Transfected Drosophila cells as a probe for defining the minimal requirements for stimulating unprimed CD8+ T cells

Stimulation of naive T cells by antigen-presenting cells (APC) is thought to involve two qualitatively different signals: signal one results from T-cell receptor (TCR) recognition of antigenic peptides bound to major histocompatibility complex (MHC) molecules, whereas signal two reflects contact with one or more costimulatory molecules. The requirements for stimulating naive T cells were studied with MHC class I-restricted CD8+ T cells from a T-cell receptor transgenic line, with defined peptides as antigen and transfected Drosophila cells as APC. Three main findings are reported. First, stimulation of naive T cells via signal one alone (MHC plus peptide) was essentially nonimmunogenic; thus T cells cultured with peptides presented by MHC class I-transfected Drosophila APC lacking costimulatory molecules showed little or no change in their surface phenotype. Second, cotransfection of two costimulatory molecules, B7-1 and intercellular adhesion molecule 1 (ICAM-1), converted class I+ Drosophila cells to potent APC capable of inducing strong T-proliferative responses and cytokine (interleukin 2) production. Third, B7-1 and ICAM-1 acted synergistically, indicating that signal two is complex; synergy between B7-1 and ICAM-1 varied from moderate to extreme and was influenced by both the dose and affinity of the peptide used and the parameter of T-cell activation studied. Transfected Drosophila cells are thus a useful tool for examining the minimal APC requirements for naive T cells.

Structural requirements for antigen presentation by mouse CD1

The structural basis for the T cell response to glycolipid antigens (Ags) remains poorly understood. T lymphocytes autoreactive for mouse CD1 (mCD1.1) or reactive for the glycosphingolipid αgalactosylceramide (α-GalCer) presented by mCD1.1 have been described previously. In this paper it is shown that mutations at the top of the α helices and in the bottom of the Ag-binding groove can disrupt both mCD1.1 autoreactivity and α-GalCer recognition. The locations of the positions that affect T cell responses indicate that recognition of mCD1.1 is not likely to be unconventional or superantigen-like. Furthermore, the effects of the bottom of the pocket mutation suggest that the autoreactive response could require an autologous ligand, and they indicate that α-GalCer binds to the groove of mCD1.1, most likely with the shorter 18-carbon hydrophobic chain in the A′ pocket. Natural killer T cell hybridomas with identical T cell antigen receptor (TCR) α chains and different β chains respond differently to α-GalCer presented by mCD1.1 mutants. This finding indicates a role for TCR β in defining natural killer T cell specificity, despite the more restricted diversity of the α chains in these cells. Overall, the data are consistent with a mode of lipoglycan recognition similar to that proposed for glycopeptides, in which the TCR α and β chains survey a surface composed of both mCD1.1 and the carbohydrate portion of α-GalCer.

Reconstitution of HIV-1 Rev nuclear export: Independent requirements for nuclear import and export

The Rev protein of HIV-1 actively shuttles between nucleus and cytoplasm and mediates the export of unspliced retroviral RNAs. The localization of shuttling proteins such as Rev is controlled by the relative rates of nuclear import and export. To study nuclear export in isolation, we generated cell lines expressing a green fluorescent protein-labeled chimeric protein consisting of HIV-1 Rev and a hormone-inducible nuclear localization sequence. Steroid removal switches off import thus allowing direct visualization of the Rev export pathway in living cells. After digitonin permeabilization of these cells, we found that a functional nuclear export sequence (NES), ATP, and fractionated cytosol were sufficient for nuclear export in vitro. Nuclear pore-specific lectins and leptomycin B were potent export inhibitors. Nuclear export was not inhibited by antagonists of calcium metabolism that block nuclear import. These data further suggest that nuclear pores do not functionally close when luminal calcium stores are depleted. The distinct requirements for nuclear import and export argue that these competing processes may be regulated independently. This system should have wide applicability for the analysis of nuclear import and export.

Temperature, template topology, and factor requirements of archaeal transcription

Although Archaea are prokaryotic and resemble Bacteria morphologically, their transcription apparatus is remarkably similar to those of eukaryotic cell nuclei. Because some Archaea exist in environments with temperatures of around 100°C, they are likely to have evolved unique strategies for transcriptional control. Here, we investigate the effects of temperature and DNA template topology in a thermophilic archaeal transcription system. Significantly, and in marked contrast with characterized eucaryal systems, archaeal DNA template topology has negligible effect on transcription levels at physiological temperatures using highly purified polymerase and recombinant transcription factors. Furthermore, archaeal transcription does not require hydrolysis of the β-γ phosphoanhydride bond of ATP. However, at lower temperatures, negatively supercoiled templates are transcribed more highly than those that are positively supercoiled. Notably, the block to transcription on positively supercoiled templates at lowered temperatures is at the level of polymerase binding and promoter opening. These data imply that Archaea do not possess a functional homologue of transcription factor TFIIH, and that for the promoters studied, transcription is mediated by TATA box-binding protein, transcription factor TFB, and RNA polymerase alone. Furthermore, they suggest that the reduction of plasmid linking number by hyperthermophilic Archaea in vivo in response to cold shock is a mechanism to maintain gene expression under these adverse circumstances.

Structural requirements for peptidic antagonists of the corticotropin-releasing factor receptor (CRFR): Development of CRFR2β-selective antisauvagine-30

Different truncated and conformationally constrained analogs of corticotropin-releasing factor (CRF) were synthesized on the basis of the amino acid sequences of human/rat CRF (h/rCRF), ovine CRF (oCRF), rat urocortin (rUcn), or sauvagine (Svg) and tested for their ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1), or mouse CRF receptor, type 2β (mCRFR2β). Furthermore, the potency of CRF antagonists to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRFR1 (HEK-rCRFR1 cells) or mCRFR2β (HEK-mCRFR2β cells) was determined. In comparison with astressin, which exhibited a similar affinity to rCRFR1 (Kd = 5.7 ± 1.6 nM) and mCRFR2β (Kd = 4.0 ± 2.3 nM), [dPhe11,His12]Svg(11–40), [dLeu11]Svg(11–40), [dPhe11]Svg(11–40), and Svg(11–40) bound, respectively, with a 110-, 80-, 68-, and 54-fold higher affinity to mCRFR2β than to rCRFR1. The truncated analogs of rUcn displayed modest preference (2- to 7-fold) for binding to mCRFR2β. In agreement with the results of these binding experiments, [dPhe11,His12]Svg(11–40), named antisauvagine-30, was the most potent and selective ligand to suppress agonist-induced adenylate cyclase activity in HEK cells expressing mCRFR2β.

Cis-acting requirements in flanking DNA for the programmed elimination of mse2.9: a common mechanism for deletion of internal eliminated sequences from the developing macronucleus of Tetrahymena thermophila

During macronuclear development in the ciliated protozoan Tetrahymena thermophila, extensive DNA deletions occur, eliminating thousands of internal eliminated sequences (IESs). Using an rDNA-based transformation assay we have analyzed the role during DNA deletion of DNA flanking mse2.9, an IES within the second intron of a gene encoding an as yet incompletely characterized protein. We establish that a cis-acting sequence for mse2.9 deletion acts at a distance to specify deletion boundaries. A complex sequence element necessary for efficient and accurate mse2.9 deletion is located in the region 47–81 bp from the right side of mse2.9. The ability of a variety of IES flanking sequences to rescue a processing deficient mse2.9 construct indicates that some cis-acting signal is shared among different IESs. In addition, the short intronic sequence that flanks mse2.9 is able to direct efficient and accurate processing. Despite no obvious sequence similarity between mse2.9 and other IESs, we suggest that a common mechanism is used to delete different families of IESs in Tetrahymena.

Structural Requirements of Tom40 for Assembly into Preexisting TOM Complexes of Mitochondria

Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex). To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein. Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly. We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus. Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex. Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro. A model is proposed for the integration of Tom40 subunits into the TOM complex.

Shifts of Intracellular pH Distribution as a Part of the Signal Mechanism Leading to the Elicitation of Benzophenanthridine Alkaloids1 : Phytoalexin Biosynthesis in Cultured Cells of Eschscholtzia californica

Cultured cells of Eschscholtzia californica (Californian poppy) respond to a yeast elicitor preparation or Penicillium cyclopium spores with the production of benzophenanthridine alkaloids, which are potent phytoalexins. Confocal pH mapping with the probe carboxy-seminaphthorhodafluor-1-acetoxymethylester revealed characteristic shifts of the pH distribution in challenged cells: within a few minutes after elicitor contact a transient acidification of cytoplasmic and nuclear areas occurred in parallel with an increase of the vacuolar pH. The change of proton concentration in the vacuole and in the extravacuolar area showed a nearly constant relation, indicating an efflux of vacuolar protons into the cytosol. A 10-min treatment with 2 mm butyric or pivalic acid caused a transient acidification of the cytoplasm comparable to that observed after elicitor contact and also induced alkaloid biosynthesis. Experimental depletion of the vacuolar proton pool reversibly prevented both the elicitor-triggered pH shifts and the induction of alkaloid biosynthesis. pH shifts and induction of alkaloid biosynthesis showed a similar dependence on the elicitor concentration. Net efflux of K+, alkalinization of the outer medium, and browning of the cells were evoked only at higher elicitor concentrations. We suggest that transient acidification of the cytoplasm via efflux of vacuolar protons is both a necessary and sufficient step in the signal path toward biosynthesis of benzophenanthridine alkaloids in Californian poppy cells.

A Benzothiadiazole Primes Parsley Cells for Augmented Elicitation of Defense Responses

Systemic acquired resistance is an important component of the disease-resistance arsenal of plants, and is associated with an enhanced potency for activating local defense responses upon pathogen attack. Here we demonstrate that pretreatment with benzothiadiazole (BTH), a synthetic activator of acquired resistance in plants, augmented the sensitivity for low-dose elicitation of coumarin phytoalexin secretion by cultured parsley (Petroselinum crispum L.) cells. Enhanced coumarin secretion was associated with potentiated activation of genes encoding Phe ammonia-lyase (PAL). The augmentation of PAL gene induction was proportional to the length of pretreatment with BTH, indicating time-dependent priming of the cells. In contrast to the PAL genes, those for anionic peroxidase were directly induced by BTH in the absence of elicitor, thus confirming a dual role for BTH in the activation of plant defenses. Strikingly, the ability of various chemicals to enhance plant disease resistance correlated with their capability to potentiate parsley PAL gene elicitation, emphasizing an important role for defense response potentiation in acquired plant disease resistance.

Determinants of the phagocytic signal mediated by the type IIIA Fc gamma receptor, Fc gamma RIIIA: sequence requirements and interaction with protein-tyrosine kinases.

The Fc gamma receptor-associated gamma and zeta subunits contain a conserved cytoplasmic motif, termed the immunoglobulin gene tyrosine activation motif, which contains a pair of YXXL sequences. The tyrosine residues within these YXXL sequences have been shown to be required for transduction of a phagocytic signal. We have previously reported that the gamma subunit of the type IIIA Fc gamma receptor (Fc gamma RIIIA) is approximately 6 times more efficient in mediating phagocytosis than the zeta subunit of Fc gamma RIIIA. By exchanging regions of the cytoplasmic domains of the homologous gamma and zeta chains, we observed that the cytoplasmic area of the gamma chain bearing a pair of the conserved YXXL sequences is important in phagocytic signaling. Further specificity of phagocytic signaling is largely determined by the two internal XX amino acids in the YXXL sequences. In contrast, the flanking amino acids of the YXXL sequences including the seven intervening amino acids between the two YXXL sequences do not significantly affect the phagocytic signal. Furthermore, the protein-tyrosine kinase Syk, but not the related kinase ZAP-70, stimulated Fc gamma RIIIA-mediated phagocytosis. ZAP-70, however, increased phagocytosis when coexpressed with the Src family kinase Fyn. These data demonstrate the importance of the two specific amino acids within the gamma subunit YXXL cytoplasmic sequences in phagocytic signaling and explain the difference in phagocytic efficiency of the gamma and zeta chains. These results indicate the importance of Syk in Fc gamma RIIIA-mediated phagocytosis and demonstrate that ZAP-70 and syk differ in their requirement for a Src-related kinase in signal transduction.

Chimeric tumor necrosis factor receptors with constitutive signaling activity.

Many hormone and cytokine receptors are crosslinked by their specific ligands, and multimerization is an essential step leading to the generation of a signal. In the case of the tumor necrosis factor (TNF) receptors (TNF-Rs), antibody-induced crosslinking is sufficient to trigger a cytolytic effect. However, the quaternary structural requirements for signaling--i.e., the formation of dimers, trimers, or higher-order multimers--have remained obscure. Moreover, it has not been clear whether the 55-kDa or 75-kDa TNF-R is responsible for initiation of cytolysis. We reasoned that an obligate receptor dimer, targeted to the plasma membrane, might continuously signal the presence of TNF despite the actual absence of the ligand. Such a molecule, inserted into an appropriate vector, could be used to project receptor-specific "TNF-like" activity to specific cells and tissues in vivo. Accordingly, we constructed sequences encoding chimeric receptors in which the extracellular domain of the mouse erythropoietin receptor (Epo-R) was fused to the "stem," transmembrane domain, and cytoplasmic domain of the two mouse TNF-Rs. Thus, the Epo-R group was used to drive dimerization of the TNF-R cytoplasmic domain. These chimeric proteins were well expressed in a variety of cell lines and bound erythropoietin at the cell surface. Both the 55-kDa and the 75-kDa Epo/TNF-R chimeras exerted a constitutive cytotoxic effect detected by cotransfection or clonogenic assay. Thus, despite the lack of structural homology between the cytoplasmic domains of the two TNF-Rs, a similar signaling endpoint was observed. Moreover, dimerization (rather than trimerization or higher-order multimerization) was sufficient for elicitation of a biological response.

Role of phosphorylation in elicitation of the oxidative burst in cultured soybean cells.

The oxidative burst is likely the most rapid defense response mounted by a plant under pathogen attack, and the generated oxidant species may be essential to several subsequent defense responses. In our effort to characterize the signal-transduction pathways leading to rapid H2O2/O2- biosynthesis, we have examined the role of protein phosphorylation in this resistance mechanism. K-252a and staurosporine, two protein-kinase inhibitors, were found to block the oxidative burst in a concentration-dependent manner. When added during H2O2 generation, the burst was observed to rapidly terminate, suggesting that continuous phosphorylation was essential for its maintenance. Importantly, phosphatase inhibitors (calyculin A and okadaic acid) were found to induce the oxidative burst in the absence of any additional stimulus. This may suggest that certain kinases required for the burst are constitutively active and that stabilization of the phosphorylated forms of their substrates is all that is required for burst activity. In autoradiographs of elicited and unstimulated cells equilibrated with 32PO4(3-), several phosphorylated polypeptide bands were revealed that could represent proteins essential for the burst.