Sadahiro Watanabe

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).

Charge-dependent regulation of NADPH oxidase activity in guinea-pig polymorphonuclear leukocytes.

The mechanism of respiratory burst was studied by modulating membrane surfaces with lipophilic ions in guinea-pig polymorphonuclear leukocytes and their subcellular membranes. Positively charged alkylamines in concentration ranges of 0.5 to 15 microM (ED50 values) inhibited the O2- generation with phorbol 12-myristate 13-acetate, N-formylmethionylleucylphenylalanine, A23187, myristate and arachidonate in intact cells, and the inhibition was relieved by negatively charged agents. A similar molecular size of alkylalcohols had no effects. A similar charge-dependent O2- generation was also observed with fatty acids in subcellular membrane fractions prepared from unstimulated control cells, and this was insensitive to H-7 and W-7. These results suggest that triggering of NADPH oxidase activation involves a reaction(s) that is regulated by membrane charges.

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.

Interaction of cytoplasmic proteins with liposomes and their cell specificity.

Numerous studies have shown that the plasma membrane has a fundamental role in the regulation of cell metabolism [l-4]. Attention has been focussed on the transmembrane control mechanism concerning the interaction between plasma membrane and cytoskeletal structures [2-121. For the transfer of biological information from external surface to cytoplasm, 3 steps are postulated: (i) Interaction of surface receptors with ligands at the external cell surface; (ii) Transmission of the information across the plasma membrane; (iii) Modulation of cytoplasmic components at the inner surface of the membrane. For example, cell agglutination by concanavalin A (con A) is accompanied by the redistribution of con A receptors on the cell surface from a random pattern to form a cap [2], and microtubules and microfilaments are involved in the transmembrane control of redistribution of lectin receptor sites in the cell membrane [2,3]. Therefore, the interaction of cytoplasmic proteins with the plasma membrane through their lipophilic nature and affinity to the membrane-integrated proteins have an important role for the regulatory mechanism of the transmembrane control system in cells. Here we report our attempts to determine the existence of an interaction between cytoplasmic proteins and artificial lipid vesicles, liposomes. We find that cytoskeletal components such as tubulin, actin and ol-actinin have a strong tendency to associate with liposomes and that this characteristic, which may be altered by several physiological conditions, is respon-

Developmental Changes in Mitochondrial Components in Liver of Newborn Rats

Chronological studies on the respiratory function and membrane components of newborn rat liver mitochondria revealed that perinatal mitochondria retained considerably well-coupled oxidative phosphorylation that functioned in sites II and III prior to site I. The ratio of protein to phospholipid contents (mg/mumol) was low in these mitochondria, in which phospholipids were characterized by their high phosphatidylethanolamine contents (43% of the total phospholipids) and low cardiolipin contents (6% of the total), as compared to those of the adult. The fatty acid composition in major phospholipid species was also different from those of the adult. These characteristics altered with development, and the carbamylphosphate synthetase contents markedly increased from 12 to 20% of the total polypeptides in 10 days postnatal time. From these results structural and functional completion of mitochondria accompanying development is discussed from a mitochondrial biogenesis viewpoint.

Vinblastine inhibits platelet aggregation by a microtubule-independent mechanism, probably by its perturbing action on the plasma membrane

Inhibitory mechanism of vinblastine on platelet activation was examined with respect to its effect on disassembly of the microtubule system. Vinblastine at 10 microM concentration caused washed platelets to become sphere with disorganized microtubule system, but did not affect aggregation induced by collagen, arachidonic acid or thrombin. Collagen-induced aggregation was inhibited by 50-100 microM of vinblastine and much higher concentration was required to inhibit arachidonic acid- and thrombin-induced aggregation. When the vinblastine (100 microM)-treated platelets were washed with albumin medium, the impaired aggregability was well recovered in response to collagen. In this case, however, both the vinblastine-induced sphered shape and disappeared microtubule system were not recovered to the normal states. Within the concentration ranges that inhibited collagen-induced aggregation, vinblastine also suppressed reversibly Ca2+ influx and arachidonic acid liberation from membrane phospholipids by phospholipase A2. Conversion of added arachidonic acid to thromboxane A2 was not inhibited even by such concentration. These results suggest that vinblastine interacts non-specifically with the cell membrane to cause reversible inhibition of arachidonic acid liberation by phospholipase A2 and Ca2+ influx and thereby aggregation through physical perturbation of membrane lipid bilayer, independent of the activity to disassemble platelet microtubule system.

Middle Ear Anomalies Induced by Hypertriazene Administration in the Mouse

The middle ear is derived from various embryonic tissues. Many experiments using teratogens have been performed, employing the difference of each tissues sensitivity to the teratogen and the tissues critical time of development. Triazene, a foliate metabolism antagonist, produced anomalies in fetuses that resembled those associated with thalidomide in humans, the so-called the first and second branchial syndrome. In our experiment, we administrated 3,3-dimethyl-1-phenyltriazene, an inductor of triazene, to pregnant mice at 7 to 14 days of gestation resulting in unique fetal anomalies. We examined the development of the stapes, stapedial artery, facial nerve, and oval window using an optical microscopic and three-dimensional reconstruction. Middle ear and facial nerve anomalies in mice depend on the gestation day when triazene is administrated. The stapedial artery, oval window, facial nerve (horizontal segment), stapes footplate, and styloid process are affected on the 9th to 11th administration day, the annular stapedialis on the 10th to 11th day, and the malleus and incus on the 9th to 11th day. The use of the Vox View/Mac, allowed us to create three-dimensional pictures from two-dimensional slides providing an improved understanding of the relationships between anatomical structures.

Establishment of a Mouse Model of Cystitis and Roles of Type 1 Fimbriated Escherichia coli in Its Pathogenesis

The role of type 1 fimbriae in promoting bladder colonization and the course of Escherichia coli cystitis were examined with type 1 fimbriated strains of clinically isolated E. coli. In the experiments of mice in vivo, intact bladder epithelium showed natural resistance to the adherence of type 1 fimbriated and non‐fimbriated E. coli. However, the exfoliation of bladder superficial cells by trypsinization before the bacterial inoculation promoted the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium. After colonization of E. coli, maximum numbers of E. coli and leukocytes were observed 3 days after inoculation. Nine days after inoculation, both of E. coli and leukocytes disappeared and the regeneration of superficial cells was observed. On the other hand, superficial cells in mice injected with phosphate‐buffered saline or non‐fimbriated E. coli regenerated 5 days after trypsinization. The present study demonstrated that the removal of superficial cells is essential for the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium in vivo and a new model of E. coli cystitis in mice was established. The model which we established is valuable for histopathological, immunological, and therapeutic studies.

Immuno-electron microscopic localization of fibronectin on rat mast cells

Rabbit anti-rat plasma fibronectin (pFN) causes histamine release from rat mast cells in the presence of complement. Fibronectin (FN) on rat mast cells, as shown by immuno-electron microscopy, is principally localized on cell folds, so they may play a role of attachment in the matrix of connective tissue.