Stereoscopic back-scattered electron imaging of silver-stained proteins in nucleoli

By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode and stereo-tilt in scanning electron microscopy (SEM), it is possible to observe the nucleus through the cell surface, the nucleolus, and the tri-dimensional distribution of the AgNOR-associated acidic proteins. In C3H10T1:2 cells and their 7-12-dimethylbenz--anthracene-treated transformants, the staining demonstrates several intranucleolar silver-staining granules (SSG), surrounded by a weakly staining region. The SSG may represent the fibrillar center (FC) and the weakly staining region, the fibrillar dense component (FD). This component can link several SSG together to form a rope-like structure. In cells with no visible nucleolus and inactive nucleolar organizer regions (NORs) the silver-staining granules are less numerous, close together and the presumed fibrillar dense components are not visible. The SSG are located more peripheraly, and the weakly staining region and the rope-like structure are less prominent in control cell nucleoli than in transformed cells with a comparatively high rate of RNA synthesis.     

Adenosine 5'-diphosphate as an allosteric effector of phosphorylase kinase from rabbit skeletal muscle

Equilibrium binding and activity studies indicate that adenosine 5'-diphosphate binds to phosphorylase kinase with high affinity at a site, or sites, distinct from the catalytic site. Equilibrium dialysis at pH 6.8 and 8.2, with and without Mg2+, and with phosphorylated and nonphosphorylated enzyme preparations revealed approximately 8 ADP binding sites per alpha 4 beta 4 gamma 4 delta 4 hexadecamer, with Kd values ranging from 0.26 to 17 microM. Decreasing the pH from 8.2 to 6.8 or removing the Mg2+ enhanced the affinity for ADP. At pH 6.8, ADP stimulated the phosphorylase conversion and autophosphorylation activities of the nonactivated enzyme. Analogs of ADP with modifications at the 2'-, 3'-, and 5'-positions allowed determination of structural requirements for the stimulation of activity. ADP seems to alter the conformation of the beta subunit because addition of the nucleotide inhibits its dephosphorylation by phosphoprotein phosphatase and its chemical cross-linking by 1,5-difluoro-2,4-dinitrobenzene. The binding affinities and effects of ADP suggest that it may function physiologically as an allosteric effector of phosphorylase kinase.     

Activation of human thymocytes via the 50KD T11 sheep erythrocyte binding protein induces the expression of interleukin 2 receptors on both T3+ and T3- populations

Recent studies have demonstrated that the 50KD T11 molecule is a surface component of a macrophage-independent alternative pathway of human T cell activation that is unrelated to the T3/Ti antigen-MHC receptor complex. Given the expression of T11 on all human thymocytes, it was of interest to determine whether they could be activated via this pathway. The triggering of T11 by monoclonal antibodies anti-T112 and anti-T113, directed at two unique epitopes on the molecule, induced IL 2 receptor expression on both T3+ and T3- thymocytes but did not induce IL 2 production. Consequently, in contrast to peripheral blood T cells, thymocytes did not proliferate in response to anti-T112 and anti-T113 in the absence of exogenous IL 2. These studies suggest that IL 2 receptor gene activation precedes IL 2 gene activation in T cell development. The ability of the alternative pathway of T cell activation to induce IL 2 receptor expression on T3- thymocytes implies that the T11 molecule may have an important role in early thymocyte ontogeny.     

Identification of the peptide bond cleaved during activation of human Clr

CNBr cleavage of unreduced proenzyme Clr yielded fragment CP2b, isolated by gel filtration and highpressure gel permeation chromatography. This fragment (˜ M_τ 55000) comprised at least 4 disulphidelinked peptides, which were separated by gel filtration after reduction and alkylation. Peptide CP2bRA4, overlapping the A- and B-chain regions in proenzyme Clr was digested by V8 staphylococcal protease, and the digest separated by reversed-phase HPLC. N-terminal sequence analysis of peptide CP2bRA4SP9 established that Clr activation involves the cleavage of a single Arg-Ile bond, located in the sequence: Gln-Arg-Gln-Arg-Ile-Ile-Gly-Gly     

Comparison of Three 18F-Labeled Butyrophenone Neuroleptic Drugs in the Baboon Using Positron Emission Tomography

The butyrophenone neuroleptics spiroperidol, benperidol, and haloperidol were radiolabeled with fluorine-18 and studied in baboon brain using positron emission transaxial tomography (PETT). Pretreatment of the baboon with a high pharmacological dose of (+)-butaclamol reduced the specifically bound component of radioactivity distribution in the striatum to approximately the radioactivity distribution found in the cerebellum. Comparative studies of brain distribution kinetics over a 4-h period indicated that either [18F]spiroperidol or [18F]benperidol may be suitable for specific labeling of neuroleptic receptors. In an 8-h study with [18F]spiroperidol, striatal radioactivity did not decline, suggesting that spiroperidol either has a very slow dissociation rate or that it binds irreversibly to these receptors in vivo. [18F]Haloperidol may not be suitable for in vivo PETT studies, because of a relatively high component of nonspecific distribution and a faster dissociation from the receptor. Analysis of 18F in plasma after injection of [18F]spiroperidol indicated rapid metabolism to polar and acidic metabolites, with only 40% of the total radioactivity being present as unchanged drug after 30 min. Analysis of the metabolic stability of the radioactively labeled compound in rat striatum indicated that greater than 95% of [18F]spiroperidol remains unchanged after 4 h.     

Some aspects of the natural history and food selection ofAvahi laniger

Two groups ofAvahi laniger were studied in the Forêt de Analamozoatra near Perinet in the eastern rainforest of Madagascar from August to October 1984. Overlap between the home ranges of neighbouring groups ofA. laniger was minimal. Group size ranged from one to four individuals with a median group size of two. In four out of ten groups a baby was born between August and September.A. laniger were most active after dusk and before dawn. They had an extended resting period around midnight. Their diet consisted mostly of leaves from at least 17 different plant species. They also ate flowers. Fruit eating was recorded twice. Leaves eaten had high contents of protein and sugar but did not contain alkaloids. The concentration of condensed tannins did not differ between food items and non-food items. There was no indication of competition with other prosimians that might explain their nocturnality.     

Use of purified antipeptide sera of selected specificity for the detection of the simian virus 40 large T antigen by western blotting

Western blotting was used as a powerful alternative to immunoprecipitation for the detection of the simian virus 40 (SV40) large tumor (T) antigen. After resolution by electrophoresis on a SDS-polyacrylamide gel of a [¹⁵S]methionine labeled crude extract from SV40 infected monkey kidney cells, the separated proteins were transferred electrophoretically on nitrocellulose paper. T antigen was detected on nitrocellulose strips by using for the first time, specific, purified antipeptide monoclonal antibodies directed against the N- and C-terminal portions of the molecule, and¹²⁵I-labeled Protein A.     

Morphogenic substances released by plant tissue cultures

The release of endogenous substances is a general phenomenon of plant tissue cultures, with some substances having significant developmental effects on the releasing tissues. Their systematic study was initiated with Nandina tissue cultures, and a yellow compound that accumulated in the culture medium was identified as the alkaloid, berberine. The rate of its release was related to the supplies of auxin, cytokinin, and nutrient salts. Addition of berberine \ HCl to nutrient media did not inhibit Nandina tissues, but suppressed shoot formation in Nicotiana stem segments. Growth of Nandina and Nicotiana callus, as well as rooting of Nicotiana stem segments, was promoted by alkaloid addenda.     

Age-related changes of the somatotrophs of the domestic fowl Gallus gallus

Summary The ultrastructure of the somatotrophs of the caudal pituitary of the domestic fowl was studied quantitatively. Two age groups of male chickens were compared: 4–6 weeks and 24–30 weeks post-hatching. With age, somatotrophs decreased from about 40% to about 30% of the pituitary cell population. Their volume density decreased similarly. Mean volume of a somatotroph was the same in young and adult animals. Because the granule volume density of the somatotrophs was unchanged, but the somatotroph volume density of the gland declined, the granule volume density of the caudal pituitary gland dropped in parallel with that of the somatotrophs. Thus the volume of the gland comprised of somatotroph granules fell about 32%: from 6.57% to 4.45%. This lowered pool of stored hormone may be linked to the lowered circulating levels of growth hormone found in older animals by other investigators.The granule volume density of the somatotrophs was unchanged but the numerical density approximately doubled; thus the mean granule size decreased by 47% with age. The relationship of the size reduction of the granules to the lowered plasma growth hormone levels is not understood at present.Supported in part by Hatch and State funds from the New Jersey State Agricultural Experimental Station and NSF grants PCM 8,0227,27 and PCM 8,302197.