells in that they must survive transplantation into a foreign host in order to perform their physiological role. The biggest hurdle to overcome is innate immune defence that will target the invaders in the female genital tract. Complement is a major player in innate immunity and is present in the female genital tract. Spermatozoa must therefore evade complement attack if they are to reach their goal. Complement evasion is achieved by the presence of complement regulators both in seminal plasma and on the spermatozoa. Here we discuss the parts played by complement and complement regulators in permitting spermatozoa to survive long enough to reach the oocyte, in clearance of the excess spermatozoa that have outlived their usefulness and in aiding activation of spermatozoa to engage the oocyte. In particular, we focus on the unique distribution patterns of complement regulators on spermatozoa, patterns that strongly suggest roles in spermatozoal development and oocyte binding. An understanding of these roles will inform studies of their contribution to fertility and infertility in man.

Animals↗

PubMed2006

[Degradation of cell wall polysaccharides during postharvest fruit ripening and softening of different apple varieties].

The cell wall material (CWM) and eight cell wall polysaccharides fractions were extracted from 'Fuji' and 'Kinsei' apples during storage at different time (0 and 42 days). The sugar composition characteristics of each fraction were determined by gas chromatography. The results showed that, during storages, the firmness of 'Kinsei' apples decreased significantly, and a significant peak of ethylene production was shown after 10 d storage, but only a little ethylene was produced in 'Fuji' apples, which had a better storability. Compare to other cell wall polysaccharide fractions, in Na(2)CO(3)-soluble pectic fractions of apple fruit, there were abundant rhamnogalacturonan I (RG-I), which branched highly in side chains due to the compositions of arabinans, galactans, arabinogalactans etc. As for cell wall polysaccharides, in 'Kinsei' apples, the decrease of pectic fractions was shown most significantly in Na(2)CO(3)-1 fraction, which was associated with a significant degradation of arabinosyl and galactosyl residues on the side chains. Further more, higher molecular mass in Na(2)CO(3)-1 pectic polysaccharides degraded and turned into ones with smaller molecular mass. From these results, the degradation of side chains in Na(2)CO(3)-1 pectic polysaccharides under the activity of enzyme was considered one of the most significant factors of apple fruit softening through modifying the network of cell wall polysaccharides.

Carbonates↗

PubMed2005

Spermatogenic cells distal to the blood-testis barrier in rats lack C3 convertase regulators and may be at risk of complement-mediated injury.

On most tissues, multiple membrane complement regulators (CReg) protect self-cells from damage by complement. An exception is the brain, where the blood-brain barrier provides a protected environment within which cells survive with little or no protection from complement. The testis has a functionally similar structure, the blood-testis barrier (BTB). Here, we have investigated the expression of C3/C5 convertase CReg and C3 in the normal rat testis at different ages and different spermatogenetic stages, as well as in rats in which spermatogenesis and the BTB were impaired due to a developmental deficit. Immature testis, prior to BTB formation at puberty, displayed broad expression of the ubiquitous rodent CReg Crry on all elements and no expression of CD46 or CD55. Within days of BTB formation, CReg expression was dramatically altered; Crry was expressed only in the spermatogenetic cells external to the BTB in basal layers of adult seminal epithelium. Spermatogenic cells immediately distal to the BTB at first expressed no C3/C5 convertase regulators but later acquired expression of CD46 and CD55. Staining for C3 was widespread pre-puberty, but absent distal to the BTB in mature rats. In rats with defects in spermatogenesis and BTB integrity, expression patterns of CReg and C3 resembled those in pre-pubertal normals. The relative paucity of CReg and absence of C3 synthesis distal to the BTB suggest the presence of a complement-protected environment analogous to that described in the brain, and suggest also that cells enclosed by the BTB may be susceptible to complement damage when the barrier is breached.

Animals↗

PubMed2005

Characterization of the Lentinula edodes exg2 gene encoding a lentinan-degrading exo-beta-1,3-glucanase.

Lentinan, an antitumor substance purified from Lentinula edodes, is degraded during post-harvest preservation as a result of increased glucanase activity. We isolated an exo-beta-1,3-glucanase encoding gene, exg2, from L. edodes which is a homologue of an exo-glucanase-encoding gene conserved in ascomycetous fungi. The exg2 gene was cloned as an approximately 2.4-kbp cDNA, and as a genomic sequence of 3.9-kbp. The product of the exg2 gene is predicted to contain 759 amino acids with a molecular weight of 79 kDa and a pI value of 4.6. The putative N-terminus of EXG2 is identical to the N-terminal sequences of lentinan-degrading enzymes, GNase I and II, and a custom-made anti-EXG2 peptide anti-serum cross-reacted with purified GNase I and II. Transcription and translation of exg2 was low in the gills of mature fruiting bodies, but increased after harvesting. We conclude that the exg2 gene is a lentinan-degrading enzyme-encoding-gene in L. edodes.

Amino Acid Sequence↗

PubMed2005

CD46 plays a key role in tailoring innate immune recognition of apoptotic and necrotic cells.

Complement is the canonical innate immune system involved in host defense and tissue repair with the clearance of cell debris. In contrast to the robust armory mounted against microbial nonself-pathogens, complement is selectively activated on altered self (i.e. apoptotic and necrotic cells) to instruct the safe demise by poorly characterized mechanisms. Our data shed new light on the role of complement C1q in sensing nucleic acids (NA) rapidly exposed on apoptotic Jurkat T cell membranes and in driving C3 opsonization but without the lytic membrane attack complex. DNA/RNase-treated apoptotic cells failed to activate complement. We found that several other apoptotic cell models, including senescent keratinocytes, ionophore-treated sperm cells, and CMK-derived platelets, stained for cleaved caspase 3 were rapidly losing the key complement regulator CD46. CD46 from nuclear and membrane stores was found to cluster into blebs and shed into microparticles together with NA, phosphatidylserine, C1q, and factor H. Classical and alternative pathways of complement were involved in the recognition of H2O2-treated necrotic cells. Membrane attack complex was detected on necrotic cells possibly as a result of CD46 and CD59 shedding into soluble forms. Our data highlight a novel and universal paradigm whereby the complement innate immune system is using two synergistic strategies with the recognition of altered self-NA and missing self-CD46 signals to instruct and tailor the efficient removal of apoptotic and necrotic cells in immunoprivileged sites.

Apoptosis↗

PubMed2005

Shiga toxin 1 causes direct renal injury in rats.

Infection with Shiga toxin (Stx)-producing Escherichia coli has been implicated to cause hemolytic uremic syndrome, which is characterized by histological abnormalities such as microvascular thrombi and tubular cell damage in the kidney. Although Stx is known to be the major virulence factor of the pathogen, it is still unclear whether Stx directly impairs renal cells in vivo to cause such histological changes and deterioration of renal function. To assess the consequence of the direct action of Stx on renal cells, left kidneys of rats were perfused with Stx1 from the renal artery through the renal vein and then revascularized. Kidneys of control animals were perfused with the vehicle alone. On day 1, apoptosis and induction of tumor necrosis factor alpha gene expression were noticed to occur in the medulla of the Stx1-perfused kidneys. On day 3, extensive tubular injuries were observed by light microscopy: aggregated platelets and monocytic infiltrates in both glomeruli and the medullary interstitium were detected by immunostaining. Tubular changes were more extensive on day 9, with areas of infarction seen in the cortex and medulla. These changes were not found to occur in the sham-operated kidneys. No obvious glomerular changes were detected by light microscopy at any time point. When nonperfused right kidneys were removed after the Stx1 perfusion of the left kidneys, the serum creatinine and blood urea nitrogen levels were increased from day 2, and acute renal failure followed on day 3. These results indicate that Stx1 caused glomerular platelet aggregation, tubular damage, and acute deterioration of renal function by acting directly on renal cells.

Animals↗

PubMed2005

Novel C5a regulators in inflammatory disease.

Complement is part of the innate immune system, acting to protect the host from microorganisms such as bacteria, and other foreign and abnormal cells. Although primarily protective, complement activation can also cause damage to the host. In a number of inflammatory diseases, including rheumatoid arthritis and dermatitis, there is excessive and inappropriate complement activation. Many of the toxic effects seen in these conditions are attributable to the excessive production of the anaphylatoxin C5a, which may contribute to both the initiation and progression of the disease. Therefore, the regulation of C5a production and modulation of its function are good pharmacological targets in these disorders. As yet, there are no effective agents for the therapeutic regulation of C5a in routine clinical practice. This review describes the role of C5a in inflammatory disease, animal models used to study C5a-related effects, and current strategies aimed at regulating C5a. There is also a discussion of the strengths and weaknesses of these approaches, and an outline of the likely progress of this class of drugs in the future.

Animals↗

PubMed2004

Expression of CD46 in developing rat spermatozoa: ultrastructural localization and utility as a marker of the various stages of the seminiferous tubuli.

Identification of the various stages of the seminal tubule epithelium that are important in spermatogenesis in humans and rodents requires considerable expertise for analysis of ultrastructural appearance under light microscopy. Few good stage-specific markers have been reported to facilitate the process. We recently described characterization of the expression of CD46 (membrane cofactor protein) in the rat using a novel monoclonal antibody. Expression of CD46 was restricted to spermatozoa and their immediate precursors in the testis. In the present study, we used a combination of morphological analyses, known acrosome markers, actin staining, direct nuclear staining, and staining for CD46 to delineate precisely the subcellular location of CD46. Staining of CD46 colocalized with known acrosome markers in late spermatids and mature spermatozoa and was confirmed by electron microscopy to be acrosome-restricted. Expression was first detected in step 7 spermatids, whereas known markers were not expressed until step 9. The CD46 staining pattern differed through spermatid development, and distinct patterns of staining could be identified that, when combined with 4'-6-diamino-2-phenylindole-2HCl nuclear staining, enabled the accurate staging of the seminiferous tubule epithelium in different profiles. This detailed description of the spatiotemporal expression patterns of CD46 provides a valuable tool for analysis of spermatogenesis in the rat. Furthermore, this information will aid ongoing studies regarding the roles of CD46 in acrosome-related spermatozoal functions.

Acrosome↗

PubMed2004

Catalase and alternative oxidase cooperatively regulate programmed cell death induced by beta-glucan elicitor in potato suspension cultures.

In potato (Solanum tuberosum L.) suspension cells, the expression of the gene encoding alternative oxidase (AOX) and H2O2 accumulation were induced by treatment with beta-glucan elicitor. The inhibition of catalase activity enhanced both AOX mRNA expression and the production of H2O2, whereas the ascorbate peroxidase inhibitor did not have any effect on these responses. Simultaneous inhibition of catalase and AOX activities in elicited cells dramatically increased H2O2 accumulation, leading to the disruption of mitochondrial membrane potential (deltapsi(m)) and programmed cell death (PCD). The results demonstrate, for the first time, that not only AOX but also catalase plays a central role in the suppression of mitochondrial deltapsi(m) breakdown and PCD induced by beta-glucan elicitor.

Apoptosis↗

PubMed2004

CD59a is the primary regulator of membrane attack complex assembly in the mouse.

Gene-deleted mice have provided a potent tool in efforts to understand the roles of complement and complement-regulating proteins in vivo. In particular, mice deficient in the membrane regulators complement receptor 1-related gene/protein y, decay-accelerating factor, or CD59 have demonstrated homeostatic relevance and backcrossing between the strains has revealed cooperativity in regulation. In mouse, genes encoding decay-accelerating factor and CD59 have been duplicated and show differential expression in tissues, complicating interpretation and extrapolation of findings to man. The first described form of CD59, CD59a, is broadly distributed and deletion of the cd59a gene causes a mild hemolytic phenotype with increased susceptibility in complement-mediated disease models. The distribution of the second form, CD59b, was originally described as testis specific, but later by some as widespread. Deletion of the cd59b gene caused a severe hemolytic and thrombotic phenotype. To apply data from these mouse models to man it is essential to know the relative distribution and functional roles of these two forms of CD59. We have generated new specific reagents and used them in sensitive quantitative analyses to comprehensively characterize expression of mRNA and protein and functional roles of CD59a and CD59b in wild-type (wt) and CD59a-negative mice. cd59b mRNA was detected only in testis and, at very low levels, in bone marrow. CD59b protein was present on mature spermatozoa and precursors and, in trace amounts, erythrocytes. Erythrocyte CD59b did not inhibit complement lysis except when CD59a was absent or blocked. These data confirm that CD59a is the primary regulator of complement membrane attack in mouse.

Animals↗

PubMed2004

Rat membrane cofactor protein (MCP; CD46) is expressed only in the acrosome of developing and mature spermatozoa and mediates binding to immobilized activated C3.

The rat analogue of the complement regulator membrane cofactor protein (MCP; CD46) was recently cloned and analysis at the mRNA level suggested that expression was restricted to testis. In light of the proposed roles of human MCP in sperm-egg interaction, we undertook to analyze rat MCP expression at the protein level in order better to address its putative role in fertilization. Recombinant fusion proteins comprising antibody Fc and specific domains of rat MCP were generated and used to develop a monoclonal antibody, MM.1, specific for rat MCP. Immunohistochemistry using these reagents confirmed the reported testis-specific expression of MCP in sexually mature rats and demonstrated that MCP was expressed only by spermatozoa and their immediate precursors in spermiogenesis, spermatids. Prepubertal male rats did not express MCP, and there was no evidence of MCP expression at any site in the embryo. Spermatozoal MCP expression was restricted to the inner acrosomal membrane, exposed only after fixation or induction of the acrosome reaction. Acrosome-reacted but not unreacted spermatozoa bound methylamine-activated C3 immobilized on plastic. The retention of MCP at this subcellular site, which is probably crucial to sperm-egg interaction, and the functional demonstration of binding to activated C3 strengthen suggestions from human studies that MCP may play an important role in fertilization. The reagents and results described here will enable studies of the role of spermatozoal MCP in sperm-egg interaction using a relevant animal model system.

Acrosome↗

PubMed2004

Lentinan from shiitake mushroom (Lentinus edodes) suppresses expression of cytochrome P450 1A subfamily in the mouse liver.

Induction of xenobiotics metabolizing enzymes is related to the formation and chemoprevention of cancer. Since cytochrome P450s (CYPs) including CYP1A subfamily metabolize certain pro-carcinogens to their ultimate forms, down-regulation of CYP1As by food factors leads to the prevention of cancer. Mushroom polysaccharides, especially beta-glucans such as lentinan from Lentinus edodes, possess the anti-tumor and immunomodulating activities through the cytokine production from immunocytes. Recent our studies have demonstrated that lentinan suppresses hepatic CYP1As expression in the both constitutive and inducible levels through the production of tumor necrosis factor-alpha and an increase in the DNA-binding activity of nuclear factor-kappaB. This paper discusses on the effective lentinan dosage and route of administration for suppression of CYP1As.

Animals↗

PubMed2004

A new southwestern chemistry-based ELISA for detection of aryl hydrocarbon receptor transformation: application to the screening of its receptor agonists and antagonists.

Halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produce a wide variety of biological and toxic effects mainly through the aryl hydrocarbon receptor (AhR)-dependent mechanism. After the binding of HAHs, the AhR subsequently transforms its form in order to interact with a specific DNA sequence, the dioxin responsive element (DRE). Thus, detection of the transformed AhR is a target for estimation of the biological and toxic potency of ligands. In this study, we have developed a simple method for quantitative assessment of the transformation state of AhR based on an enzyme-linked immunosorbent assay (ELISA) combined with southwestern chemistry technique (SW-ELISA) that detects the complex of transformed AhR:fluorescein isothiocyanate (FITC)-labeled DRE probe. SW-ELISA has shown the response to HAHs including TCDD and other known agonists in a dose-dependent manner. In the case of TCDD, SW-ELISA has revealed a minimum detection limit (MDL) of 2 pM (0.026 pg/assay), a median effective concentration (EC(50)) value of 0.125 nM (1.6 pg/assay), and a maximum response at 10 nM (129 pg/assay). Furthermore, SW-ELISA provides the confirmation that flavonoids, the potent antagonists for AhR as reported previously, show the inhibitory effects on TCDD-induced AhR transformation. These results indicate that SW-ELISA is a new and straightforward method for the detection of AhR transformation and will be useful in screening of agonists or antagonists for AhR.

Animals↗

PubMed2004

Synthesis and evaluation of 1-arylsulfonyl-3-piperazinone derivatives as factor Xa inhibitors III. Effect of ring opening of piperazinone moiety on inhibition.

Compounds containing an ethylenediamine structure in place of the piperazine ring of M55113 (1) and M55551 (2) were synthesized to investigate the effects of a piperazine moiety and evaluated for activity as factor Xa (FXa) inhibitors. Most such compounds, however, exhibited lower activity (1/10-1/100) than that of M55113 and M55551 as FXa inhibitors.

Factor Xa Inhibitors↗

PubMed2004

The expression profile of lectin differs from that of seed storage proteins in Castanea crenata trees.

Using Northern blot analysis, the expression of the Japanese chestnut (Castanea crenata Sieb. et Zucc.) agglutinin (CCA) gene was compared with that of its seed storage protein (SSP) gene. After cDNA cloning of SSP, the expression profile of SSP mRNA and CCA mRNA were compared. SSP mRNA was seed-specific, while CCA mRNA was expressed in the stems and flowers (both male and female) as well as in the seeds. Whereas extracts from all organs observed using Western blot analysis exhibited positive signals, in seeds, large expressions of SSP mRNA were restricted to the late maturation and harvest stages. Levels were maintained during the dormant period. No expression was observed during the germination stage. In contrast, CCA mRNA expression was maintained at a high level during development, was at a relatively low level during dormancy, and showed subsequent high expression during germination. These results suggest that one of the physiological roles of CCA is to act as a vegetative storage protein. But since protein expression did not coincide with that of mRNA, the expression of CCA may be regulated both at the transcription and the translation levels.

Agglutinins↗