The human topoisomerase 1B Arg634Ala mutation results in camptothecin resistance and loss of inter-domain motion correlation

Human topoisomerase 1B, the unique target of the natural anticancer compound camptothecin, catalyzes the unwinding of supercoiled DNA by introducing transient single strand nicks and providing covalent protein–DNA adducts. The functional properties and the drug reactivity of the single Arg634Ala mutant have been investigated in comparison to the wild type enzyme. The mutant is characterized by an identical relaxation and cleavage rate but it displays resistance to camptothecin as indicated by a viability assay of the yeast cells transformed with the mutated protein. The mutant also displays a very fast religation rate that is only partially reduced by the presence of the drug, suggesting that this is the main reason for its resistance. A comparative analysis of the structural–dynamical properties of the native and mutant proteins by molecular dynamics simulation indicates that mutation of Arg634 brings to a loss of motion correlation between the different domains and in particular between the linker and the C-terminal domain, containing the catalytic tyrosine residue. These results indicate that the loss of motion correlation and the drug resistance are two strongly correlated events.     

Euphorbia characias Latex Amine Oxidase and Peroxidase: Interacting Enzymes?

This minireview deals the enzymatic transformation of some amino acids as arginine and ornithine, amines as tyramine, putrescine, spermine and spermidine, and other substances as nitric oxide and thiocyanate. These reactions, catalyzed by two proteins purified from the latex of Euphorbia characias, a copper/quinone containing amine oxidase and a cationic peroxidase, show enzymatic activity interactions probably occurring between these proteins in Euphorbia latex.     

Circulating miRNAs expression as potential biomarkers of mild traumatic brain injury

Molecular Biology Reports - TBI is the main cause of death and disability in individuals aged 1–45 in Western countries. One of the main challenges of TBI at present is the lack of specific...     

Psychiatric disorders and SLC6A4 gene variants: possible effects on alcohol dependence and alzheimer’s disease

Molecular Biology Reports - Serotoninergic system is one of the most important neurotransmission systems investigated in the field of psychiatry. Extensive evidence reveals how alterations of this...     

Long-term survival and immunological parameters in metastatic melanoma patients who responded to ipilimumab 10 mg/kg within an expanded access programme

Background

Ipilimumab can result in durable clinical responses among patients with advanced melanoma. However, no predictive marker of clinical activity has yet been identified. We provide preliminary data describing the correlation between immunological parameters and response/survival among patients with advanced melanoma who received ipilimumab 10 mg/kg in an expanded access programme.

Methods

Patients received ipilimumab 10 mg/kg every 3 weeks (Q3W) for four doses (induction) and Q12W from week 24 (W24) as maintenance therapy. Tumor assessments were conducted Q12W. Expression of inducible T cell costimulator (ICOS) on CD4⁺ and CD8⁺ T cells was assessed at baseline, W7, W12 and W24, and the ratio between absolute neutrophils (N) and lymphocytes (L) determined at baseline, W4, W7 and W10.

Results

Median overall survival among 27 patients was 9.6 months (95 % CI 3.2–16.1), with 3- and 4-year survival rates of 20.4 %. Five patients survived >4 years. Patients with an increase in the number of circulating ICOS⁺ T cells at W7 were more likely to experience disease control and have improved survival. An N/L ratio below the median at W7 and W10 was also associated with better survival compared with an N/L ratio above the median.

Conclusions

Ipilimumab can induce long-term survival benefits in heavily pretreated patients with metastatic melanoma. Changes in the number of circulating ICOS⁺ T cells or N/L ratio during ipilimumab treatment may represent early markers of response. However, given the limited sample size, further investigation is required.     

A Novel EPS-Producing Strain of Bacillus licheniformis Isolated from a Shallow Vent Off Panarea Island (Italy)

A haloalkaliphilic, thermophilic Bacillus strain (T14), isolated from a shallow hydrothermal vent of Panarea Island (Italy), produced a new exopolysaccharide (EPS). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain T14 was highly related (99 % similarity) to Bacillus licheniformis DSM 13^T and Bacillus sonorensis DSM 13779^T. Further DNA–DNA hybridization analysis revealed 79.40 % similarity with B. licheniformis DSM 13^T and 39.12 % with B. sonorensis DSM 13779^T. Sucrose (5 %) was the most efficient carbon source for growth and EPS production. The highest EPS production (366 mg l^?1) was yielded in fermenter culture at 300 rpm after 48 h of incubation. The purified fraction EPS1 contained fructose/fucose/glucose/galactosamine/mannose in a relative proportion of 1.0:0.75:0.28:tr:tr and possessed a molecular weight of 1,000 kDa displaying a trisaccharide unit constituted by sugars with a β-manno-pyranosidic configuration. Screening for biological activity showed anti-cytotoxic effect of EPS1 against Avarol in brine shrimp test, indicating a potential use in the development of novel drugs.     

Mesenchymal stem cells from patients to assay bone graft substitutes

Bio‐engineered scaffolds used in orthopedic clinical applications induce different tissue responses after implantation. In this study, non‐stoichiometric Mg²⁺ ions and stoichiometric apatites, which are used in orthopedic surgery as bone substitutes, have been assayed in vitro with human adult mesenchymal stem cells (hMSC) to evaluate cytocompatibility and osteoconductivity. hMSCs from the bone marrow aspirates of orthopedic patients were isolated and analyzed by flow cytometry for the surface markers Stro1, CD29, CD44, CD71, CD73, CD90, CD105 (positive) and CD45, CD235 (negative). The hMSC were analyzed for self‐renewal capacity and for differentiation potential. The hMSC, which were grown on different biomaterials, were analyzed for (i) cytotoxicity by AlamarBlue metabolic assay, (ii) osteoconductivity by ELISA for activated focal adhesion kinase, (iii) cytoskeleton organization by fluorescence microscopy, and (iv) cell morphology which was investigated by scan electron microscopy (SEM). Results indicate that isolated cell populations agree with minimal criteria for defining hMSC cultures. Non‐stoichiometric Mg²⁺ and stoichiometric apatites, in granular form, represent a more favorable environment for mesenchymal stem cell adhesion and growth compared to the non‐stoichiometric Mg²⁺ apatite, in nano‐structured paste form. This study indicates that different forms of biomaterials modulate osteoconductivity and cellular growth by differential activation focal adhesion kinase. J. Cell. Physiol. 228: 1229–1237, 2013. © 2012 Wiley Periodicals, Inc.     

Electrophysiological characterization of the Cyclophilin D-deleted mitochondrial permeability transition pore

Mitochondria isolated from engineered mice lacking Cyclophilin D (CypD), a component of the Permeability Transition Pore (PTP) complex, can still undergo a Ca^2?+?-dependent but Cyclosporin A-insensitive permeabilization of the inner membrane. Higher Ca^2?+? concentrations are required than for wild-type controls. The characteristics of the pore formed in this system were not known, and it has been proposed that they might differ substantially from those of the normal PTP. To test this hypothesis, we have characterized the PTP of isogenic wild-type and CypD^? mouse liver mitochondria in patch clamp experiments, which allow biophysical characterization. The pores observed in the two cases, very similar to those of rat liver mitochondria, are indistinguishable according to a number of criteria. The only clear difference is in their sensitivity to Cyclosporin A. CypD is thus shown to be an auxiliary, modulatory component of the “standard” PTP, which forms and has essentially the same properties even in its absence. The observations suggest that Ca^2?+?, CypD, and presumably other inducers and inhibitors act at the level of an activation or assembly process. Activation is separate and upstream of the gating observable on a short or medium-term time scale. Once the pore is activated, its molecular dynamics and biophysical properties may thus be predicted not to depend on the details of the induction process.     

Variability of the mc1r Gene in Melanic and Non-Melanic Podarcis lilfordi and Podarcis pityusensis from the Balearic Archipelago

The association between polymorphism at the mc1r locus and colour variation was studied in two wall lizard species (Podarcis lilfordi and P. pityusensis) from the Balearic archipelago. Podarcis lilfordi comprises several deep mitochondrial lineages, the oldest of which originated in the Pliocene, while much shallower mitochondrial lineages are found in P. pityusensis. Here, we examined whether specific substitutions were associated with the melanic colouration found in islet populations of these species. Homologous nuclear sequences covering most of the mc1r gene were obtained from 73 individuals from melanic and non-melanic Podarcis from different populations (the entire gene was also sequenced in six selected individuals). MtDNA gene trees were also constructed and used as a framework to assess mc1r diversity. Mc1r showed greater polymorphism in P. lilfordi than in P. pityusensis. However, we observed no substitutions that were common to all melanic individuals across the two species. Only one significant association was detected in the mc1r partial sequence, but this was a synonymous A/G mutation with A alleles being more abundant in melanic populations. In addition, there were no associations between the main dominant phenotypes (green and brown, blue and yellow spots and ventral colour) and synonymous or non-synonymous substitutions in the mc1r gene. There was no statistical evidence of selection on mc1r. This study suggests no relationship between mc1r polymorphism and colour variation in Balearic Podarcis.     

The TNF-Family Cytokine TL1A Inhibits Proliferation of Human Activated B Cells

Death receptor (DR3) 3 is a member of the TNFR superfamily. Its ligand is TNF-like ligand 1A (TL1A), a member of the TNF superfamily. TL1A/DR3 interactions have been reported to modulate the functions of T cells, NK, and NKT cells and play a crucial role in driving inflammatory processes in several T-cell-dependent autoimmune diseases. However, TL1A expression and effects on B cells remain largely unknown. In this study, we described for the first time that B cells from human blood express significant amounts of DR3 in response to B cell receptor polyclonal stimulation. The relevance of these results has been confirmed by immunofluorescence analysis in tonsil and spleen tissue specimens, which showed the in situ expression of DR3 in antigen-stimulated B cells in vivo. Remarkably, we demonstrated that TL1A reduces B-cell proliferation induced by anti-IgM-antibodies and IL-2 but did not affect B-cell survival, suggesting that TL1A inhibits the signal(s) important for B-cell proliferation. These results revealed a novel function of TL1A in modulating B-cell proliferation in vitro and suggest that TL1A may contribute to homeostasis of effector B-cell functions in immune response and host defense, thus supporting the role of the TL1A/DR3 functional axis in modulating the adaptive immune response.