Demonstration of five major glycoproteins in myelin and myelin subfractions.

Myelin was found to contain five major glycoproteins with molecular weights of 120000, 95000, 88000, 43000 and 38000. Light myelin contained only 5-7% of the amount of these glycoproteins in whole myelin, whereas heavy myelin and the membrane fraction contained amounts nearly identical with whole myelin. Since all the major and minor glycoproteins, with the exception of 120000-mol-wt. glycoprotein, were detected only after treating the myelin membrane with neuraminidase, N-acetylneuraminic acid is a terminal sugar residue in these glycoproteins.     

An Auxin-Responsive Promoter Is Differentially Induced by Auxin Gradients during Tropisms

We constructed a chimeric gene consisting of a soybean small auxin up RNA (SAUR) promoter and leader sequence fused to an Escherichia coli [beta]-glucuronidase (GUS) open reading frame and a 3[prime] untranslated nopaline synthase sequence from Agrobacterium tumefaciens. This chimeric gene was used to transform tobacco by Agrobacterium-mediated transformation. In R2 etiolated transgenic tobacco seedlings, GUS expression occurred primarily in elongation regions of hypocotyls and roots. In green plants, GUS was expressed primarily in the epidermis and cortex of stems and petioles, as well as in elongation regions of anther filaments in developing flowers. GUS expression was responsive to exogenous auxin in the range of 10-8 to 10-3 M. During gravitropism and phototropism, the GUS activity became greater on the more rapidly elongating side of tobacco stems. Auxin transport inhibitors and other manipulations that blocked gravitropism also blocked the asymmetric distribution of GUS activity in gravistimulated stems. Light treatment of dark-grown seedlings resulted in a rapid decrease in GUS activity. Light-induced decay in GUS activity was fully reversed by application of auxin. Taken together, our results add support for the formation of an asymmetric distribution of auxin at sites of action during tropism.     

Retinol-induced modification of the extracellular matrix of endothelial cells: Its role in growth control

Summary The growth of the endothelial cell (EC) is tightly regulated throughout the body. Many factors have been implicated in modulating EC growth including diffusible compounds, cell-to-cell interactions, and the extracellular matrix (ECM). Retinol, or vitamin A alcohol, has recently been shown to inhibit the growth of bovine capillary ECs, in vitro. Retinoids are known to modify ECM in other cell systems, and pure ECM components have been shown to effect EC growth rates. We, therefore, examined the role of the matrix in the retinol-induced inhibition of ECs. Cell-free matrices from control and vitamin A-treated ECs were prepared by removing cells with EGTA treatment after 7 d of culture. Matrix proteins were analyzed by solubilizing the matrices in 5M quanidine-HCl and performing Western blot analysis using specific antibodies to matrix proteins. In isolating the ECM, we observed that retinol-treated cultures of ECs were resistant to EGTA removal; retinol-treated ECs required twice the exposure time to EGTA to detach from their matrix than did controls cells. Western blot analysis of matrix proteins derived from control and retinol-treated EC cultures demonstrated a 1.6-fold increase in lamininβ chains and a 2.5-fold increase in fibronectin in the ECM of retinol-treated EC compared to control cell matrix. Functional properties of these matrices were assessed by plating control and Day 6 retinol-treated ECs onto the matrices and measuring attachment and growth by determining cell numbers at 24, 72, and 144 h. These studies revealed that control cells attached in greatest numbers to a control matrix whereas retinol-treated ECs preferentially attached to a matrix derived from retinol-treated cells. Furthermore, control ECs which grew rapidly on a control matrix were growth inhibited on a retinol-derived matrix. These data indicate that vitamin A treatment of ECs effects both their phenotype and influences the composition and the functional properties of their underlying ECM. These studies also demonstrate that alterations of the matrix are at least in part responsible for the growth inhibition of EC by retinol.     

The influence of flanking sequence on the O-glycosylation of threonine in vitro.

To investigate the influence of flanking amino acid sequence on the O-glycosylation of a single threonine residue in vitro, we have examined a series of 52 related peptides. The substrates were based upon a sequence from human von Willebrand factor which is known to be glycosylated in vivo (-6PHMAQVTVGPGL+5). Each residue of the parent peptide was substituted, in turn, with isoleucine, alanine, proline, glutamic acid, or arginine. Peptides were glycosylated using a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase purified 15,000-fold from bovine colostrum by chromatography on DEAE-Sephacel, SP-Sephadex, Sephacryl S-300, Affi-Gel Blue, and 5-mercuri-UDP-GalNAc thiopropyl-Sepharose. Single amino acid changes in the sequences flanking the threonine could profoundly alter the glycosylation of the substrate peptides. Substitution of any amino acid tested at positions +3, -3, and -2 markedly decreased O-glycosylation, as did the presence of a charged residue at position -1. The substitution of amino acids at the other positions of the peptide substrate had little effect on the incorporation of GalNAc. Statistical analysis of sequences flanking known glycosylated threonine and serine residues suggests that they should be glycosylated with equal efficiency in the same sequence context (O'Connell et al., 1991). However, the bovine colostrum transferase failed to glycosylate a peptide derived from human erythropoietin which contains a serine that is glycosylated in vivo (-5PPDAASAAPLR+5). When a threonine was substituted for the serine in this peptide (-5PPDAATAAPLR+5), the substrate proved to be an excellent acceptor of GalNAc. These observations indicate that although flanking amino acid sequence is important for the O-glycosylation of specific hydroxyamino acids, discrete threonine- and serine-specific transferases may exist.     

Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines.

Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ID13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromosomal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis of BPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPV DNA and of BPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachromosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism.     

The third subunit of desulfoviridin-type dissimilatory sulfite reductases.

In addition to the 50-kDa (alpha) and 40-kDa (beta) subunits, an 11-kDa polypeptide has been discovered in highly purified Desulfovibrio vulgaris (Hildenborough) dissimilatory sulfite reductase. This is in contrast with the hitherto generally accepted alpha 2 beta 2 tetrameric subunit composition. Purification, high-ionic-strength gel-filtration, native electrophoresis and isoelectric focussing do not result in dissociation of the 11-kDa polypeptide from the complex. Densitometric scanning of SDS gels and denaturing gel-filtration indicate a stoichiometric occurrence. A similar 11-kDa polypeptide is present in the desulfoviridin of D. vulgaris oxamicus (Monticello), D. gigas and D. desulfuricans ATCC 27774. We attribute an alpha 2 beta 2 gamma 2 subunit structure to desulfoviridin-type sulfite reductases. N-terminal sequences of the alpha, beta and gamma subunits are reported.     

Inwardly rectifying potassium current in rabbit osteoclasts: A whole-cell and single-channel study

Summary Ionic conductances of rabbit osteoclasts were investigated using both whole-cell and cell-attached configurations of the patch-clamp recording technique. The predominant conductance found in these cells was an inwardly rectifying K⁺ conductance. Whole-cell currents showed an N-shaped current-voltage (I–13;V) relation with inward current activated at potentials negative to E_K. When external K⁺ was varied, I-V curves shifted 53 mV/10-fold change in [K⁺]_out, as predicted for a K⁺-selective channel. Inward current was blocked by Ba²⁺ and showed a time-dependent decline at negative potentials, which was reduced in Na⁺-free external solution. Inward single-channel currents were recorded in the cell-attached configuration. Single-channel currents were identified as inward-rectifier K⁺ channels based on the following observations: (i) Unitary I-V relations rectified, with only inward current resolved. (ii) Unitary conductance () was 31 pS when recorded in the cell-attached configuration with 140 mm K⁺ in the pipette and was found to be dependent on [K⁺]. (iii) Addition of Ba²⁺ to the pipette solution abolished single-channel events. We conclude that rabbit osteoclasts possess inwardly rectifying K⁺ channels which give rise to the inward current recorded at negative potentials in the whole-cell configuration. This inwardly rectifying K⁺ current may be responsible for setting the resting membrane potential and for dissipating electrical potential differences which arise from electrogenic transport of protons across the osteoclast ruffled border.This work was supported by The Arthritis Society and the Medical Research Council of Canada. M.E.M.K. was supported by a fellowship, S.J.D. a development Grant and S.M.S. a scholarship from the Medical Research Council. We thank Dr. Zu Gang Zheng for help with scanning microscopy.     

Alternate splicing pathways of the immunoglobulin heavy chain transcript of a teleost fish,Ictalurus punctatus

Previously we sequenced a partial cDNA clone encoding the 3' region of the message for the membrane receptor form of the heavy (mu) chain of the channel catfish which indicated that the first transmembrane (TM1) exon is spliced directly to the C mu 3 exon and not into a cryptic site within the CH4 exon, as occurs in other vertebrates. Studies utilizing polymerase chain reaction analysis of mRNA and further analysis of cDNA clones now confirm that the only detectable splicing pattern used in micron production by the channel catfish utilizes this C mu 3----TM1 pathway of pre-mRNA splicing.