The effect of alkaline borohydride treatment onN-linked carbohydrates of glycoproteins

The effects of treatments of the glycoprotein ribonuclease-B, the proteins ribonuclease-A and myoglobin, and the glyco-amino acid GlcNAc(1-N) Asn with alkalil alkaline sodium borohydride, and aqueous sodium borohydride were systematically studied as a function of the concentration of the reagents, the temperature, and the length of the treatment. High-field¹H-NMR spectroscopy, chromatographic methods and amino-acid analysis were used to characterize products of the treatments of the various compounds. Our results indicate that mild alkaline borohydride treatment, as well as aqueous borohydride treatment alone, is capable of extensively degrading polypeptides and of partially releasing theN-linked glycans from ribonuclease-B. Initially, glycopeptides are produced, the peptide portion of which consists of several amino acids, which are further hydrolyzed to yield a mixture of glyco-asparagines and oligosaccharide-alditols in the ratio of 4:1. Strong alkaline borohydride treatment of ribonuclease-B is capable of completely releasing theN-linked carbohydrates as oligosaccharide-alditols.Abbreviation RNase ribonuclease     

Vaccination againstBoophilus microplus: Localization of antigens on tick gut cells and their interaction with the host immune system

Cattle have been vaccinated againstBoophilus microplus with antigens derived from partially fed female ticks. The immune response of the host lyses the gut cells of adult ticks, causing a reduction in the number, weight and reproductive capacity of engorging ticks. This response is different from the immunity that cattle acquire after repeated tick infestation. Evidence is presented that the antigens used in vaccination are located on the plasma membrane of the gut cells and it is unlikely that these antigens are secreted into the host during feeding. Vaccination using such concealed antigens may not encounter the mechanisms of immune evasion that parasites usually demonstrate.In-vitro assays suggest that vaccination immunity is not dependent on the need to stimulate cell-mediated responses. Immunoglobulin G alone, or with the aid of complement, is enough to damage tick gut.The normal function of the one protein antigen isolated so far is unknown but we speculate that it serves some vital function on the cell plasma membrane.     

Rapid detection of chromosome 16 inversion in acute nonlymphocytic leukemia, subtype M4: regional localization of the breakpoint in 16p

The pericentric inversion of chromosome 16 characteristic for acute nonlymphocytic leukemia, subtype M4, was detected in five patients by means of nonradioactive in situ hybridization of complete cosmids. First, five cosmids situated along the short arm of chromosome 16 were used to map the breakpoint of the inversion distal to the rare folate-sensitive fragile site FRA16A. Then, the use of two cosmids on either side of the breakpoint, combined with a probe specific for the centromeric region of chromosome 16, readily detected the inversion, even in poor metaphase spreads.     

Local anesthetic action of phentolamine on insect mechanoreceptors

1. The effect of phentolamine on the response properties of insect mechanoreceptors and on the conduction in their axons was examined using electrophysiological techniques. 2. Phentolamine blocked conduction of action potentials along axons, an effect which exhibited 3 characteristics typical of local anesthetics: the effect was frequency-dependent, reversible and varied for nerves with different diameters. 3. The concentration of phentolamine required to block axonal conduction (1-2 x 10(-3) M) was significantly higher than that required to abolish the response of receptors to mechanical stimulation (3-5 x 10(-4) M). 4. All mechanoreceptors that were examined in Locusta migratoria and Periplaneta americana were inactivated by phentolamine (Table 1). The type I receptors (chordotonal, campaniform and hair sensilla) were inactivated within 5-15 min following phentolamine application. The only type II receptor examined (forewing stretch-receptor) underwent a phase of repetitive discharge before being inactivated. 5. Tolazoline and metoclopramide inactivated, like phentolamine, mechanoreceptors at lower concentrations than necessary to block axonal conduction. However, yohimbine and chlorpromazine inactivated mechanoreceptors and blocked axonal conduction at similar concentrations. 6. These findings suggest that phentolamine affects sense-organ specific ionic processes that are more sensitive to the drug than the ionic processes along the axons.     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

Analysis of the Escherichia coli glycogen gene cluster suggests that catabolic enzymes are encoded among the biosynthetic genes

The nucleotide sequences of the Escherichia coli genome between the glycogen biosynthetic genes glgB and glgC, and 1170 bp of DNA which follows glgA have been determined. The region between glgB and glgC contains an open reading frame (ORF) of 1521 bp which we call glgX. This ORF is capable of coding for an M_r 56 684 protein. The deduced amino acid (aa) sequence for the putative product shows significant similarity to the E. coli glycogen branching enzyme, and to several different glucan hydrolases and transferases. The regions of sequence similarity include residues which have been reported to be involved in substrate binding and catalysis by taka-amylase. This suggests that the proposed product may catalyze hydrolysis or glycosyltransferase reactions. The cloned region which follows glgA contains an incomplete ORF (1149 bp), glgY, which appears to encode 383 aa of the N terminus of glycogen phosphorylase, based upon sequence similarity with the enzyme from rabbit muscle (47% identical aa residues) and with maltodextrin phosphorylase from E. coli (37% identical aa residues). Results suggest that neither ORF is required for glycogen biosynthesis. The localization of glycogen biosynthetic and degradative genes together in a cluster may facilitate the regulation of these systems in vivo.     

Tenascin interferes with fibronectin action

Primary chick embryo fibroblasts attach to a tenascin substrate, but remain rounded and do not spread out. The proportion between tenascin and fibronectin in mixtures used to coat the substrate determines the shape of the cells. Tenascin inhibits integrin-mediated chick fibroblast attachment to fibronectin, laminin, and the GRGDS peptide. Rat fibroblast attachment to fibronectin, but not to laminin, is inhibited by tenascin. A monoclonal antibody against tenascin, as well as its Fab fragments, is able to neutralize the inhibitory activity on cell attachment and is therefore assumed to mask the cell-binding site of tenascin. On electron micrographs showing this monoclonal antibody bound to tenascin, its epitope can be localized to the terminal knob at the distal ends of the tenascin arms.     

Serological recognition of HLA-DR allodeterminant corresponding to DNA sequence involved in gene conversion

HLA class 11 molecules were isolated from mouse L cells transfected with a DR gene and an allele, 52a, of locus DR III from an HLA-homozygous cell line, AVL, of the DR3 haplotype. The isolated molecules were found to possess a new allospecificity, named TR81. This specificity behaved allelic to the previously described DR III locus. The TR81 specificity was also present on the DR I gene product of the DR3 haplotype. The nucleotide sequence of the gene encoding TR81 differs from TR81-negative DR genes of the DRw52 family in only two codons, both located in the regions known to be involved in a gene conversion event. Consequently, the following conclusions can be formulated. (a) TR81 is a bi-locus specificity and allelic to TR22 only in its DR III locus localization. (b) The TR81 specificity is the phenotypic counterpart of the gene conversion event which led to the generation of the DR I gene of the DR3 haplotype. (c) One or both individual amino acid substitutions in the first domain of the DR chain are responsible for the TR81 allospecificity. (d) Since TR81 is expressed on the DR I chain of the DR3 haplotype, it is possible that TR81 and DR3 represent the same serological specificity.