Hormone-dependent processing of the avian progesterone receptor

Avian progesterone receptor exists as two forms, A and B, with molecular weights of 79,000 and 110,000 daltons, respectively. The origin and significance of these two forms is an area of active investigation and debate. Monoclonal antibodies produced against these two forms were used to examine receptor stability in cytosol and changes in the receptor forms induced by hormone binding. The lability of hormone binding at elevated temperatures is well documented. Analysis by Western blotting showed the receptor was stable in freshly prepared oviduct cytosol for 2 hr at 37°C, while hormone binding was lost within 30 min. However, loss of receptor through degradation was seen when cytosol was prepared from frozen tissue or when homogenization was excessive. Progesterone was injected into diethylstilbestrol-stimulated chicks to examine, in vivo, effects of hormone treatment on receptor forms in the cytosol and nuclear fractions. Progesterone treatment caused a time- and dose-dependent conversion of the A receptor to a form (A′) with a slower electrophoretic mobility. The cytosolic progesterone receptor was divided equally between the B and A forms, while the nuclear receptor was predominantly A′. The amount of nuclear receptor was consistently less than cytosolic receptor. Receptor phosphorylation was analyzed by incubating tissue minces with [³²P]orthophosphate with or without progesterone followed by immune isolation of receptor forms. Progesterone treatment caused a time-dependent increase in cytosol receptor phosphorylation which was evident after 5 min of treatment. This phosphorylation was observed with both the A and B receptor forms. The results indicate that receptor phosphorylation is a very early event during progesterone action.     

Defective production of anti-herpes simplex virus antibody by neonatal mice. Reconstitution with Ia+ macrophages and T helper lymphocytes from nonimmune adult syngeneic mice

Both neonatal humans and mice are exquisitely susceptible to severe HSV infection. We have now documented a profound defect in the ability of neonatal C57BL/6 mice to produce anti-HSV ADCC antibody. This ability is acquired over the first 2 to 4 wk of life. Reconstitution of neonatal mice by i.p. injection of peritoneal cells from adult nonimmune syngeneic mice both affords dose-dependent protection against lethal HSV infection and reconstitutes the antibody-production defect. By cell-separation techniques (adherence, nylon wool column purification, B cell panning) and cell ablation techniques (silica treatment, irradiation, anti-T cell, anti-Ia, anti-Lyt-1.2 and anti-Lyt-2.2 monoclonal antibodies plus complement treatment) the subpopulations involved in the antibody production reconstitution of neonatal mice by adult cells were identified. These include both an Ia+, radioresistant, adherent, silica-sensitive macrophage population and a nylon wool column-purified, radiosensitive, anti-T, anti-Lyt-1.2-sensitive helper T cell population. The latter cell may be substituted for by concanavalin A-stimulated lymphokine-containing spleen cell supernatants or human recombinant IL 2. In addition to reconstitution of ADCC antibody production, the same cell populations, or cells plus lymphokine-containing supernatants or IL 2, protected the newborn mice from lethal HSV infection. Further characterization of this system and of soluble replacement factors has implications for therapy or immunoprophylaxis of human neonates with, or at risk of, HSV infection.     

Analysis of fidelity mechanisms with eukaryotic DNA replication and repair proteins

We are investigating the mechanisms for producing or avoiding errors during DNA synthesis catalyzed by DNA replication and repair proteins purified from eukaryotic sources. Using assays that monitor the fidelity of a single round of DNA synthesis in vitro, we have defined the error frequency and mutational specificity of the four classes of animal cell DNA polymerases (alpha, beta, delta, gamma), and the fidelity of an SV40 origin-dependent DNA replication complex in extracts of HeLa cells.     

Rock-Climbing Injuries in Yosemite National Park

This study was presented in part at the annual meeting of the Wilderness Medical Society at Aspen, Colorado, in August 1986 and at “Mountain Medicine 1987,” Leavenworth, Washington, in November 1987.We questioned 220 injured rock climbers or their partners seen consecutively at the Yosemite (California) Medical Clinic over 3 ½ years regarding details of their accidents. Injury type and location were extracted from medical records and severity quantified. The National Park Service rescued 27% of the climbers. Injured climbers were characteristically male (88%) and experienced (mean 5.9 years) and typically fell while leading climbs (66%). Among 451 injuries, 50% were to the skin or subcutaneous tissues, while 28% involved the lower extremity and were predominantly fractures. In terms of each climber''s most severe injury (n = 220), 45% involved the lower extremities (30% from the ankle alone). Head injury or hypothermia caused 12 of 13 fatalities, showing the lowest case-fatality rate reported to date among injured climbers (6%). Rescue personnel successfully managed airways in victims of head injuries, anticipated and treated complications of hypothermia, and stabilized fractures. Victims requiring immediate extensive surgical intervention or blood transfusion usually died before rescue could be effected.     

Metal-free and metal-substituted cytochromes c. Use in characterization of the cytochrome c binding site.

The luminescent properties of metal-free, tin(IV) and zinc(II) cytochromes c have been used to characterize the interaction of cytochrome c with mitochondria and cytochrome oxidase. Diminution in the fluorescence yields of tin and zinc cytochrome c occur when these derivates bind to cytochrome oxidase or mitochondria. Based upon spectral overlap and quantum yield, the distance between the porphyrin rings of cytochrome a and cytochrome c is estimated according to Forster theory to be in the neighborhood of 3.5 nm. Measurements of the polarized emission of metal-free 'porphyrin' cytochrome c when bound to oriented layers of cytochrome c oxidase indicate that the porphyrin is bound obliquely to the plane of the oxidase layers with an angle of about 70 degrees C from heme plane to membrane plane. It is proposed that these data have significance for elucidation of electron transfer mechanisms.     

Histochemical classification based upon reaction types and its application to carbohydrate histochemistry

Summary A method of classification is presented, which divides histochemical visualization reactions into categories based on general reaction types. This scheme is dependent upon the reaction between two elements, the substrate and the probe. The substrate represents a tissue component(s) with one or more reactive groups that can combine directly with the probe. Alternatively, the substrate reactive groups are chemically modified (activation) with a suitable reagent before reaction with the probe. Probes are of three types: those that yield a coloured product, those that result in a colourless product, and those that produce a coloured product only after a further reaction.Methods used in carbohydrate histochemistry are divided into one, two and three probe reactions. Two probe reactions are further subdivided into sequences involving one or two coloured products (one and two dye sequences); three probe reactions into sequences involving one, two or three coloured products (one, two and three dye sequences). This classification permits the rationalization and organization of methods, and provides a framework for the examination of existing methods and the development of new ones.     

CHROMOSOME NUMBERS IN THE GENUS ANTHURIUM (ARACEAE) II

Chromosome numbers were determined for 86 Anthurium species. Fifty-one of these were newly determined with counts ranging from 2n = 24 to 66 and 30 being the most common. All known Anthurium chromosome numbers were summarized, and 43 taxonomic changes were made in the previous reports to reflect current taxonomy. In terms of somatic chromosome numbers, the numbers form four polyploid series of 20–40–60, 24–30–48–84, 28–56 and 30–60–90–ca. 124. Paleoaneuploidy, polyploidy and B-chromosomes are basic features of the genus, but subsequent recent aneuploidy is not. The exact nature of chromosome evolution in Anthurium remains to be elucidated.