Isolation and characterization of a human T lymphocyte-associated glycoprotein (gp40)

The biosynthetic and structural characteristics of the human thymocyte/T cell antigen defined by the monoclonal antibody WT1 have been studied. WT1 identified a monomeric cell surface glycoprotein of Mr = 40,000 ( gp40 ). Cross-absorption experiments and two-dimensional gel analyses indicate that WT1 and another monoclonal antibody, 3A1, react with the same structure. This glycoprotein was asymmetrically inserted into the rough endoplasmic reticulum as a transmembrane structure. At this stage, the polypeptide chain possessed two N-linked, "high-mannose" type glycans; these were subsequently processed into endo-H-insensitive, complex oligosaccharides during intracellular transport to the cell surface. Inhibition of N-linked glycosylation with tunicamycin failed to block the processing of the nonglycosylated Mr = 29,000 polypeptide to a glycoprotein of Mr = 33,000. Cleavage of the mature Mr = 40,000 form with endo-F yielded a similar Mr = 33,000 product. The kinetics of synthesis of the Mr = 33,000 intermediate in conjunction with gal-NAc oligosaccharidase digestion indicated the presence of O-linked glycans in the mature cell surface WT1 antigen. The fully processed cell surface form of the polypeptide also contains covalently associated fatty acid, and was labeled by 32P phosphate, the predominantly labeled phosphoamino acid being phosphoserine. We also demonstrate biochemically that the reactivity of WT1 with cells from a few patients with acute myeloid leukemia reflects genuine expression of the gp40 structure on myeloid cells.     

A radiation hybrid panel for human Chromosome 6q

A panel of 63 radiation-reduced hybrids has been derived from a mouse cell line containing a neo-marked human Chromosome (Chr) 6, primarily to provide a resource for higher resolution localization of new markers. Hybrids were generated with radiation doses of 40–400 Gy, selected in G418, and were shown by PCR to contain the neo gene. PCR was also used to score the retention of 15 loci that map from 6q13 to q25.2 of the current consensus map plus six other loci assigned to 6q26-q27. An average retention frequency of 27.8% was observed, with the highest frequencies at D6S313 and D6S280 (63.5%) located near the centromere at 6q13, and at D6S283 (68.5%) at 6q16.3-q21, presumably close to the neo integration site. Lowest frequencies (4.8%) were observed for telomeric markers. All markers segregated independently except D6S297 and D6S193. Agreement and some improvement to the current consensus map of 6q was made by mapping 12 loci by the non-parametric statistical method of Falk. In addition, deletion mapping with informative hybrids allowed the ordering of six loci from 6q26 to q27 and permitted some integration of maps of this region.     

Effects of Storage on Viability and Efficacy of Granular Formulations of the Microbial Herbicides Alternaria alternata and Trematophoma lignicola

Alternaria alternata Pesta (gluten matrix) granules stored at 12% relative humidity were still viable and infective, giving total control of Amaranthus retroflexus after 24 months storage. Viability of the propagule in the granule was an important factor of the evaluation test and not just the viability of the granule. The concentration of inoculum in the granule was important, with 10 5 to 10 6 conidia g -1 being the ideal, since lower concentrations may not be able to out-compete the soil micro-flora. Even with soil application, the effect of local humidity was still important. Trematophoma lignicola was also formulated as microbial herbicide granules but the conidia were severely damaged in the production process.     

Behaviourally and genetically distinct populations of an invasive ant provide insight into invasion history and impacts on a tropical ant community

Populations of invasive species often exhibit a high degree of spatial and temporal variability in abundance and hence their effects on resident communities. Here, we examine behavioural, genetic and environmental factors that influence variation in populations of the yellow crazy ant, Anoplolepis gracilipes, on the remote Nukunonu Atoll of Tokelau, Pacific Ocean. Behavioural assays revealed high levels of aggression between two groups of yellow crazy ants from different islands, and genetic analysis confirmed the presence of two distinct populations with unique mitochondrial (mt)DNA haplotypes, designated A and D. The two populations likely resulted from two separate invasion events. The populations exhibited significant differences in abundance of A. gracilipes, with a mean sevenfold difference in relative abundance between the two main haplotypes. The higher density haplotype D population coexisted with 50% fewer other ant species and altered ant community composition. Vegetation composition was also significantly different on islands harbouring the two populations. The results suggest genetic differences could play a role in the spatial and temporal variation in the effect of the yellow crazy ant on a small oceanic atoll. We could not differentiate between genetic effects and effects of vegetation. However, our results indicate that spatial variability in behaviour and impacts within populations of invasive species could be in part due to genetic differences, and play a substantial role in influencing the outcome of biological invasions.     

Membrane-bound CC chemokine inhibitor 35K provides localized inhibition of CC chemokine activity in vitro and in vivo

CC chemokines mediate mononuclear cell recruitment and activation in chronic inflammation. We have shown previously that gene transfer using recombinant adenoviruses, encoding a soluble CC chemokine-binding protein of vaccinia virus 35K, can dramatically reduce atherosclerosis and vein graft remodeling in apolipoprotein E knockout mice. In this study, we report the development of a membrane-bound form of 35K (m35K), tagged with GFP, which allows for localized, broad-spectrum CC chemokine blockade. In vitro experiments indicate that m35K-expressing cells no longer undergo CC chemokine-induced chemotaxis, and m35K-expressing cells can locally deplete the CC chemokines RANTES (CCL5) and MIP-1alpha (CCL3) from supernatant medium. This sequestration of CC chemokines can prevent chemotaxis of bystander cells to CC, but not CX(3)C chemokines. Intraperitoneal injection of mice with an adenovirus-encoding m35K leads to a significant (44%) decrease in leukocyte recruitment into the peritoneal cavity in a sterile peritonitis model. Intravenous adenovirus-encoding m35K delivery leads to m35K expression in hepatocytes, which confers significant protection against liver damage (75% reduction in liver enzymes) in a Con A-induced hepatitis model. In summary, we have generated a membrane-bound CC chemokine-binding protein (m35K) that provides localized broad-spectrum CC chemokine inhibition in vitro and in vivo. m35K may be a useful tool to study the role of CC chemokines in leukocyte trafficking and block the recruitment of monocytes in chronic inflammation.     

Palmitoylation of the SNAP25 Protein Family: SPECIFICITY AND REGULATION BY DHHC PALMITOYL TRANSFERASES*

SNAP25 plays an essential role in neuronal exocytosis pathways. SNAP25a and SNAP25b are alternatively spliced isoforms differing by only nine amino acids, three of which occur within the palmitoylated cysteine-rich domain. SNAP23 is 60% identical to SNAP25 and has a distinct cysteine-rich domain to both SNAP25a and SNAP25b. Despite the conspicuous differences within the palmitoylated domains of these secretory proteins, there is no information on their comparative interactions with palmitoyl transferases. We report that membrane association of all SNAP25/23 proteins is enhanced by Golgi-localized DHHC3, DHHC7, and DHHC17. In contrast, DHHC15 promoted a statistically significant increase in membrane association of only SNAP25b. To investigate the underlying cause of this differential specificity, we examined a SNAP23 point mutant (C79F) designed to mimic the cysteine-rich domain of SNAP25b. DHHC15 promoted a marked increase in membrane binding and palmitoylation of this SNAP23 mutant, demonstrating that the distinct cysteine-rich domains of SNAP25/23 contribute to differential interactions with DHHC15. The lack of activity of DHHC15 toward wild-type SNAP23 was not overcome by replacing its DHHC domain with that from DHHC3, suggesting that substrate specificity is not determined by the DHHC domain alone. Interestingly, DHHC2, which is closely related to DHHC15, associates with the plasma membrane in PC12 cells and can palmitoylate all SNAP25 isoforms. DHHC2 is, thus, a candidate enzyme to regulate SNAP25/23 palmitoylation dynamics at the plasma membrane. Finally, we demonstrate that overexpression of specific Golgi-localized DHHC proteins active against SNAP25/23 proteins perturbs the normal secretion of human growth hormone from PC12 cells.