Takako Nomura

Identification and characterization of functional subunits of Clostridium botulinum type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes

Clostridium botulinum type A hemagglutinin‐positive progenitor toxin consists of three distinct components: neurotoxin (NTX), hemagglutinin (HA), and non‐toxic non‐HA (NTNH). The HA consists of four subcomponents designated HA1, 2, 3a and 3b. By employing purified toxin and GST‐fusion proteins of each HA subcomponent, we found that the HA‐positive progenitor toxin, GST‐HA1 and GST‐HA3b bind to human erythrocytes and microvilli of guinea pig upper small intestinal sections. The HA‐positive progenitor toxin and GST‐HA1 bind via galactose moieties, GST‐HA3b binds via sialic acid moieties. GST‐2 and GST‐3a showed no detectable binding.

Detection of manganese superoxide dismutase mRNA in the theca interna cells of rat ovary during the ovulatory process by in situ hybridization

To determine the possible involvement of reactive oxygen species in ovulation, dynamic aspects of superoxide dismutase (SOD) isozyme were studied in the ovaries of rats by in situ hybridization histochemistry. Previously, mRNA levels of ovarian manganese superoxide dismutase (Mn-SOD) were reported markedly to increase whilst enzymic activity of Mn-SOD decreased during the ovulatory process after treating immature rats with 10 and 5 Units, respectively, of pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG). Levels of Cu/Zn-SOD activity and Cu/Zn-SOD mRNA were reported to remain unchanged throughout ovulation. This increase in the Mn-SOD mRNA level was shown in the present study by in situ hybridization to be localized to the theca interna cells throughout the PMSG/HCG-induced ovulatory process. The observations suggest that the turnover rate of Mn-SOD but not Cu/Zn-SOD increases specifically in the mitochondria of these cells. SOD has been postulated to play important roles in steroidogenesis. The relationship is discussed between mitochondrial functions in steroid-secreting cells and superoxide radicals and related metabolite(s).

Expression of manganese superoxide dismutase mRNA in reproductive organs during the ovulatory process and the estrous cycle of the rat

Expression of managanese superoxide dismutase (Mn-SOD) mRNA during the pregnant mare serum gonadotrophin (PMSG)/human chorionic gonadotrophin (HCG)-induced ovulatory process, and during the estrous cycle was examined in rat female reproductive organs. Mn-SOD mRNA levels in theca interna cells markedly increased in PMSG-primed rats and high levels of the transcripts were maintained after HCG injection. The PMSG-enhanced expression of Mn-SOD mRNA in follicular epithelial cells increased concomitantly with luteinization of these cells. The levels of Mn-SOD mRNA remained high and became equivalent in both granulosa and theca lutein cells 24 h after HCG injection. Neither luteinization nor the expression of Mn-SOD mRNA was observed in the epithelial cells of unovulated follicles. Luteal bodies had formed 3 days after HCG injection, and the same level of Mn-SOD mRNA expression continued in lutein cells, but not in stromal cells. During the estrous cycle, Mn-SOD mRNA was localized to theca interna cells on proestrus, to the epithelial cells of luteinizing follicles on estrus, and to newly formed luteal bodies on diestrus. The epithelial cells in the oviduct did not express Mn-SOD mRNA throughout the ovulatory process or the estrous cycle. Expression of Mn-SOD mRNA in the luminal epithelial cells of the uterus increased after PMSG injection, reaching a maximum after 24 h, and became relatively negative 3 days after HCG injection when corpora lutea had formed in the ovary. During the estrous cycle, uterine epithelial cells and leukocytes showed marked increases in Mn-SOD mRNA expression on estrus and on proestrus, respectively. Expression in the vaginal epithelium became apparent 3 days after HCG injection and continued for at least 12 days after HCG injection. The expression was localized to the superficial layer of the epithelium. During the estrous cycle, expression occurs in the basal layer on proestrus and estrus, transferring to the superficial layer on diestrus day 1, and expression stops on diestrus day 2. The relationship between the expression of Mn-SOD mRNA and hormone-induced metabolic changes, including steroidogenesis, is discussed.

Quantification of In Situ Hybridization Signals in Rat Testes

We performed basic research into quantifying in situ hybridization (ISH) signals in rat testis, a suitable organ for the quantification because germ cells undergo synchronized development and show stage-specific gene expression. In this model experiment, rRNA was selected as the hybridizable RNA in paraffin sections. Specimens fixed with Bouins fixative and hybridized with digoxygenin-labeled probes could easily be analyzed quantitatively through “posterization” of the images. The amount of rRNA hybridized with the probe was greatest in early primary spermatocytes, followed by pachytene primary spermatocytes, then diplotene spermatocytes, and finally by secondary spermatocytes and spermatids. The amounts reached low levels in metaphase, anaphase, and telophase of meiotic division and early step 1 spermatids, and then slightly increased during spermiogenesis. ISH rRNA staining was a useful parameter for evaluation of the quantitative analysis of mRNA and the levels of hybridizable RNA in tissue sections. (J Histochem Cytochem 52:813–820, 2004)

Characterization of the inner enamel epithelium in the enamel-free area based on the ability to secrete enamel protein demonstrated by in situ hybridization and immunohistochemistry.

Both the expression of amelogenin mRNA and secretion of amelogenin were investigated in rat molars by in situ hybridization and immunohistochemistry. Probes were designed by multiple-labeling of oligonucleotide probes for in situ hybridization. Amelogenin mRNA first appeared in differentiating ameloblasts of the distal region and some inner enamel epithelial cells of enamel-free area (EFA cells) of the second cusp at postnatal day 0. At the same time, amelogenin protein was detected in the extracellular matrix between dentin and differentiating ameloblasts and in some EFA cells of the second cusp. At postnatal day 1-3, amelogenin was expressed in the secretory ameloblasts, and in the matrix beneath these cells. Both amelogenin mRNA and amelogenin were detected in the EFA cells and their extracellular matrix. After postnatal day 5, amelogenin mRNA and amelogenin were detected in the secretory ameoloblasts and extracellular matrix in the enamel-forming region, respectively. At this time, amelogenin mRNA was not detected in the EFA cells, but a small amount of amelogenin was found in the matrix beneath the EFA cells. These findings suggest that EFA cells differentiate into amelogenin-secreting cells, i.e. ameloblasts, but that the secretion lasts for only a short period at the early stage of tooth development.

Expression of managanese superoxide dismutase in rat submandibular gland demonstrated by in situ hybridization and immunohistochemistry

Superoxide, an active oxygen species, plays an important role in protecting against bacterial infection. However, it also has adverse effects on health. Manganese superoxide dismutase (Mn-SOD) is a scavenger of superoxide. Antioxygen enzymes such as Mn-SOD exist in various tissues, and provide protections against oxidative injury. We investigated both the expression of Mn-SOD mRNA and the localization of Mn-SOD in adult rat submandibular glands using in situ hybridization and immunohistochemistry. Both Mn-SOD mRNA and Mn-SOD were detected in striated duct cells, and in some granular duct cells and excretory duct cells. With immunoelectron microscopy, many immunolabelings were observed on the mitochondria. These findings suggest that the expression of Mn-SOD mRNA and the localization of Mn-SOD in submandibular glands correlate with the number of mitochondria in cells.

Histochemical and immunohistochemical changes in rat hepatocytes after halothane exposure

AbstractPurpose. Histochemical and immunohistochemical changes were observed in hepatocytes to study the developing and recovery processes of halothane-induced hepatic injury from 0 to 7 days after halothane exposure. Methods. A total of 330 7-week-old male Sprague-Dawley rats, with or without phenobarbital preteatment, were exposed to halothane in 100%, 21%, 10% oxygen or oxygen alone for 2 h. Results. In the phenobarbital group, degenerated hepatocytes were observed immediately after exposure to 10% oxygen, both with and without halothane: glycogen and ribosomal ribonucleic acid (rRNA) disappeared immediately and 6 h after exposure, respectively, and necrosis developed in zones 3 to 2 at 6 h after exposure. From 12 h to day 1, the necrosis was more marked and more widely observed in the cells with halothane than those without halothane. However, all tissues returned to normal by day 7. Conclusion. Disappearance of glycogen at 0 h and rRNA at 6 h after exposure to halothane under 10% oxygen is considered to be one of the factors inducing necrosis around the central vein. Recovery of the hepatolobular structure was attributed to the rearrangement of the remaining hepatocytes in the portal vein area.