Purification and characterization of human liver dehydroepiandrosterone sulphotransferase.

A BAL17 B lymphoma cell line bearing mu and delta chains on its surface behaves in a similar manner to normal mature B cells in terms of initial biochemical transmembrane signalling [Mizuguchi, Beaven, Ohara & Paul (1986) J. Immunol. 137, 2162-2167; Mizuguchi, Yong-Yong, Nakabayashi, Huang, Beaven, Chused & Paul (1987) J. Immunol. 139, 1054-1059]. Therefore the effects of protease inhibitors on increases in inositol phospholipid metabolism and intracellular free calcium concentration ([Ca2+]i) were examined. We show that the serine protease inhibitors Tos-Phe-CH2Cl (1-chloro-4-phenyl-3-L-tosylamidobutan-2-one-, TPCK) and Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one; TLCK) inhibit anti-IgM-mediated accumulation of inositol phosphates in a dose-dependent manner. InsP3 production induced by anti-IgM is also inhibited by pretreatment with Tos-Lys-CH2Cl or Tos-Phe-CH2Cl. Tos-Lys-CH2Cl- Tos-Phe-CH2Cl-mediated inhibition is not overcome by high concentrations of anti-IgM. Moreover, anti-IgM-mediated increases in [Ca2+]i are inhibited by pretreatment of the cells with these inhibitors. However, increases in inositol phospholipid metabolism caused by NaF, an activator of guanine-nucleotide-binding proteins (G-proteins), are approx. 10-fold more resistant to Tos-Lys-CH2Cl and Tos-Phe-CH2Cl inhibition compared with anti-IgM-induced changes. Furthermore, NaF-induced increases in [Ca2+]i are not inhibited by Tos-Lys-CH2Cl or Tos-Phe-CH2Cl pretreatment, suggesting that the inhibitors act at a step proximal to phospholipase C activation. The Tos-Lys-CH2Cl or Tos-Phe-CH2Cl treatment does not change the membrane IgM density as measured by flow cytometry, indicating that the active site of the inhibitors is distal to the membrane IgM molecule. These results indicate that serine proteases may be involved in coupling the receptor cross-linkage to G-protein.     

Comparative 113Cd-n.m.r. studies on rabbit 113Cd7-, (Zn1,Cd6)- and partially metal-depleted 113Cd6-metallothionein-2a.

Rabbit 113Cd7-metallothionein-2a (MT) contains two metal-thiolate clusters of three (cluster B) and four (cluster A) metal ions. The 113Cd-n.m.r. spectrum of 113Cd6-MT, isolated from 113Cd7-MT upon treatment with EDTA, is similar to that of 113Cd7-MT, but the cluster B resonances are lower in intensity, suggesting its co-operative metal depletion. (Zn1,113Cd6)-MT, formed upon addition of the Zn(II) ions to 113Cd6-MT, shows 113Cd-n.m.r. features characteristic of cluster B populations containing both Cd(II) and Zn(II) ions. The overall intensity gain of the mixed cluster B resonances per Cd as to those in 113Cd6- and 113Cd7-MT suggests a stabilization effect of the bound Zn(II) ions upon the previously established intramolecular 113Cd exchange within this cluster.     

Androgen metabolism in the human epididymis. Effect of in vivo estrogen administration

Androgen metabolism in human epididymis was studied by incubating tissue fragments with isotopically labeled testosterone (T) and androstenedione (A) under batch and superfusion conditions. Epididymides were obtained from 16 patients with prostatic cancer, 5 of them treated with diethylstilbestrol (2.5 mg/d) for several months prior to castration. Results from batch incubations with [3H]T (100 nM) for 2 h at 25 degrees C indicated a markedly lower 5 alpha-reductase activity in tissues from estrogen-treated patients, as evaluated by measuring the amounts of radioactive 5 alpha-dihydrotestosterone, 5 alpha-androstanediols and 5 alpha-androstanedione present in tissue and medium at the end of the incubation period. Superfusion experiments confirmed this estrogen effect and also showed a shift of the interconversion between A and T towards the reductive direction and a diminished tissue retention of DHT after estrogen treatment. These effects may contribute to the marked regression of the epididymal epithelium that was noted in the estrogen-treated patients, which is thought to be mainly the result of the inhibition of androgen biosynthesis caused by chemical hypophysectomy.     

Sequences required for enhancer blocking activity of scs are located within two nuclease-hypersensitive regions.

The Drosophila 87A7 heat shock locus is bordered, on the proximal and distal sides, by two special chromatin structures, scs and scs'. Each structure is characterized by two sets of nuclease-hypersensitive sites, located within moderately G/C-rich DNA, flanking an A/T-rich nuclease-resistant region. scs and scs' have been shown to insulate a white reporter gene from position effects and to prevent enhancer-promoter interactions. These and other properties suggest scs and scs' might function as chromatin domain boundaries. To identify the DNA sequences which are essential for the insulating activity of scs we used an enhancer blocking assay based on the white gene. Sequences capable of suppressing activation of white by its upstream enhancer elements reside within a 900 bp DNA fragment corresponding to the scs chromatin structure. Within this region, DNA fragments associated with the two nuclease-hypersensitive regions are essential for full enhancer blocking activity, while the central A/T-rich region is dispensable. Deletions which remove part of the hypersensitive regions result in intermediate levels of white activity. Insulating activity can, however, be reconstituted by multimerizing DNA fragments from either hypersensitive region. Our results suggest that the scs boundary is assembled from a discrete number of functionally redundant DNA sequences located within both hypersensitive regions and that boundaries act by decreasing the frequency of enhancer-promoter interactions. We also show that certain types of position effects, like those involved in dosage compensation, are not efficiently blocked by scs.     

The automating skeletal and muscular mechanisms of the avian wing (Aves)

The avian wing possesses the ability to synchronize flexion or extension of the elbow and wrist joints automatically. Skeletal and muscular mechanisms are involved in generating this phenomenon. The drawing-parallels action of the radius and ulna coordinates the movements of the forearm with the carpus. Movement of the radius along the length of the forearm isnot dependent on the shape disparity between the dorsal and ventral condyles of the humerus, nor is it generated by the shape of the dorsal condyle itself. Instead, shifting of the radius toward the wrist occurs during humeroulnar flexion when the radius, being pushed by muscles toward the ulna, is deflected off theIncisura radialis toward the wrist. Movement of the radius toward the elbow occurs during the latter stages of humeroulnar extension when, as the dorsal condyle of the humerus and the articular surface of the ulna's dorsal cup roll apart, the radius gets pulled by the humerus and its ligaments away from the wrist. Synchronization of the forearm with the manus is accomplished by twojoint muscles and tendons.M. extensor metacarpi radialis and the propatagial tendons act to extend the manus in unison with the forearm, whileM. extensor metacarpi ulnaris helps these limb segments flex simultaneously.M. flexor carpi ulnaris, in collaboration with the drawing-parallels mechanisms, flexes the carpus automatically when the elbow is flexed, thereby circumducting the manus from the plane of the wing toward the body. In a living bird, these skeletal and muscular coordinating mechanisms may function to automate the internal kinematics of the wing during flapping flight. A mechanized wing may also greatly facilitate the initial flight of fledgling birds. The coordinating mechanisms of the wing can be detected in a bird's osteology, thereby providing researchers with a new avenue by which to gauge the flight capabilities of avian fossil taxa.     

Polypeptide synthesis catalyzed by p-hydroxymercuribezoate-modified ribosomes

The stimulation of poly(U)-directed polyphenylalanine synthesis produced by modification ofEscherichia coli ribosomes withp-hydroxymercuribenzoate, at low molar ratios of reagent to ribosomes, is due to an increase in the average chain length of polyphenylalanine synthesized, and not to the activation of inactive ribosomes. At a higher molar ratio ofp-hydroxymercuribenzoate to ribosomes, which produces no overall change in activity, approximately 50% of the active ribosomes present in the untreated preparation have been completely inactivated, and the remaining active ones, like the ribosomes of the stimulated preparation, synthesize polyphenylalanine at an increased rate as compared with the untreated ribosomes.Abbreviations pHMB p-hydroxymercuribenzoate - SucNBr N-bromosuccinimide     

Structure of the yeast ribosomes. Proteins associated with the rRNA.

Polyamines have been shown to bind to doubled stranded regions of rRNA [3]. Therefore, ribosomal proteins that can be cross linked to these molecules in the ribosomes structure must be bound to or located in the vicinity of the RNA. This technique is the first to yield results on the proteins associated with the rRNA in the eukaryotic ribosome where the lack of purified ribosomal proteins does not allow the use of direct binding studies as in bacterial systems. Proteins S7, S10, S13, S21, S22 and S27 in the small subunit and L2/3, L5, L10/12, L19/20, L22, L23, L36/37, L42 and L43' in the large subunit are labelled when cross linked to [14C]spermidine using 1,5-difluoro 2,4-dinitrobenzene and are good candidates to be RNA-binding proteins in ribosomes from Saccharomyces cerevisiae.