UNC-11, a Caenorhabditis elegans AP180 Homologue, Regulates the Size and Protein Composition of Synaptic Vesicles

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.     

Decreased severity of potato common scab after foliar sprays of 3,5 -dichlorophenoxyacetic acid, a possible antipathogenic agent

Glasshouse tests on the potato cultivars Majestic and Maris Bard measured the effects of single early foliar sprays of ring-substituted phenoxyacetic acids (0.9 × 10–³ M) on the incidence of potato common scab, caused by soil-borne Streptomyces scabies. The most effective compound was 3,5-dichlorophenoxyacetic acid (3,5-D), which decreased scab by about 90%; its action was preventative, early sprays being more effective than late However, it slightly decreased yield, and increased the number of tubers per plant and the proportion of deformed tubers. The trichloro- and tetrachloro- acids containing the 3,5-dichloro- group also decreased scab, though to lesser extents; the most active of these was the 2,3,4,5-tetrachloro- acid, which had the same side effects as 3,5-D. The herbicide 2,4-D almost halved scab incidence, but seriously decreased yield. Other acids tested had little or no effect on scab. In tests against S. scabies in culture, 3,5-D was no more toxic than other disubstituted acids which were inactive, or weakly active, against the disease in vivo. 3,5-D may be an ‘antipathogenic agent’, preventing scab development by altering tuber metabolism.     

Quantifying and imaging NY-ESO-1/LAGE-1-derived epitopes on tumor cells using high affinity T cell receptors

Presentation of intracellular tumor-associated Ags (TAAs) in the context of HLA class I molecules offers unique cancer-specific cell surface markers for the identification and targeting of tumor cells. For most peptide Ags, the levels of and variations in cell surface presentation remain unknown, yet these parameters are of crucial importance when considering specific TAAs as targets for anticancer therapy. Here we use a soluble TCR with picomolar affinity for the HLA-A2-restricted 157-165 epitope of the NY-ESO-1 and LAGE-1 TAAs to investigate presentation of this immunodominant epitope on the surface of a variety of cancer cells. By single molecule fluorescence microscopy, we directly visualize HLA-peptide presentation for the first time, demonstrating that NY-ESO-1/LAGE-1-positive tumor cells present 10-50 NY-ESO-1/LAGE-1(157-165) epitopes per cell.     

Ribonucleic acid-protein cross-linking within the intact Escherichia coli ribosome, utilizing ethylene glycol bis[3-(2-ketobutyraldehyde) ether], a reversible, bifunctional reagent: synthesis and cross-linking within 30S and 50S subunits

We have used the reversible, bifunctional reagent ethylene glycol bis[3-(2-ketobutyraldehyde) ether] to cross-link RNA to protein within intact ribosomal subunits from Escherichia coli. Here we describe the synthesis of this compound (termed bikethoxal) and demonstrate its ability to form covalent attachments between RNA and protein in the 5S RNA-L18 complex and within 30S and 50S ribosomal subunits. The reagent is a symmetrical dicarbonyl compound and reacts with guanine in single-stranded RNA and with arginine in protein. RNA-protein cross-links generated with this reagent are stable, as demonstrated by the comigration of 35S-labeled ribosomal proteins with ribosomal RNA on neutrally buffered sodium dodecyl sulfate (SDS)-agarose gels. However, the cross-linked product is unstable in mildly basic conditions, allowing the identification of the linked macromolecules by conventional techniques. The reagent is potentially capable of cross-linking any combination of single-stranded RNA, single-stranded DNA, or protein; it should prove a useful probe of the RNA-protein proximities within the E. coli ribosome, since the SDS-agarose gel system we describe provides a rapid method of optimizing this RNA--protein cross-linking reaction.     

Characterization of temperature-sensitive mutants of bacteriophage PM2: Membrane mutants

Summary In an effort to understand the genetic regulation of membrane morphogenesis, twenty-nine temperature-sensitive mutants of the membrane-containing bacteriophage PM2 were isolated. Characterization at restrictive temperature revealed groups showing no lysis (Groups I–IV), partial lysis (Groups V–VIII), and full lysis (Groups IX–XII) of the host Pseudomonas BAL-31. When the cell lysis data are considered in conjunction with data on stimulation of viral DNA synthesis, at least six mutant groups are defined. Analysis by gel electrophoresis of the pattern of viral proteins synthesized under restrictive conditions further divides the mutants into twelve groups. Temperature shift experiments delineate early, intermediate and late mutants. Complementation data support some of these groupings. The observed low levels of complementation and recombination are discussed in terms of gene product/genome restriction, bound to the membrane at the site of infection.It is of particular interest to membrane morphogenesis that under restrictive conditions late mutants in Groups II, III and IV make empty-appearing vesicles inside the cell that are the size of virus membranes as seen in thin sections of cells in the electron microscope. Mutants ts 1 (Group II) and ts 12 (Group III) show defects in their ability to incorporate into membranes viral structural proteins sp 13 and sp 6.6. The possibility is discussed that either of these proteins control the size and shape of the viral membrane.     

Hydrolysis of beta-D-glucopyranosyl fluoride to alpha-D-glucose catalyzed by Aspergillus niger alpha-D-glucosidase

Aspergillus niger alpha-D-glucosidase, crystallized and free of detectable activity for beta-D-glucosides, catalyzes the slow hydrolysis of beta-D-glucopyranosyl fluoride to form alpha-D-glucose. Maximal initial rates, V, for the hydrolysis of beta-D-glucosyl fluoride, p-nitrophenyl alpha-D-glucopyranoside, and alpha-D-glucopyranosyl fluoride are 0.27, 0.75, and 78.5 mumol.min-1.mg-1, respectively, with corresponding V/K constants of 0.0068, 1.44, and 41.3. Independent lines of evidence make clear that the reaction stems from beta-D-glucosyl fluoride and not from a contaminating trace of alpha-D-glucosyl fluoride, and is catalyzed by the alpha-D-glucosidase and not by an accompanying trace of beta-D-glucosidase or glucoamylase. Maltotriose competitively inhibits the hydrolysis, and beta-D-glucosyl fluoride in turn competitively inhibits the hydrolysis of p-nitrophenyl alpha-D-glucopyranoside, indicating that beta-D-glucosyl fluoride is bound at the same site as known substrates for the alpha-glucosidase. Present findings provide new evidence that alpha-glucosidases are not restricted to alpha-D-glucosylic substrates or to reactions providing retention of configuration. They strongly support the concept that product configuration in glycosylase-catalyzed reactions is primarily determined by enzyme structures controlling the direction of approach of acceptor molecules to the reaction center rather than by the anomeric configuration of the substrate.