CD8+ cytolytic T cell clones derived against the Plasmodium yoelii circumsporozoite protein protect against malaria

Immunization of BALB/c mice with radiation-attenuated Plasmodium yoelli sporozoites induces cytotoxic T lymphocytes (CTL) specific for an epitope located within the amlno acid sequence 277-288 of the P. yoellicircumsporozoite (CS) protein. Several CD8 + CTL clones were derived from the spleen cells of sporozolte-immunlzed mice, all displaying an apparently identical epitope specificity. All the clones Induced high levels of cytotysls in vitro upon exposure to peptide-incubated MHC-compatlble target cells. The adoptive transfer of two of these clones conferred complete protection against sporozotte challenge to naive mice. This protection Is species and stage specific. Using P. yoelli specific ribosomal RNA probes to monitor the in vivo effects of the CTL clones, we found that their target was the intrahepatocytic stage of the parasite. The protective clones completely Inhibited the development of the liver stages of P. yoelli Some CTL clones were only partially Inhibitory in vivo, while others failed completely to alter liver stage development and to confer any detectable degree of protection. The elucidation of the effector mechanism of this CTL mediated protection against rodent malaria should facilitate the design of an effective malaria vaccine. From a broader perspective this model may provide further insight into the mechanlsm(s) of CTL mediated killing of intracellular non-viral pathogens in genera

P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34 � hematopoietic progenitor cells

Abstract. Selectins play a critical role in initiating leukocyte binding to vascular endothelium. In addition, in vitro experiments have shown that neutrophils use L-selectin to roll on adherent neutrophils, suggesting that they express a nonvascular L-selectin ligand. Using a L-selectin/IgM heavy chain (Ix) chimeric protein as an immunocytological probe, we show here that L-selectin can bind to neutrophils, monocytes, CD34 ÷ hematopoietic progenitors, and HL-60 and KG-1 myeloid cells. The interaction between L-selectin and teukocytes was protease sensitive and calcium dependent, and abolished by cell treatment with neuraminidase, chlorate, or O-sialoglycoprotein endopeptidase. These results revealed common features between leukocyte L-selectin ligand and the mucin-like P-selectin glycoprotein ligand 1 (PSGL-1), which mediates neutrophil rolling on P- and E-selectin. The possibility that PSGL-1 could be a ligand for L-selectin was further supported by the ability of P-selectin/Ix chimera to inhibit L-selectin/Ix S EVERAL adhesion molecules are involved in the regulation of leukocyte homing into tissues. Selectins initiate neutrophil rolling along vascular endothelium at sites of inflammation, whereas integrins and immunoglobulin-like adhesion molecules have a more important role in subsequent steps of leukocyte migration int

Type-I myosins promote actin polymerization to drive membrane bending in endocytosis

Clathrin-mediated endocytosis in budding yeast requires the formation of a dynamic actin network that produces the force to invaginate the plasma membrane against the intracellular turgor pressure. The type-I myosins Myo3 and Myo5 are important for endocytic membrane reshaping, but mechanistic details of their function remain scarce. Here, we studied the function of Myo3 and Myo5 during endocytosis using quantitative live-cell imaging and genetic perturbations. We show that the type-I myosins promote, in a dose-dependent way, the growth and expansion of the actin network, which controls the speed of membrane and coat internalization. We found that this myosin-activity is independent of the actin nucleation promoting activity of myosins, and cannot be compensated for by increasing actin nucleation. Our results suggest a new mechanism for type-I myosins to produce force by promoting actin filament polymerization

Effect of Regulatory Element DNA Methylation on Tissue-Type Plasminogen Activator Gene Expression

<div><p>Expression of the tissue-type plasminogen activator gene (t-PA; gene name <i>PLAT</i>) is regulated, in part, by epigenetic mechanisms. We investigated the relationship between <i>PLAT</i> methylation and <i>PLAT</i> expression in five primary human cell types and six transformed cell lines. CpG methylation was analyzed in the proximal <i>PLAT</i> gene promoter and near the multihormone responsive enhancer (MHRE) -7.3 kilobase pairs upstream of the <i>PLAT</i> transcriptional start site (TSS, -7.3 kb). In Bowes melanoma cells, the <i>PLAT</i> promoter and the MHRE were fully unmethylated and t-PA secretion was extremely high. In other cell types the region from -647 to -366 was fully methylated, whereas an unmethylated stretch of DNA from -121 to +94 was required but not sufficient for detectable t-PA mRNA and t-PA secretion. DNA methylation near the MHRE was not correlated with t-PA secretion. Specific methylation of the <i>PLAT</i> promoter region -151 to +151, inserted into a firefly luciferase reporter gene, abolished reporter gene activity. The region -121 to + 94 contains two well-described regulatory elements, a PMA-responsive element (CRE) near -106 and a GC-rich region containing an Sp1 binding site near +59. Methylation of double-stranded DNA oligonucleotides containing the CRE or the GC-rich region had little or no effect on transcription factor binding. Methylated CpGs may attract co-repressor complexes that contain histone deacetylases (HDAC). However, reporter gene activity of methylated plasmids was not restored by the HDAC inhibitor trichostatin. In conclusion, efficient <i>PLAT</i> gene expression requires a short stretch of unmethylated CpG sites in the proximal promoter.</p></div

The In Vivo Architecture of the Exocyst Provides Structural Basis for Exocytosis

The structural characterization of protein complexes in their native environment is challenging but crucial for understanding the mechanisms that mediate cellular processes. We developed an integrative approach to reconstruct the 3D architecture of protein complexes in vivo. We applied this approach to the exocyst, a hetero-octameric complex of unknown structure that is thought to tether secretory vesicles during exocytosis with a poorly understood mechanism. We engineered yeast cells to anchor the exocyst on defined landmarks and determined the position of its subunit termini at nanometer precision using fluorescence microscopy. We then integrated these positions with the structural properties of the subunits to reconstruct the exocyst together with a vesicle bound to it. The exocyst has an open hand conformation made of rod-shaped subunits that are interlaced in the core. The exocyst architecture explains how the complex can tether secretory vesicles, placing them in direct contact with the plasma membrane

Effect of DNA methylation on reporter gene activity from mutated <i>PLAT</i> promoters.

<p>Reporter gene activity was compared after transfection of HT1080 cells with pCpGLprom151 and promoter mutants. MutA: CpGs -121 and -106 mutated; mut B: CpGs -81, -51 and + 94 mutated; mutC: CpGs +27, +42, +50 and +59 mutated. Results are expressed as means ± SEM of four independent experiments.</p

Influence of DNA methylation on transcription factor binding to a double-stranded DNA containing the Sp1 site.

<p>The effect of oligonucleotide methylation on transcription factor binding was assessed using nuclear extracts from HeLa or HT1080 cells and by comparing the effect of different concentrations of unmethylated (C, concentrations given in nM) or methylated competitor oligonucleotides (MC) on the binding of transcription factors to a double-stranded IRD700-labeled unmethylated oligonucleotide GCCAC<b>CG</b>A<u>CCCCACCCCC</u>TGCCTGGA, which includes the Sp1 site at +62 (underlined) immediately preceded by one CpG (in bold). The analysis was done for cells cultured for 24h in culture medium in the absence or presence of 20 nM PMA. The effect of oligonucleotide methylation on transcription factor binding was assessed using nuclear extracts from HeLa cells (A) or HT1080 cells (B) and by comparing the effect of different concentrations of unmethylated (C, concentrations given in nM) or methylated competitor oligonucleotide (MC) on band intensities. Note that the methylated and unmethylated oligonucleotides had comparable efficacies in preventing transcription factor binding to the IRD700 labeled unmethylated Sp1 probe.</p

Methylation of the <i>PLAT</i> promoter and t-PA secretion in HT1080 cell clones.

<p>The methylation state of the promoter region was analyzed for eight HT1080 cell clones differing in their t-PA secretion and in non-cloned HT1080 cells. The figure gives the percentage methylation of the CpGs in the <i>PLAT</i> promoter region (between -647 and + 94, with respect to TSS1). Bar length’s are proportional to the percentage methylation for each position, with the dotted lines indicating 100% methylation. The position of TSS1 and of the promoter interaction sites with CRE, Sp1 and NF1 is indicated at the top of the figure. Secretion of t-PA expressed in ng per million cells over a 24h period by cells cultured in medium alone is given at the left of the figure.</p