Helicobacter pyloril-asparaginase: a promising chemotherapeutic agent

Bacterial l-asparaginases are amidohydrolases that catalyse the conversion of l-asparagine to l-aspartate and ammonia and are used as anti-cancer drugs. The current members of this class of drugs have several toxic side effects mainly due to their associated glutaminase activity. In the present study, we report the molecular cloning, biochemical characterisation and in vitro cytotoxicity of a novel l-asparaginase from the pathogenic strain Helicobacter pylori CCUG 17874. The recombinant enzyme showed a strong preference for l-asparagine over l-glutamine and, in contrast to most l-asparaginases, it exhibited a sigmoidal behaviour towards l-glutamine. The enzyme preserved full activity after 2 h incubation at 45 °C. In vitro cytotoxicity assays revealed that different cell lines displayed a variable sensitivity towards the enzyme, AGS and MKN28 gastric epithelial cells being the most affected. These findings may be relevant both for the interpretation of the mechanisms underlying H. pylori associated diseases and for biomedical applications.     

Coupling of fully automated chip-based electrospray ionization to high-capacity ion trap mass spectrometer for ganglioside analysis

NanoMate robot was coupled to a high-capacity ion trap (HCT) mass spectrometer to create a system merging automatic chip-based electrospray ionization (ESI) infusion, ultrafast ion detection, and multistage sequencing at superior sensitivity. The interface between the NanoMate and HCT mass spectrometer consists of an in-laboratory constructed mounting device that allows adjustment of the robot position with respect to the mass spectrometer inlet. The coupling was optimized for ganglioside (GG) high-throughput analysis in the negative ion mode and was implemented in clinical glycolipidomics for identification and structural characterization of anencephaly-associated species. By NanoMate HCT mass spectrometry (MS), data corroborating significant differences in GG expression in anencephalic versus age-matched normal brain tissue were collected. The feasibility of chip-based nanoESI HCT multistage collision-induced dissociation (CID MSⁿ) for polysialylated GG fragmentation and isomer discrimination was tested on a GT1 (d18:1/18:0) anencephaly-associated structure. MS²-MS⁴ obtained by accumulating scans at variable fragmentation amplitudes gave rise to the first fragmentation patterns from which the presence of GT1b structural isomer could be determined unequivocally without the need for supplementary investigation by any other analytical or biochemical methods.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5-vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed.     

Mouse Sertoli cells display phenotypical and functional traits of antigen-presenting cells in response to interferon gamma

The testis is regarded as an immunologically privileged site because it tolerates either autoantigenic germ cells or allografts. Because the blood testis barrier represents an incomplete immunological barrier, we have explored whether Sertoli cells, the somatic cells of the seminiferous epithelium, might play an active role in immune evasion. We report data indicating that B7-H1(officially known as CD274)-mediated co-inhibition, an immunomodulatory mechanism based on cell-cell interaction, can be activated in Sertoli cell-lymphocyte cocultures. We have found that, in response to interferon gamma (IFNG), mouse Sertoli cells strongly up-regulate the negative co-stimulatory ligand B7-H1 but remain devoid of positive co-stimulatory molecules. Blockade of B7-H1 on the Sertoli cell surface resulted in enhanced proliferation of CD8(+) T cells cocultured with Sertoli cells. Moreover, IFNG-stimulated Sertoli cells were found to express, concurrent with B7-H1, MHC class II. Therefore, we have hypothesized that Sertoli cells could function as nonprofessional tolerogenic antigen-presenting cells by inducing enrichment in regulatory T cells (Tregs) in a mixed T lymphocyte population. Interestingly, we found that coculturing T cells with Sertoli cells can indeed induce an increase in CD4(+)CD25(+)(officially known as IL2RA)FOXP3(+) Tregs and a decrease in CD4(+)CD25(-) T cells, suggesting Sertoli cell-mediated Treg conversion; this process was found to be B7-H1-independent. Altogether these data show that Sertoli cells are potentially capable of down-regulating the local immune response, on one hand by directly inhibiting CD8(+) T cell proliferation through B7-H1 and, on the other hand, by inducing an increase in Tregs that might suppress other bystander T cells.     

Regulation of mammalian nitric oxide synthases by electrostatic interactions in the linker region of calmodulin

Calmodulin (CaM), the ubiquitous Ca(2+)-sensing protein, consists of two globular domains separated by a flexible central linker that properly orients CaM's globular domains to bind and regulate various intracellular proteins, including the nitric oxide synthase (NOS) enzymes. In the present study we determined that the charge and length of the central linker of CaM has an effect on the binding and activation of the NOS isozymes by using a variety of charge CaM mutants (T79D, S81D, T79D/S81D, S101D and E84R/E87K) and CaM mutants with residues removed (Delta84, Delta83-84, and Delta81-84). Our kinetic and spectropolarimetry results demonstrate that the NOS enzymes are not adversely affected by the CaM mutants with the exceptions of S101D, E84R/E87K and the deletion of residue 84. Electrostatic interactions in the central linker between residues 82-87 in combination with hydrophobic interactions in the globular domains of CaM are important for its tight association to inducible NOS.     

Immune activation driven by CTLA-4 blockade augments viral replication at mucosal sites in simian immunodeficiency virus infection

The importance of chronic immune activation in progression to AIDS has been inferred by correlative studies in HIV-infected individuals and in nonhuman primate models of SIV infection. Using the SIV(mac251) macaque model, we directly address the impact of immune activation by inhibiting CTLA-4, an immunoregulatory molecule expressed on activated T cells and a subset of regulatory T cells. We found that CTLA-4 blockade significantly increased T cell activation and viral replication in primary SIV(mac251) infection, particularly at mucosal sites, and increased IDO expression and activity. Accordingly, protracted treatment with anti-CTLA-4 Ab of macaques chronically infected with SIV(mac251) decreased responsiveness to antiretroviral therapy and abrogated the ability of therapeutic T cell vaccines to decrease viral set point. These data provide the first direct evidence that immune activation drives viral replication, and suggest caution in the use of therapeutic approaches for HIV infection in vivo that increase CD4(+) T cell proliferation.     

Jakmip1 is expressed upon T cell differentiation and has an inhibitory function in cytotoxic T lymphocytes

Jakmip1 belongs to a family of three related genes encoding proteins rich in coiled-coils. Jakmip1 is expressed predominantly in neuronal and lymphoid cells and colocalizes with microtubules. We have studied the expression of Jakmip1 mRNA and protein in distinct subsets of human primary lymphocytes. Jakmip1 is absent in naive CD8(+) and CD4(+) T lymphocytes from peripheral blood but is highly expressed in Ag-experienced T cells. In cord blood T lymphocytes, induction of Jakmip1 occurs upon TCR/CD28 stimulation and parallels induction of effector proteins, such as granzyme B and perforin. Further analysis of CD8(+) and CD4(+) T cell subsets showed a higher expression of Jakmip1 in the effector CCR7(-) and CD27(-) T cell subpopulations. In a gene expression follow-up of the development of CMV-specific CD8(+) response, Jakmip1 emerged as one of the most highly up-regulated genes from primary infection to latent stage. To investigate the relationship between Jakmip1 and effector function, we monitored cytotoxicity of primary CD8(+) T cells silenced for Jakmip1 or transduced with the full-length protein or the N-terminal region. Our findings point to Jakmip1 being a novel effector memory gene restraining T cell-mediated cytotoxicity.     

Dominant TEL1-hy mutations compensate for Mec1 lack of functions in the DNA damage response

Eukaryotic genome integrity is safeguarded by two highly conserved protein kinases that are called ATR and ATM for humans and Mec1 and Tel1 for Saccharomyces cerevisiae. Although they share sequence similarities and substrates, these protein kinases perform different specialized functions. In particular, Mec1 plays a key role in the DNA damage checkpoint response, whereas Tel1 primarily is involved in telomere homeostasis, and its checkpoint function is masked by the prevailing activity of Mec1. In order to understand how this specificity is achieved, we searched for TEL1 mutations able to compensate for the lack of Mec1 functions. Here, we describe seven independent dominant TEL1-hy alleles that are able to suppress, to different extents, both the hypersensitivity to genotoxic agents and the checkpoint defects of Mec1-deficient cells. Most of these alleles also cause telomere overelongation. In vitro kinase activity was increased compared to that of wild-type Tel1 in the Tel1-hy385, Tel1-hy394, Tel1-hy680, and Tel1-hy909 variants, but its activity was not affected by the TEL1-hy184 and TEL1-hy628 mutations and was slightly reduced by the TEL1-hy544 mutation. Thus, the phenotypes caused by at least some Tel1-hy variants are not simply the consequence of improved catalytic activity. Further characterization shows that Tel1-hy909 not only can sense and signal a single double-stranded DNA break, unlike wild-type Tel1, but also contributes more efficiently than Tel1 to single-stranded DNA accumulation at double-strand ends, thus enhancing Mec1 signaling activity. Moreover, it causes unscheduled checkpoint activation in unperturbed conditions and upregulates the checkpoint response to small amounts of DNA lesions. Finally, Tel1-hy544 can activate the checkpoint more efficiently than wild-type Tel1, while it causes telomere shortening, indicating that the checkpoint and telomeric functions of Tel1 can be separable.     

In vivo evaluation of (+)-MR200 as a new selective sigma ligand modulating MOP, DOP and KOP supraspinal analgesia

The compound (+)-MR200 [(+)-methyl (1R,2S)-2-{[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]methyl}-1-phenylcyclopropanecarboxylate] is a sigma ligand with increased affinity and selectivity compared to the structurally related ligand haloperidol. From the results of a previous study on the modulation of a systemically injected KOP opioid agonist analgesia by (+)-MR200, we analysed the influence of this sigma ligand on the antinociceptive effect of centrally injected MOP, DOP, and KOP selective agonists using the tail-flick test in rats. The results obtained confirmed that systemic administration of (+)-MR200 (1mg/Kg s.c.) did not modify basal tail-flick latency. Pre-treatment with 1mg/Kg s.c. of (+)-MR200 provided a significant increase in the antinociceptive effect of DAMGO (100ng/rat i.c.v.) and DPDPE (20 microg/rat i.c.v.). Conversely to previous reports, pre-treatment with (+)-MR200 reversed, in these experimental conditions, U-50488H (100 microg/rat i.c.v.) analgesia. The mechanism involved in these effects was not clear, but provided additional data on a diverging modulator role of selective sigma-1 antagonists on KOP analgesia.