Activation of a Microtubule-Associated Protein-2 Kinase by Insulin-Like Growth Factor-I in Bovine Chromaffin Cells

Treatment of bovine chromaffin cells with insulin-like growth factor-I (IGF-I) caused the activation of a protein kinase that phosphorylates microtubule-associated protein-2 (MAP-2) in vitro. Activation of MAP-2 kinase by IGF-I varied with the time of treatment (maximal at 10-15 min) and the concentration of IGF-I (maximal at 10 nM). The IGF-I-activated MAP-2 kinase was localized to the soluble fraction of chromaffin cell extracts and required Mg2+ for activity. The IGF-I-activated kinase also phosphorylated myelin basic protein, but had little or no activity toward histones or ribosomal S6 protein. To examine the role of protein tyrosine phosphorylation in the activation of the MAP-2 kinase, we isolated phosphotyrosine (PTyr)-containing proteins from chromaffin cells by immunoaffinity adsorption on anti-PTyr-Sepharose beads. Anti-PTyr-Sepharose eluates from IGF-I-treated cells showed increased MAP-2 kinase activity; thus, the MAP-2 kinase (or a closely associated protein) appears to be a PTyr-containing protein. Treatment of anti-PTyr-Sepharose eluates or crude chromaffin cell extracts with alkaline phosphatase significantly decreased kinase activity toward myelin basic protein, indicating that phosphorylation of the IGF-I-activated kinase is required for its activity.     

Purification of endogenous inhibitors of [3H]flunitrazepam binding from bovine brain

Endogenous substances which inhibited the binding of [³H]flunitrazepam ([³H]FNZ) to bovine synaptosomal membranes have been purified from the hot acetic acid extracts of the bovine brain. Three peaks of inhibitory activity were obtained by Sephadex G-10 gel chromatography. Two of the peaks (Peak 2, and Peak 3) which had lower molecular weights that that of peak 1 were identified as inosine and hypoxanthine by TLC methods. Another peak (Peak 1) was further purified to homogeneity using both cation and anion ion-exchange chromatography and the following two-step reversed-phase HPLC. The purified substance inhibited the [³H]FNZ binding dose-dependently and competitively but did not have an effect on the binding of the peripheral-type BZ ligand [³H]Ro 5-4864. It was also shown that the substance was heat-stable and resistant to proteolytic degradation (trypsin, -chymotrypsin, pronase). However, a significant loss of inhibitory activity to [³H]FNZ binding was observed after acid hydrolysis. Molecular weight estimates based on gel filtration methods were less than 500 dalton, and the maximal ultraviolet absorption peak was at 314 nm. These results suggest that this substance is a new endogenous ligand for the central BZ receptor and may play an important role in regulating the GABAergic tone in the central nervous system.     

Birth of lambs after in vitro maturation, fertilization, and coculture with oviductal cells.

Control ovine oocytes matured and fertilized in vitro were transferred to intermediate recipient ewes. After 5 days, 59% of eggs were recovered. Thirty-one (38%) reached morula/blastocyst stage. Twenty-one embryos at the morula or blastocyst stage were transferred to six recipient ewes, resulting in five pregnancies, of which four were maintained. Nine lambs were born (43%). In the experiment, 72 ooctyes matured and fertilized in vitro were cocultured for 5 days with sheep oviductal epithelial cells. Thirty-one eggs (43%) developed to the noncompacted morula stage. Transfer of 26 embryos to 11 recipient ewes resulted in two pregnancies (18%). Two male lambs were born. The result indicates that the coculture of in vitro matured and fertilized ovine eggs with sheep oviductal epithelial cells throughout the preimplantation period is compatible with further development to term.     

Stimulation of proliferation of bovine placental cells by products of activated mononuclear leukocytes

Summary Culture medium conditioned with concanavalin A-stimulated mononuclear leukocytes was tested for its ability to stimulate in vitro proliferation of bovine placental cells. The crude preparation of cytokines caused a dose-dependent increase in [³H]thymidine uptake into cells obtained by trypsinization of fresh bovine placentae and placental cell lines established from cellular outgrowths of long-term bovine placental cultures, but had no effect on growth of 3T3 fibroblasts. Growth of trypsinized placental cells was not enhanced by culture in the presence of interleukin-2, interferon-β₂, interferon-γ, or tumor necrosis factor-α. These results corroborate those of murine studies, suggesting a growth-promoting role for cytokines released into the maternal-fetal interface. Supported in part by a grant from th e Florida Dairy Farmers Milk Checkoff Fund and a grant from CIBA-GEIGY. This is Journal Series R-01079 of the Florida Agricultural Experiment Station.     

Laparoscopic investigation of the bovine ovary in the periovulatory phase of the cycle

Laparoscopy, in combination with a rapid radioimmunoassay for plasma-LH determination, has been used to predict and observe ovulation in heifers. Experiments on three animals with typical progesterone levels, LH levels and apex formation are described. Ovulation was observed from 25 h to 29 h after the beginning of LH rise and from 17 h to 19 h after LH peak. The LH peak lasted for 9 h to 11 h. Cow ovulation was observed and photographed. The preovulatory follicle, apex formation, ovulation and freshly ruptured follicles are illustrated. The results presented here demonstrate that laparoscopy could offer valuable diagnostic assistance in clinical veterinary medicine.     

FSH receptors in the bovine corpus luteum

Binding of follicle stimulating hormone (FSH) to a crude membrane fraction of bovine corpus luteum (CL) has been detected. This binding meets the usual criteria for a receptor based on specificity, time course of reaction and association constant (Ka = 8.5 x 10(10)M(-1)). Physiological studies with CL removed from heifers at specific times after estrus indicate that day-6 CL had the highest FSH binding. However, a correlation with physiological function was not obvious since some functional mid-cycle CL were high in progesterone and luteinizing hormone (LH) receptor but had nondetectable FSH receptor. Conversely, some late-cycle CL had low progesterone and LH receptor but significant quantities of FSH receptor.     

ATP and other purine nucleotides stimulate the inactivation of ornithine transcarbamylase by broken lysosomes

Calpain II, a high Ca2+-requiring form of Ca2+-dependent cysteine proteinase (EC 3.4.22.17), isolated from bovine lens was found to cleave actin and vimentin, two major cytoskeletal elements of the lens. Polyacrylamide gel electrophoresis revealed that actin (Mr 43 000) was broken down through intermediary products of approximate Mr 42 000 and 40 000, while vimentin (Mr 57 000) was rapidly cleaved into several fragments ranging from Mr 44 000 to 20 000. The cleavage was dependent on Ca2+ and could be blocked by calpastatin , a calpain-specific inhibitor. These findings suggest that calpain might play a role in age-related degradation of the lens cytoskeleton.     

Electrophoresis '82 : Advanced methods. Biochemical & clinical applications. Proceedings of the International Conference on Electrophoresis,Athens, April 1982 Edited by D. Stathakos Walter de Gruyter; Berlin, 1983 xvi + 867 pages. DM 260.00

A competitive solid-phase immunoassay for the determination of testosterone in serum samples using time-resolved fluorescence is described. The solid phase is a testosterone-3-(O-carboxymethyl)-oxime-ovalbumin conjugate coated to polystyrene microtiter strips. Europium-labelled polyclonal and monoclonal antibodies against testosterone-3-(O-carboxymethyl)-oxime-bovine serum albumin were compared. Their behavior was quite similar although the polyclonal antibody was more sensitive, giving a detection limit of 15 fmol testosterone per assay. Correlation with RIA was very good (r = 0.982 and y = ?0.150 + 0.969x).     

Effect of temperature on the reproduction of Glossina pallidipes, with reference to G. m. morsitans

The reproductive biology of G. pallidipes Austen was studied at 28°, 25° and 22° C. Experiments showed that incubation of puparia at 28° C resulted in sterility of both males and females. Incubation at 22° C resulted in a reduced fecundity of the females due to egg retention; the fertility of the males was not affected.Comparative studies with G. m. morsitans Westw. showed that G. m. morsitans puparia are less affected by a temperature of 28° C than are G. pallidipes puparia.

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Effet de la température sur la reproduction de Glossina pallidipes, avec référence à G. m. morsitans

Résumé Les productivités de G. pallidipes Austen élevés au laboratoire pendant tout leur cycle à 22, 25 et 28° C, ont été comparées.A 28° C, la vie intrapupale est réduite à environ 23 jours, contre 30 jours environ à 25° C; la survie des adultes est plus brève qu'à 25° C et les mouches ne s'accouplent pas. Les ovaires présentent une rétention d'oeufs et seulement 1/3 des mâles contient des spermatozoïdes mobiles. A 22° C, le cycle est considérablement prolongé, la vie intrapupale durant environ 40 jours. Les femelles s'accouplaient environ 14 jours après l'émergence. Les ovaires présentaient une rétention d'oeufs, bien que moins souvent qu'à 28° C. Les mâles contenaient des spermatozoïdes mobiles.Des expériences avec changements de température à différents moments du cycle ont montré que la stérilité des mâles et des femelles est provoquée par l'incubation de pupes de G. pallidipes à 28° C. La mensuration des ovocytes montre à 28° C un effet nocif sur leur maturation. Des observations sur les testicules dans les pupes révèlent, par comparaison avec 25° C, que l'enroulement des testicules et des spermatozoïdes est retardé à 28° C, tandis que la pigmentation des testicules est retardée à 22° C. Les pupes de G. m. morsitans sont moins affectées à 28° C que celles de G. pallidipes.

     

Detection of the messenger RNA coding for preproenkephalin A in bovine adrenal by in situ hybridization

The messenger RNA (mRNA) coding for the adrenal precursor of enkephalins (preproenkephalin-A) has been detected in bovine adrenal medulla cells using in situ hybridization with 32P-labelled preproenkephalin A (PPA) complementary DNA. In formaldehyde- and Carnoy-fixed tissue sections, an intense elective labelling restricted to the cells located at the periphery of the adrenal medulla can be detected after hybridization procedure, using X-ray film and classical autoradiographic procedure. Adequate controls show that this labelling is obtained only using PPA complementary DNA, inserted or not in its vector. Distribution of PPA mRNA appears identical to that of its immunoreactive end products, namely Met-enkephalin and BAM22 peptide, detected by immunohistochemistry. Norepinephrine, detectable using monoamine histofluorescence, appears restricted to the cells of the center of the gland unlabelled for PPA mRNA and its end-products. Cultured bovine adrenomedullary cells that exhibited enkephalin immunoreactivity also contain PPA mRNA located in their cytoplasm.