Leishmania donovani: cell-surface heparin receptors of promastigotes are recruited from an internal pool after trypsinization

With the use of [3H]heparin, we recently demonstrated that Leishmania donovani promastigotes express a cell-surface receptor that is specific for the glycosaminoglycan heparin (Mukhopadhyay et al. 1989, The Biochemical Journal, 264, 517-525.). Treatment of the parasite with trypsin abolishes 75-90% of this [3H]heparin-binding activity. When trypsinized promastigotes were resuspended in fresh culture medium in the absence and presence of cycloheximide (10 micrograms/ml), approximately 25-30% of the original heparin-binding capacity was restored within 1 hr, indicating that recruitment of receptors from an internal pool occurred without de novo protein synthesis. Scatchard analysis of the regenerated receptor revealed that the number of regenerated binding sites per cell was 2.3 x 10(5); these sites have a binding affinity of 6.7 x 10(-7) M. Like the native heparin receptors on the surface of freshly isolated cells, the receptors recruited after trypsinization are also highly specific for heparin, as a 25-fold excess of four other glycosaminoglycans displaced less than 10% of bound [3H]heparin from the trypsinized cells. The structural requirements of the ligand heparin, namely the number of monosaccharide units and degree of sulfation, were compared for both the native and regenerated receptor: for both receptors, oversulfated polysaccharide heparin fragments of at least six to eight sugar residues were most efficient at displacing [3H]heparin. The concentrations of oligosaccharide fragments required to displace 50% of [3H]heparin were 0.32 and 0.035 microM for the hexa- and octasaccharides, respectively. Colloidal gold-labeled heparin was bound to promastigotes and visualized by electron microscopy. This analysis revealed that the heparin bound almost exclusively to the flagella of control cells (not subjected to trypsin) and those which had regenerated receptor after trypsinization. The physiological significance of this heparin-binding activity on the surface of promastigotes is discussed.     

PDGF stimulates transient phosphorylation of 180,000 dalton protein

Cell-free extracts of platelet-derived growth factor (PDGF) treated, density-arrested, quiescent BALB/c-3T3 cells are capable of phosphorylating a 180,000 dalton protein (PP180). The phosphorylation of PP180 was observed in SDS polyacrylamide gel electrophoresis profiles of Nonidet P-40 solubilized cell preparations that had been incubated with [gamma-32P]ATP. When quiescent BALB/c-3T3 cell cultures were incubated at 37 degrees C with PDGF, phosphorylation of PP180 in cell extracts could be detected after a 3-min exposure of the intact cells to PDGF, which was maximal after 10-15 minutes and had diminished by 30-60 min. PDGF stimulation of PP180 phosphorylation also was observed in extracts of cells that had been incubated with PDGF at 4 degrees C; however, in contrast to PDGF exposure at 37 degrees C, the ability of cell extracts to phosphorylate PP180 did not decrease even after 4 hr of cell exposure to PDGF at 4 degrees C. When cells exposed to PDGF at 4 degrees C were transferred to 37 degrees C for 30 min, the ability of cell extracts to phosphorylate PP180 decreased to a nonstimulated level. After cells stimulated by PDGF showed a diminished ability to phosphorylate PP180, immediate restimulation with PDGF did not induce the ability to phosphorylate PP180. Incubation for 11 hr at 37 degrees C was required before readdition of PDGF allowed observable phosphorylation of PP180 in cell extracts, but maximum PDGF stimulation of the phosphorylation of PP180 was found after the cells were incubated for 24 hr in culture conditions. The amount of the stimulation of PP180 phosphorylation was dependent on the concentration of PDGF. The stimulation of DNA synthesis by PDGF was correlated to the phosphorylation of PP180. This phosphorylation activity was not observed in extracts of cells that had been treated with epidermal growth factor (EGF), somatomedin C, insulin, plasma, or fibroblast growth factor (FGF). This novel experimental approach allows the investigation of a PDGF-stimulated phosphorylation activity in relation to the cell cycle and growth regulation.     

A large‐area,spatially continuous assessment of land cover map error and its impact on downstream analyses

Land cover maps increasingly underlie research into socioeconomic and environmental patterns and processes, including global change. It is known that map errors impact our understanding of these phenomena, but quantifying these impacts is difficult because many areas lack adequate reference data. We used a highly accurate, high‐resolution map of South African cropland to assess (1) the magnitude of error in several current generation land cover maps, and (2) how these errors propagate in downstream studies. We first quantified pixel‐wise errors in the cropland classes of four widely used land cover maps at resolutions ranging from 1 to 100 km, and then calculated errors in several representative “downstream” (map‐based) analyses, including assessments of vegetative carbon stocks, evapotranspiration, crop production, and household food security. We also evaluated maps’ spatial accuracy based on how precisely they could be used to locate specific landscape features. We found that cropland maps can have substantial biases and poor accuracy at all resolutions (e.g., at 1 km resolution, up to ～45% underestimates of cropland (bias) and nearly 50% mean absolute error (MAE, describing accuracy); at 100 km, up to 15% underestimates and nearly 20% MAE). National‐scale maps derived from higher‐resolution imagery were most accurate, followed by multi‐map fusion products. Constraining mapped values to match survey statistics may be effective at minimizing bias (provided the statistics are accurate). Errors in downstream analyses could be substantially amplified or muted, depending on the values ascribed to cropland‐adjacent covers (e.g., with forest as adjacent cover, carbon map error was 200%–500% greater than in input cropland maps, but ～40% less for sparse cover types). The average locational error was 6 km (600%). These findings provide deeper insight into the causes and potential consequences of land cover map error, and suggest several recommendations for land cover map users.     

Empirical Bayes Estimation and Prediction Using Summary-Level Information From External Big Data Sources Adjusting for Violations of Transportability

Large external data sources may be available to augment studies that collect data to address a specific research objective. In this article we consider the problem of building regression models for prediction based on individual-level data from an “internal” study while incorporating summary information from an “external” big data source. We extend the work of Chatterjee et al. (J Am Stat Assoc 111(513):107–117, 2006) by introducing an adaptive empirical Bayes shrinkage estimator that uses the external summary-level information and the internal data to trade bias with variance for protection against departures in the conditional probability distribution of the outcome given a set of covariates between the two populations. We use simulation studies and a real data application using external summary information from the Prostate Cancer Prevention Trial to assess the performance of the proposed methods in contrast to maximum likelihood estimation and the constrained maximum likelihood (CML) method developed by Chatterjee et al. (J Am Stat Assoc 111(513):107–117, 2006). Our simulation studies show that the CML method can be biased and inefficient when the assumption of a transportable covariate distribution between the external and internal populations is violated, and our empirical Bayes estimator provides protection against bias and loss of efficiency.     

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants.

Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negative-stain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VPI/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VPl, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 single-layered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.     

Multiple response elements in the Sex-lethal early promoter ensure its female-specific expression pattern.

The choice of sexual identity in somatic tissues of the fruit fly Drosophila melanogaster is determined early in embryogenesis by the X-chromosome-to-autosome (X/A) ratio. The system that signals the X/A ratio selects the sexual development pathway by determining the activity state of the binary switch Sex-lethal (Sxl). In 2X/2A animals, the X/A signalling system turns the Sxl gene on, ultimately activating an RNA-splicing autoregulatory feedback loop which serves to maintain the female state during the remainder of development. In 1X/2A animals, this autoregulatory feedback loop is not activated and the male state is subsequently maintained by the default splicing machinery. In the studies reported here, we have examined how the X/A signalling system controls the initial choice of sexual identity through its action on a special early embryonic Sxl promoter, Sxl-Pe. We show that in the early embryo, the activity of Sxl-Pe is controlled in a highly dose-sensitive fashion by the genes on the X chromosome that function as numerator elements and by genes located on the autosomes that function as denominator elements. Functional dissection of Sxl-Pe indicates that activating the promoter in females requires the cumulative action of multiple numerator genes which appear to exert their effects through reiterated cis-acting target sites in the promoter. Conversely, maintaining the promoter silent in males requires the repressive activities of denominator genes, and at least one of the denominator genes also appears to function through target sequences within the promoter.     

Inhibitory effects of tamoxifen and tumor necrosis factor α on human glioblastoma cells

We reported previously that tumor necrosis factor α (TNFα) inhibited proliferation and invasiveness of human malignant glial cells. Because tamoxifen, an estrogen antagonist, has also been shown to inhibit growth of such cells, we hypothesized that a combination of tamoxifen and TNFα might be more effective than either reagent alone. TNFα (1–100 ng/ml) or tamoxifen (80 ng/ml-2 μg/ml) alone inhibited proliferation of a human glioblastoma cell line (WITG3) in a dose-dependent fashion; in combination, tamoxifen and TNFα yielded additive growth inhibition. Apoptotic cells characterized by nuclear fragmentation were detectable after 48 h of TNFα or tamoxifen exposure and were significantly increased by combination treatment. In non-neoplastic human astroglia and fibroblasts, proliferation was unaffected by tamoxifen, and enhanced by TNFα as previously reported. Staurosporine (2–50 nM), which has been reported to augment the effects of TNFα, was less effective than tamoxifen against WITG3 and, in addition, was markedly inhibitory to non-neoplastic glial cells. Binding studies yielded no evidence of WITG3 estrogen or progesterone receptors, nor of tamoxifen effects on TNFα receptors. Data suggest that TNFα and tamoxifen in combination display growth-regulatory properties, which (a) are more inhibitory to human glioblastoma cells than either agent alone, (b) do not affect non-neoplastic glia, (c) do not require either estrogen/ progesterone receptors or alteration of external TNFα receptors, and (d) may involve apoptosis.     

Three-dimensional structural analysis of recombinant rotavirus-like particles with intact and amino-terminal-deleted VP2: implications for the architecture of the VP2 capsid layer.

Rotaviruses are the leading cause of severe infantile gastroenteritis worldwide. These viruses are large, complex icosahedral particles consisting of three concentric capsid layers enclosing a genome of eleven segments of double-stranded RNA (dsRNA). The amino terminus of the innermost capsid protein VP2 possesses a nonspecific single-stranded RNA and dsRNA binding activity, and the amino terminus is also essential for the incorporation of the polymerase enzyme VP1 and guanylyltransferase VP3 into the core of the virion. Biochemical and structural studies have suggested that VP2, and especially the amino terminus, appears to act as a scaffold for proper assembly of the components of the viral core. To locate the amino terminus of VP2 within the core, we have used electron cryomicroscopy and image reconstruction to determine the three-dimensional structures of recombinant virus-like particles that contain either full-length or amino-terminal-deleted forms of VP2 coexpressed with the intermediate capsid protein VP6. A comparison of these structures indicates two significant changes along the inner surface of VP2 in the structure lacking the amino terminus: a loss of mass adjacent to the fivefold axes and a redistribution of mass along the fivefold axes. Examination of the VP2 layer suggests that the proteins are arranged as dimers of 120 quasi-equivalent molecules, with each dimer extending between neighboring fivefold axes. Our results indicate that the amino termini of both quasi-equivalent VP2 molecules are located near the icosahedral vertices.     

Biochemical characterization of a smaller form of recombinant Norwalk virus capsids assembled in insect cells.

The expression of the single capsid protein of Norwalk virus (NV) in Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus results in the assembly of virus-like particles (VLPs) of two sizes, the predominant 38-nm, or virion-size VLPs, and smaller, 23-nm VLPs. Here we describe the purification and biochemical characterization of the 23-nm VLPs. The 23-nm VLPs were purified to 95% homogeneity from the medium of Sf9 cultures by isopycnic CsCl gradient centrifugation followed by rate-zonal centrifugation in sucrose gradients. The compositions of the purified 23- and 38-nm VLPs were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblots. VLPs of both sizes showed a doublet at 58 kDa, the size of the full-length capsid protein. Upon alkaline treatment, the 23-nm VLPs underwent dissociation into soluble intermediates that were able to reassemble into 23- and 38-nm VLPs upon dialysis, suggesting that the assembly of both types of structures has a common pathway. Antigenic and biochemical properties of the 38- and 23-nm VLPs were examined and found to be conserved. Immunoprecipitation assays using polyclonal and monoclonal antibodies indicated that immunodominant epitopes on the capsid protein as well as conformational epitopes are conserved in the two types of particles. The trypsin cleavage site at residue 227 was protected in the assembled particles of both sizes but exposed after alkaline dissociation. These results, and the conservation of the binding activity of both forms of recombinant NV VLPs to cultured cells (L. J. White, J. M. Ball, M. E. Hardy, T. N. Tanaka, N. Kitamoto, and M. K. Estes, J. Virol. 70:6589-6597, 1996), suggest that the tertiary folding of the capsid protein responsible for these properties is conserved in the two structures. We hypothesize that the 23-nm VLPs are formed when 60 units of the NV capsid protein assembles into a structure with T=1 symmetry.