Localization and identification of nuclear radioactivity in the pituitary gland and genital tract after administering 3H-testosterone, 3H-dihydrotestosterone,or 3H-estradiol to male rhesus monkeys

Summary Target cells for testosterone, dihydrotestosterone, and estradiol in the pituitary gland and genital tract of the male primate were localized by thaw-mount autoradiography, and high performance liquid chromatography was used to identify the metabolites of these steroids in cell nuclei. Castrated rhesus monkeys were injected with ³H-testosterone, ³H-dihydrotestosterone, or ³H-estradiol and killed 60 min later. In the anterior pituitary gland, fewer cells were labeled and less radioactivity was taken up by cell nuclei following the administration of either ³H-testosterone (4% of pars distalis cells and 5 dpm/g DNA) or ³H-dihydrotestosterone (5% of cells and 13 dpm/g DNA) than following the administration of ³H-estradiol (43% of cells and 214 dpm/g DNA). Most of the radioactivity in nuclei was in the form of the unmetabolized parent compound (78–94%). In prostate, seminal vesicles, and penis, ³H-dihydrotestosterone was the predominant form of nuclear radioactivity following both ³H-testosterone (67–90%) and ³H-dihydrostestosterone (94–97%) administration, and both androgens labeled epithelial and smooth muscle cells. In contrast, ³H-estradiol was taken up in unchanged form, by cell nuclei of the genital tract and it labeled connective tissue fibroblasts, but not epithelial cells. Thus, the distributions of target cells for androgens and estrogens were clearly different in all these tissues, and the uptake of testosterone resembled that of its androgenic rather than that of its estrogenic metabolite.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

V region sequences of anti-DNA and anti-RNA autoantibodies from NZB/NZW F1 mice

The V region sequences of two anti-DNA (A52, D42) and two anti-RNA (D44, D444) autoantibodies, derived from lupus prone NZB/NZW F1 female mice, were determined by mRNA sequencing. The sequences had the following features: 1) there was no clear sequence relationship between anti-DNA and anti-RNA antibodies; 2) there were no major similarities between any of the L chain sequences and each VL gene segment belonged to a different mouse VK subgroup; 3) the H chains of the two anti-RNA antibodies showed closely related sequences of VH gene segments and very similar third complementarity determining regions (CDR3); 4) the H chains of the two anti-DNA antibodies had VH segments belonging to different VH gene families but had a unique and similar combination of D segments and junctional sequences, suggesting a common recognition element for Ag and/or for idiotypic regulation in the H chain CDR3; and 5) the VH gene segment of one anti-DNA antibody (D42) was found to be very similar to the VH gene segment of a CBA mouse hybridoma antibody (6G6) which binds to the environmental Ag phosphocholine. The three-dimensional structure of the Fv-region of the anti-DNA antibody (D42) was modeled by computer and a stretch of poly(dT), ssDNA was docked to a cleft in the antibody combining site, formed by the three H chain CDR and by CDR1 and CDR3 of the L chain. The cleft is characterized by a preponderance of arginine and tyrosine residues, lining both the walls and base of the cleft.     

The effect of established plants on recruitment in the annual forb Sinapis arvensis

Summary The germination response of Sinapis arvensis to the presence of established plants was investigated in a greenhouse experiment. Established conspecific and heterospecific plants were found to inhibit germination and reduce the probability of recruitment of those seeds that germinate. Established plants have no effect on seed mortality in the soil. Using a simple recruitment model, it is demonstrated that the combination of variance in germination time coupled with the interaction between buried seeds and established plants can generate density dependence. The implications of these results for community processes, such as succession, are discussed.     

X-ray crystal structures of the oxidized and reduced forms of the rubredoxin from the marine hyperthermophilic archaebacterium Pyrococcus furiosus.

The structures of the oxidized and reduced forms of the rubredoxin from the archaebacterium, Pyrococcus furiosus, an organism that grows optimally at 100 degrees C, have been determined by X-ray crystallography to a resolution of 1.8 A. Crystals of this rubredoxin grow in space group P2(1)2(1)2(1) with room temperature cell dimensions a = 34.6 A, b = 35.5 A, and c = 44.4 A. Initial phases were determined by the method of molecular replacement using the oxidized form of the rubredoxin from the mesophilic eubacterium, Clostridium pasteurianum, as a starting model. The oxidized and reduced models of P. furiosus rubredoxin each contain 414 nonhydrogen protein atoms comprising 53 residues. The model of the oxidized form contains 61 solvent H2O oxygen atoms and has been refined with X-PLOR and TNT to a final R = 0.178 with root mean square (rms) deviations from ideality in bond distances and bond angles of 0.014 A and 2.06 degrees, respectively. The model of the reduced form contains 37 solvent H2O oxygen atoms and has been refined to R = 0.193 with rms deviations from ideality in bond lengths of 0.012 A and in bond angles of 1.95 degrees. The overall structure of P. furiosus rubredoxin is similar to the structures of mesophilic rubredoxins, with the exception of a more extensive hydrogen-bonding network in the beta-sheet region and multiple electrostatic interactions (salt bridge, hydrogen bonds) of the Glu 14 side chain with groups on three other residues (the amino-terminal nitrogen of Ala 1; the indole nitrogen of Trp 3; and the amide nitrogen group of Phe 29). The influence of these and other features upon the thermostability of the P. furiosus protein is discussed.     

Immunoprecipitation of human adrenal microsomal antigen

Human adrenal microsomes have been labelled with 125I and immunoprecipitated with sera from patients with Addison's disease. The immunoprecipitates were then analysed by SDS-PAGE and autoradiography. 13 of the 23 sera from the Addison patients studied contained antibodies which reacted with a 55 kDa adrenal microsomal protein. The same 13 sera were also positive for adrenal antibodies as judged by immunofluorescence. The 55 kDa protein was not immunoprecipitated from placenta or thyroid microsomes by Addison sera. Furthermore, patients with Graves' disease or rheumatoid arthritis did not immunoprecipitate the 55 kDa protein from adrenal microsomes. Our studies suggest therefore that Addison sera contain antibodies to a 55 kDa adrenal specific protein which may well be the antigen observed on immunofluorescence.     

31P-NMR saturation transfer studies of aerobic Escherichia coli cells

31P-NMR measurements of saturation transfer have been used to measure the flux between Pi and ATP in Escherichia coli cells respiring on an endogenous carbon source. Measurements were made in the wild type and in cells genetically modified to give a 5-fold higher concentration of the F1F0-ATP synthase. The flux in the two cell types was not significantly different. This, together with studies using inhibitors specific for the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase and the ATP synthase, suggests that the observed flux arises predominantly from glycolytic rather than ATP synthase activity. Although this conclusion is in disagreement with previous experiments on E. coli, it is in agreement with recent experiments on yeast.     

Nucleotide sequences of five anti-lysozyme monoclonal antibodies.

The nucleotide sequences of the heavy and light chain immunoglobulin mRNAs derived from five hybridomas (Gloop 1-5) secreting IgGs specific for the loop region of hen egg lysozyme were determined. These monoclonal antibodies recognise three distinct but overlapping epitopes within the loop region. The sequences of two pairs of antibodies with indistinguishable fine specificities were similar in both chains whereas the sequences of antibodies of non-identical specificities were very different. It is proposed that the D-segments expressed in two of the antibodies (Gloop3 and Gloop4) are the products of one, or perhaps two, previously unidentified germ line D-genes. Gloop1 and Gloop2 use a D-segment previously identified in antibodies specific for the hapten 2-phenyloxazolone; however it is recombined in a different reading frame in the anti-lysozyme antibodies, producing a different amino acid sequence.     

The mechanism of action of 4-hydroxynonenal in cell injury

The effect of the C9 ketoaldehyde, 4-hydroxynonenal (HNE), a cytotoxic product of lipid peroxidation, on DNA, RNA and protein synthesis has been investigated in cells in culture. Macromolecular synthesis is powerfully inhibited by this agent which readily enters the lipid-rich membranes and is considerably more toxic than the polar ketoaldehyde, methyl glyoxal (MG). The entry of HNE into membranes lowers their glutathione GSH content. This is associated with an increased lipid peroxidation measured in vitro which is blocked by added GSH or alpha-tocopherol. It is proposed that this latter sequence of events is the underlying cause of the cytopathic effect of HNE in cells in culture.