A new method of the immunohistochemical detection of cellular antigens for light and electron microscopy

Summary A new immunohistochemical method for light and electron microscopy of tissue- and cell-specific antigens by using ferric colloid-labeled antibody is presented. The antibodies labeled with the cationic cacodylate ferric colloid are stable and bind specifically to the target antigens to show clearly the site of antigens in tissue sections and on free cells by Prussian blue reaction for light microscopy and by the specific figure of electron opaque ferric colloid particles for electron microscopy. The staining procedure is very simple and it gives clear picture. So the method will be of beneficial for general laboratory use in immuno-histochemical researches.     

Rat oocyte tissue plasminogen activator is a catalytically efficient enzyme in the absence of fibrin. Endogenous potentiation of enzyme activity

Rat oocytes synthesize tissue plasminogen activator (tPA) in response to stimuli which initiate meiotic maturation. Purified tPA exhibits optimal activity only in the presence of fibrin or fibrin substitutes. Because oocytes are not exposed to fibrin in situ, we investigated the possible stimulation of rat oocyte tPA activity by other endogenous factor(s). Oocytes were obtained from immature female rats which were induced to ovulate with gonadotropins. tPA activity was measured by the plasminogen-dependent cleavage of a chromogenic substrate. Measurements of kinetic parameters with Glu- or Lys-plasminogen revealed a Km for the rat oocyte enzyme of 1.3-2.1 microM compared with 23-24 microM for purified human tPA. Inclusion of the soluble fibrin substitute polylysine lowered the Km of human tPA by 30-fold (0.8 microM) but had no effect on the oocyte tPA Km. Polylysine had no significant effect on the Vmax values. The rate of plasminogen activation catalyzed by oocyte tPA was increased only 4.3-fold by fibrin while fibrin stimulated purified human tPA activity by 15.2-fold. After fractionation of oocyte extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polylysine enhanced oocyte tPA activity as seen by casein zymography. tPA activity in the conditioned medium of a rat insulinoma cell line was also not stimulated with polylysine prior to fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that extravascular cells which elaborate tPA may produce stimulatory factor(s) which allow for full tPA activity at physiological concentrations of plasminogen in the absence of fibrin.     

Reaction mechanism of isomaltooligosaccharides synthesis by α-glucosidase fromAspergillus carbonarious

Summary An -glucosidase fromAspergillus carbonarious CCRC 30414 was employed for investigating the enzymatic synthesis of isomaltooligosaccharides from maltose. The enzyme transferred a glucose unit from the nonreducing end of maltose and other -linked glucosyl oligosaccharides to glucose and other glucosyl oligosaccharides which function as accepting co-substrates. The transfer of a glucose unit occurs most frequently to the 6-OH position of the nonreducing end of acceptor, but transfer to 4-OH position also occurs. Treatment of 30 % (w/v) maltose with the enzyme under optimum conditions afforded more than 50% isomaltooligosaccharides.     

Mutagenesis analysis of a hepatitis delta virus genomic ribozyme.

We conducted extensive mutagenesis analysis on a hepatitis delta virus (HDV) genomic ribozyme to study the sequence specificity of certain region and to derive the secondary structure associated with the catalytic core. The results confirmed that the autocatalytic domain of HDV genomic RNA contained four base-pairing regions as predicted in the 'pseudo-knot' model [Perrotta & Been (1990) Nature 350, 434-436]. The size and sequence of one of the base-pairing regions, i. e. stem-and-loop, could be flexible. Helix 3 and the first basepair of helix 1 required specific sequence to retain self-cleavage activity. The structural requirement of helix 2 was less stringent than the other base-pairing regions. Moreover, the size of helix 1 affected self-cleavage whereas the length of hinge could be variable even though the first three residues of hinge had stringent sequence requirement.     

Estrogens augment the stimulation of ovarian aromatase activity by follicle-stimulating hormone in cultured rat granulosa cells

The effects of estrogens on ovarian aromatase activity were investigated in vitro using granulosa cells from immature hypophysectomized estrogen-primed rats. The cells were cultured for 3 days in an androgen-free medium in the presence of follicle-stimulating hormone (FSH), with or without the specified estrogen. After washing, the cells were reincubated for 5 h with 10(-7) M androstenedione, and the formation of estrogens was measured. Estrogen production by control and diethylstilbestrol-treated cells was negligible, while FSH stimulated aromatase activity. Furthermore, concomitant treatment with diethylstilbestrol led to dose-dependent increases in the FSH-induced aromatase activity with an ED50 value of 4 X 10(-9) M and an apparent Vmax value 12- to 16-fold higher than those induced by FSH alone. The direct stimulatory effect of estrogens was time-dependent and was not accounted for by increases in cell protein. Various native and synthetic estrogens also augmented the FSH induction of aromatases (native estrogens: estradiol-17 beta = estrone greater than estradiol-17 alpha greater than estriol; synthetic estrogens: hexestrol greater than moxestrol greater than ethinyl estradiol much greater than chlorotrianisene and mestranol). The effect of estradiol-17 beta was dose-dependent with an ED50 value of 9 X 10(-9) M, which is within the physiological levels of follicular estradiol-17 beta. Although treatment with androgens also enhanced the FSH-induced aromatases, treatment with a progestin (R5020) or a mineralocorticoid (aldosterone) was without effect. Thus, estrogens directly augment the stimulation of granulosa cell aromatase activity by FSH. Follicular estrogens may activate intraovarian autoregulatory positive feedback mechanisms to enhance their own production, resulting in selective follicle maturation and the preovulatory estrogen surge.     

Glucocorticoid inhibition of fsh-induced estrogen production in cultured rat granulosa cells

The effects of glucocorticoids on the steroidogenesis of ovarian granulosa cells were investigated. Cortisol and dexamethasone inhibited the increase in aromatase activity induced by FSH in cultured rat granulosa cells. In the same cultures progesterone production was stimulated to a maximum of 167% of the control level. This differential effect of glucocorticoids on estrogen and progesterone production by the granulosa cells indicates that glucocorticoids exert specific inhibition of the induction of aromatase by FSH and do not cause a general suppression of granulosa cell activity. In contrast to their inhibition of the FSH induction of aromatase enzymes, glucocorticoids did not interfere with the activity of pre-existing aromatase enzymes. In granulosa cells containing fun aromatase activity, treatment with cortisol and dexamethasone did not inhibit aromatization of androstenedione to estrogens whereas two known aromatase inhibitors (dihydrotestosterone and 4-androstene-3, 6, 17-trione) were effective. These results indicate that the glucocorticoids exert a selective inhibition of the FSH-induction of aromatase activity in rat granulosa cells by a mechanism other than directly interfering with the aromatization reaction.     

Equine granulosa-theca cell tumors express inhibin alpha- and beta A-subunit messenger ribonucleic acids and proteins

The association of equine granulosa-theca cell tumors with atrophied contralateral ovaries and abnormal estrous cycles suggests that these tumors produce hormones that affect pituitary gonadotropin production. Because inhibin, a heterodimer protein secreted by granulosa cells, decreases FSH production, we examined the presence of inhibin alpha- and beta A-subunits and their mRNAs in ovarian tumors obtained from three mares. These tumors contained neoplastic cords and nodules, multiple fluid-filled cysts, and a predominance of neoplastic granulosa cells. Reduced proteins from tumor-conditioned media were analyzed by electrophoresis and immunoblotting using antibodies directed against peptide fragments of the alpha- and beta A-chains of porcine inhibin. Specific bands at 50-kDa and 36-kDa for the inhibin alpha-subunit and at 44 kDa and 13 kDa for the inhibin beta A-subunit were observed in these tumors. Northern blot hybridization of 32P-labeled rat inhibin alpha- and beta A-subunit complementary RNAs to total RNA from each tumor revealed predominant bands of activity in all three tumors at 1.5 and 7 kb for the alpha- and beta A-subunit mRNAs, respectively. These results demonstrate that equine granulosa-theca cell tumors express the mRNAs for inhibin alpha- and beta A-subunits and also secrete inhibin subunits that could potentially affect gonadotropin production in afflicted mares. Furthermore, cells derived from these tumors may provide a useful model for understanding inhibin gene regulation and ovarian tumorigenesis.     

Gonadotropin stimulation of testosterone production in primary culture of adult rat testis cells

Testis cells from adult hypophysectomized rats were cultured in serum-free medium. Treatment with human chorionic gonadotropin caused an initial increase and a subsequent decline in testosterone production, followed by a recovery in steroidogenesis on day 10 of culture. The recovery in testosterone production was inhibited by the addition of serum in culture media. Luteinizing hormone, dibutyryl adenosine-3′,5′-monophosphate or cholera toxin, but not follicle stimulating hormone or prolactin, stimulated testosterone production which was potentiated by a phosphodiesterase inhibitor. This is the first report of a primary culture of adult testis cells with retention of androgen synthetic capacity.     

Effects of serum on mammary epithelial proliferation in vitro during mammary gland development

The proliferative response of mammary gland epithelium from nonpregnant, pregnant, and lactating mice to mammary serum factor and insulin was studied in vitro. Mammary gland epiithelium from nonpregnant and lactating animals has a delayed proliferative response to mammary serum factor and insulin when compared to the response of epithelium from pregnant animals. The results show that as the animals go through pregnancy into lactation the mammary gland epithelium becomes less responsive to mammary serum factor while it retains its responsiveness to insulin. The concentration of mammary serum factor in sera from animals at various physiological stages is constant. Sera from hypophysectomized rats, on the other hand, show a 50% drop in mammary serum factor activity. This loss of activity cannot be reversed by injecting prolactin, 17-beta-estradiol, or growth hormone into the hypophysectomized animals. A hypothesis that the mammary gland is composed of two proliferative epithelial populations is developed, and the possible role of prolactin in stimulating DNA synthesis is discussed.