Synthetic peptides mimicking the interleukin-6/gp130 interaction: a two-helix bundle system. Design and conformational studies.

The objective of our study was to mimic in a typical reductionist approach the molecular interactions observed at the interface between the gp130 receptor and interleukin-6 during formation of their complex. A peptide system obtained by reproducing some of the interleukin-6/gp130 molecular interactions into a two-helix bundle structure was investigated. The solution conformational features of this system were determined by CD and NMR techniques. The CD titration experiments demonstrated that the interaction between the designed peptides is specific and based on a well-defined stoichiometry. The NMR data confirmed some of the structural features of the binding mechanism as predicted by the rational design and indicated that under our conditions the recognition specificity and affinity can be explained by the formation of a two-helix bundle. Thus, the data reported herein represent a promising indication on how to develop new peptides able to interfere with formation of the interleukin-6/gp130 complex.     

Vγ9/Vδ2 T lymphocytes in Italian patients with Beh?et's disease: evidence for expansion, and tumour necrosis factor receptor II and interleukin-12 receptor β1 expression in active disease

Beh?et's disease is a multisystem disease in which there is evidence of immunological dysregulation. It has been proposed that γ/δ T cells are involved in its pathogenesis. The aim of the present study was to assess the capacity of γ/δ T cells with phenotype Vγ9/Vδ2, from a group of Italian patients with Beh?et's disease, to proliferate in the presence of various phosphoantigens and to express tumour necrosis factor (TNF) and IL-12 receptors. Twenty-five patients and 45 healthy individuals were studied. Vγ9/Vδ2 T cells were analyzed by fluorescence activated cell sorting, utilizing specific monoclonal antibodies. For the expansion of Vγ9/Vδ2 T cells, lymphocytes were cultured in the presence of various phosphoantigens. The expression of TNF receptor II and IL-12 receptor β₁ was evaluated with the simultaneous use of anti-TNF receptor II phycoerythrin-labelled (PE) or anti-IL-12 receptor β₁ PE and anti-Vδ2 T-cell receptor fluorescein isothiocyanate. There was a certain hierarchy in the response of Vγ9/Vδ2 T cells toward the different phosphoantigens, with the highest expansion factor obtained with dimethylallyl pyrophosphate and the lowest with xylose 1P. The expansion factor was fivefold greater in patients with active disease than in those with inactive disease or in control individuals. TNF receptor II and IL-12 receptor β₁ expressions were increased in both patients and control individuals. The proportion of Vγ9/Vδ2 T cells bearing these receptors was raised in active disease when Vγ9/Vδ2 T cells were cultured in the presence of dimethylallyl pyrophosphate. These results indicate that Vγ9/Vδ2 T cell activation is correlated with disease progression and probably involved in the pathogenesis.     

Constitutive heterochromatin: a surprising variety of expressed sequences

The organization of chromosomes into euchromatin and heterochromatin is amongst the most important and enigmatic aspects of genome evolution. Constitutive heterochromatin is a basic yet still poorly understood component of eukaryotic chromosomes, and its molecular characterization by means of standard genomic approaches is intrinsically difficult. Although recent evidence indicates that the presence of transcribed genes in constitutive heterochromatin is a conserved trait that accompanies the evolution of eukaryotic genomes, the term heterochromatin is still considered by many as synonymous of gene silencing. In this paper, we comprehensively review data that provide a clearer picture of transcribed sequences within constitutive heterochromatin, with a special emphasis on Drosophila and humans.     

Inhibition of translesion DNA polymerase by archaeal reverse gyrase

Reverse gyrase is a unique DNA topoisomerase endowed with ATP-dependent positive supercoiling activity. It is typical of microorganisms living at high temperature and might play a role in maintenance of genome stability and repair. We have identified the translesion DNA polymerase SsoPolY/Dpo4 as one partner of reverse gyrase in the hyperthermophilic archaeon Sulfolobus solfataricus. We show here that in cell extracts, PolY and reverse gyrase co-immunoprecipitate with each other and with the single strand binding protein, SSB. The interaction is confirmed in vitro by far-western and Surface Plasmon Resonance. In functional assays, reverse gyrase inhibits PolY, but not the S. solfataricus B-family DNA polymerase PolB1. Mutational analysis shows that inhibition of PolY activity depends on both ATPase and topoisomerase activities of reverse gyrase, suggesting that the intact positive supercoiling activity is required for PolY inhibition. In vivo, reverse gyrase and PolY are degraded after induction of DNA damage. Inhibition by reverse gyrase and degradation might act as a double mechanism to control PolY and prevent its potentially mutagenic activity when undesired. Inhibition of a translesion polymerase by topoisomerase-induced modification of DNA structure may represent a previously unconsidered mechanism of regulation of these two-faced enzymes.     

The common Scandinavian human leucocyte antigen ancestral haplotype 62.1 as prognostic factor in patients with advanced malignant melanoma

Purpose  We have previously demonstrated an association of the human leukocyte antigen (HLA), HLA-A2 allele with ovarian and prostate cancer mortality as well as a segregation of the ancestral HLA haplotype (AHH) 62.1 [(A2) B15 Cw3 DRB1*04] in patients with stage III–IV serous ovarian cancer. The objective of the present study was to determine the role of the HLA phenotype on the prognosis in stage III–IV malignant melanoma patients. Patients and methods  A cohort of metastatic malignant melanoma patients (n = 91), in stage III (n = 26) or IV (n = 65) were analysed for HLA-A, -B, -Cw and -DRB1 types by PCR/sequence-specific primer method. The frequencies of HLA alleles in the patients were compared to that of healthy Swedish bone marrow donors. The effect of HLA types on prognosis was defined by Kaplan–Meier and Cox analysis. Results  The presence of the AHH 62.1 in clinical stage IV patients was significantly and independently associated with the worst survival rate recorded from the appearance of metastasis (HR = 2.14; CI = 1.02–4.4; P = 0.04). In contrast, the period from the primary diagnosis to metastasis was the longest in patients with this haplotype (HR = 0.40; CI = 0.17–0.90; P = 0.02). Conclusions  Melanoma patients in our cohort with 62.1 AHH which is associated with autoimmune diseases have an initial strong anti-tumour control with longer metastasis-free period. These patients have rapid progression after the appearance of metastasis, responding poorly to chemo- or/and immunotherapy. This apparently paradoxical clinical process could be due to the interplay between tumour clones escape and immune surveillance ending up with a rapid disease progression. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enzyme solid-state support assays: a surface plasmon resonance and mass spectrometry coupled study of immobilized insulin degrading enzyme

Solid-support based assays offer several advantages that are not normally available in solution. Enzymes that are anchored on gold surfaces can interact with several different molecules, opening the way to high throughput array format based assays. In this scenario, surface plasmon resonance (SPR) and mass spectrometry (MS) investigations have often been applied to analyze the interaction between immobilized enzyme and its substrate molecules in a tag-free environment. Here, we propose a SPR-MS combined experimental approach aimed at studying insulin degrading enzyme (IDE) immobilized onto gold surfaces and its ability to interact with insulin. The latter is delivered by a microfluidic system to the IDE functionalized surface and the activity of the immobilized enzyme is verified by atmospheric pressure/matrix assisted laser desorption ionization (AP/MALDI) MS analysis. The SPR experiments allow the calculation of the kinetic constants involved for the interaction between immobilized IDE and insulin molecules and evidence of IDE conformational change upon insulin binding is also obtained.     

F281, synthetic agonist of the sigma-2 receptor,induces Ca2+ efflux from the endoplasmic reticulum and mitochondria in SK-N-SH cells

We demonstrate that F281, a synthetic agonist of the sigma-2 receptor (s2R), induces a non transient increase in intracellular [Ca²⁺] ([Ca²⁺]_i) and cell death in SK-N-SH cells. Sigma receptors are classified into two subtypes, with different molecular weight and tissue distribution. While the sigma-1 receptor has been cloned, the s2r is less characterized and its physiological ligand and role need further investigation. In tumour cell lines, synthetic agonists of the s2R trigger apoptosis and modulate [Ca²⁺]_i. In particular, CB-64D induces a Ca²⁺ response while PB28 supresses Ca²⁺ signalling. We have recently synthesized F281, by replacing the 5-methoxytetraline moiety of PB28 with a carbazole nucleus. Although this bioisosteric substitution should not affect the ligand affinity at the receptor, F281 (after 24 h incubation) was more cytotoxic than PB28 (EC₅₀ values 65.4 nM and 8.13 μM, respectively) in SK-N-SH cells. We used the fluorescent probes fura-2, rhod-2 and JC-1. F281 mobilizes Ca²⁺ from mitochondria and from the endoplasmic reticulum, by opening its inositol 1,4,5-trisphosphate receptor; Ca²⁺-entry through the channels activated by store depletion was also observed. After the increase in [Ca²⁺]_i and within 10 min, we observed a sudden drop in metabolic activity and intracellular [ATP] leading to cell death.