Mechanism of target cell lysis by cytotoxic T lymphocytes (CTL) employing RSV-induced H-2 congenic and recombinant mouse tumor cells: demonstration of soluble mediators released from H-2-restricted CTL clones

The secretion and the specificity of cytotoxic mediators from H-2-restricted cytotoxic T lymphocytes (CTL) were examined using non-virus-producing target tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. By using rat concanavalin A supernatant, two H-2-restricted CTL clones were established from cytotoxic effector cells of B10.A(5R) mice primed with SR-RSV-induced syngeneic tumor Cell-free supernatants from the H-2-restricted CTL clones cocultured with syngeneic tumor cells had selectively high cytotoxic activity for syngeneic and H-2-compatible tumor cells, but not for H-2-incompatible tumor cells. YAC-1 cells, and B10.A(5R) blasts as defined in the 5-hr 51Cr-release assay. The cytotoxic activity was detected in the cell-free supernatants from the CTL clones cocultured with the CTL-sensitive syngeneic and H-2-compatible tumor cells, but not with the CTL-insensitive tumor cells and YAC-1 cells. The cytotoxic activity of the cell-free supernatant could be adsorbed by the syngeneic tumor cells, but not by YAC-1 and L(s) cells. Thus, the H-2-restricted CTL clones against SR-RSV-induced tumor cells were capable of releasing cytotoxic mediators by coculturing with syngeneic or H-2-compatible tumor cells, and the cytotoxic mediators showed a certain H-2-restricted manner in killing the target cells. These results suggest that the lysis of RSV-induced tumor cells by H-2-restricted CTL can at least in part be mediated by cytotoxic factors.     

The lysis function of RNA bacteriophage Qbeta is mediated by the maturation (A2) protein

Complete or partial cDNA sequences of the RNA bacteriophage Qbeta were cloned in plasmids under the control of the lambdaP(L) promoter to allow regulated expression in Escherichia coli harbouring the gene for the temperature-sensitive lambdaCI857 repressor. Induction of the complete Qbeta sequence leads to a 100-fold increase in phage production, accompanied by cell lysis. Induction of the 5'-terminal sequence containing the intact maturation protein (A2) cistron also causes cell lysis. Alterations of the A2 cistron, leading to proteins either devoid of approximately 20% of the C-terminal region or of six internal amino acids, abolish the lysis function. Expression of other cistrons in addition to the A2 cistron does not enhance host lysis. Thus, in Qbeta, the A2 protein, in addition to its functions as maturation protein, appears to trigger cell lysis. This contrasts with the situation in the distantly related group I RNA phages such as f2 and MS2 where a small lysis polypeptide is coded for by a region overlapping the end of the coat gene and the beginning of the replicase gene.     

The soluble mammalian sperm factor protein that triggers Ca2+ oscillations in eggs: evidence for expression of mRNA(s) coding for sperm factor protein(s) in spermatogenic cells

At fertilisation in mammals the sperm initiates a series of Ca2+ oscillations that activate development. One theory of signalling at fertilisation suggests that the sperm contains a soluble protein factor that causes these Ca2+ oscillations by entering the egg after sperm-egg membrane fusion. This theory is supported by the finding that, in some species, injection of sperm protein extracts into eggs triggers a pattern of Ca2+ oscillations similar to those seen at fertilisation. So far, all the direct evidence for a sperm factor has been based upon the injection of soluble proteins from mature sperm. Here, we demonstrate that injection of mRNA extracted from hamster spermatogenic cells also leads to generation of prolonged Ca2+ oscillations in mouse eggs. The ability of spermatogenic cell mRNA to induce Ca2+ oscillations is dependent upon translation into protein and also appears to be specific to spermatogenic cells since injection of mRNA isolated from somatic tissues into eggs was ineffective. These data support the hypothesis that sperm contain a soluble, cytosolic protein factor that induces Ca2+ oscillations in eggs at fertilisation. These data are discussed in the light of our recent findings that suggest that the sperm factor possesses a phospholipase C activity.     

Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8+ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies.

It was previously demonstrated that the vaccinia virus recombinants expressing the respiratory syncytial virus (RSV) F, G, or M2 (also designated as 22K) protein (Vac-F, Vac-G, or Vac-M2, respectively) induced almost complete resistance to RSV challenge in BALB/c mice. In the present study, we sought to identify the humoral and/or cellular mediators of this resistance. Mice were immunized by infection with a single recombinant vaccinia virus and were subsequently given a monoclonal antibody directed against CD4+ or CD8+ T cells or gamma interferon (IFN-gamma) to cause depletion of effector T cells or IFN-gamma, respectively, at the time of RSV challenge (10 days after immunization). Mice immunized with Vac-F or Vac-G were completely or almost completely resistant to RSV challenge after depletion of both CD4+ and CD8+ T cells prior to challenge, indicating that these cells were not required at the time of virus challenge for expression of resistance to RSV infection induced by the recombinants. In contrast, the high level of protection of mice immunized with Vac-M2 was completely abrogated by depletion of CD8+ T cells, whereas depletion of CD4+ T cells or IFN-gamma resulted in intermediate levels of resistance. These results demonstrate that antibodies are sufficient to mediate the resistance to RSV induced by the F and G proteins, whereas the resistance induced by the M2 protein is mediated primarily by CD8+ T cells, with CD4+ T cells and IFN-gamma also contributing to resistance.     

Capacitative Ca2+ entry is involved in regulating soluble amyloid precursor protein (sAPPalpha) release mediated by muscarinic acetylcholine receptor activation in neuroblastoma SH-SY5Y cells

Previous studies have demonstrated that stimulation of phospholipase C-linked G-protein-coupled receptors, including muscarinic M1 and M3 receptors, increases the release of the soluble form of amyloid precursor protein (sAPPalpha) by alpha-secretase cleavage. In this study, we examined the involvement of capacitative Ca2+ entry (CCE) in the regulation of muscarinic acetylcholine receptor (mAChR)-dependent sAPPalpha release in neuroblastoma SH-SY5Y cells expressing abundant M3 mAChRs. The sAPPalpha release stimulated by mAChR activation was abolished by EGTA, an extracellular Ca2+ chelator, which abolished mAChR-mediated Ca2+ influx without affecting Ca2+ mobilization from intracellular stores. However, mAChR-mediated sAPPalpha release was not inhibited by thapsigargin, which increases basal [Ca2+]i by depletion of Ca2+ from intracellular stores. While these results indicate that the mAChR-mediated increase in sAPPalpha release is regulated largely by Ca2+ influx rather than by Ca2+ mobilization from intracellular stores, we further investigated the Ca2+ entry mechanisms regulating this phenomenon. CCE inhibitors such as Gd3+, SKF96365, and 2-aminoethoxydiphenyl borane (2-APB), dose dependently reduced both Ca2+ influx and sAPPalpha release stimulated by mAChR activation, whereas inhibition of voltage-dependent Ca2+ channels, Na+/Ca2+ exchangers, or Na+-pumps was without effect. These results indicate that CCE plays an important role in the mAChR-mediated release of sAPPalpha.     

A role of the Ca2+/Mg2+-dependent endonuclease in apoptosis and its inhibition by Poly(ADP-ribose) polymerase

Apoptosis is characterized by various cell morphological and biochemical features, one of which is the internucleosomal degradation of genomic DNA. The role of the human chromatin-bound Ca(2+)- and Mg(2+)-dependent endonuclease (CME) DNAS1L3 and its inhibition by poly(ADP-ribosyl)ation in the DNA degradation that accompanies apoptosis was investigated. The nuclear localization of this endonuclease is the unique feature that distinguishes it from other suggested apoptotic nucleases. Purified recombinant DNAS1L3 was shown to cleave nuclear DNA into both high molecular weight and oligonucleosomal fragments in vitro. Furthermore, exposure of mouse skin fibroblasts expressing DNAS1L3 to inducers of apoptosis resulted in oligonucleosomal DNA fragmentation, an effect not observed in cells not expressing this CME, as well as in a decrease in cell viability greater than that apparent in the control cells. Recombinant DNAS1L3 was modified by recombinant human poly(ADP-ribose) polymerase (PARP) in vitro, resulting in a loss of nuclease activity. The DNAS1L3 protein also underwent poly(ADP-ribosyl)ation in transfected mouse skin fibroblasts in response to inducers of apoptosis. The cleavage and inactivation of PARP by a caspase-3-like enzyme late in apoptosis were associated with a decrease in the extent of DNAS1L3 poly(ADP-ribosyl)ation, which likely releases DNAS1L3 from inhibition and allows it to catalyze the degradation of genomic DNA.     

A specific role for Ca2+ in the oxidation of exogenous NADH by Jerusalem-artichoke (Helianthus tuberosus) mitochondria.

1. The addition of chelators to a suspension of mitochondria in a low-cation medium containing 9-aminoacridine caused a decrease in 9-aminoacridine fluorescence. The chelators removed bivalent cations from the membranes and allowed more 9-aminoacridine to move into the diffuse layer. The relative effect of EGTA and EDTA on the fluorescence suggested that the mitochondria are isolated with about equal amounts of Ca2+ and Mg2+ on the membranes. 2. The removal of the bivalent ions by chelators resulted in the inhibition of NADH oxidation. The inhibition could not be removed by adding sufficient decamethylenebistrimethylammonium ion (DM2+) to screen the fixed charges on the membranes and restore the fluorescence of 9-aminoacridine. This observation suggests that bivalent metal ions have a specific role in the oxidation of NADH. 3. Ca2+ and not Mg2+ reversed the inhibition of NADH oxidation caused by EGTA, whereas both reversed the inhibition caused by EDTA. This suggests that Ca2+ plays a specific role and that Mg2+ reverses the inhibition caused by EDTA by displacing the bound calcium from the chelator. 4. The results are interpreted as showing that Ca2+ plays a specific role in the oxidation of external NADH in addition to its ability to screen electrostatically or bind to the fixed charges associated with the surface of the membrane.     

Resistance against syncytium-inducing human immunodeficiency virus type 1 (HIV-1) in selected CD4(+) T cells from an HIV-1-infected nonprogressor: evidence of a novel pathway of resistance mediated by a soluble factor(s) that acts after virus entry.

A panel of CD4(+) T-cell clones were generated from peripheral blood lymphocytes from a patient with a nonprogressing infection of human immunodeficiency virus type 1 (HIV-1) by using herpesvirus saimiri as described recently. By and large, all of the clones expressed an activated T-cell phenotype (Th class 1) and grew without any further stimulation in interleukin-2-containing medium. None of these clones produced HIV-1, and all clones were negative for HIV-1 DNA. When these clones were infected with primary and laboratory (IIIB) strains of HIV-1 with syncytium-inducing (SI) phenotypes, dramatic variation of virus production was observed. While two clones were highly susceptible, other clones were relatively or completely resistant to infection with SI viruses. The HIV-resistant clones expressed CXCR4 coreceptors and were able to fuse efficiently with SI virus env-expressing cells, indicating that no block to virus entry was present in the resistant clones. Additionally, HIV-1 DNA was detectable after infection of the resistant clones, further suggesting that HIV resistance occurred in these clones after virus entry and probably after integration. We further demonstrate that the resistant clones secrete a factor(s) that can inhibit SI virus production from other infected cells and from a chronically infected producer cell line. Finally, we show that the resistant clones do not express an increased amount of ligands (stromal-derived factor SDF-1) of CXCR4 or other known HIV-inhibitory cytokines. Until now, the ligands of HIV coreceptors were the only natural substances that had been shown to play antiviral roles of any real significance in vivo. Our data from this study show that differential expression of another anti-HIV factor(s) by selected CD4(+) T cells may be responsible for the protection of these cells against SI viruses. Our results also suggest a novel mechanism of inhibition of SI viruses that acts at a stage after virus entry.     

The evolutionary differentiation of two histone H2A.Z variants in chordates (H2A.Z-1 and H2A.Z-2) is mediated by a stepwise mutation process that affects three amino acid residues

Background  

The histone H2A family encompasses the greatest number of core histone variants of which the replacement variant H2A.Z is currently one of the most heavily studied. No clear mechanism for the functional variability that H2A.Z imparts to chromatin has yet been proposed. While most of the past studies have referred to H2A.Z generically as a single protein, in vertebrates it is a mixture of two protein forms H2A.Z-1 (previously H2A.Z) and H2A.Z-2 (previously H2A.F/Z or H2A.V) that differ by three amino acids.     

A conditional two‐hybrid (C2H) system for the detection of protein–protein interactions that are mediated by post‐translational modification

The original bacterial two‐hybrid system is widely used but does not permit the study of interactions regulated by PTMs. Here, we have built a conditional two‐hybrid (C2H) system, in which bait and prey proteins can be co‐expressed in the presence of a modifying enzyme such as a methyltransferase, acetyltransferase, or kinase. Any increase or decrease in interaction due to the modification of the proteins can be measured by an increased or decreased level of reporter gene expression. The C2H system is comprised of eight new vectors based on the Novagen Duet co‐expression plasmids. These vectors include two multiple cloning sites per vector as well as a hexahistidine tag or S‐tag to aid in purification, if desired. We demonstrate the use of the C2H system to study the dimerization of the yeast protein Npl3, which is increased when methylated by the methyltransferase Hmt1.