Structural analysis of the Sulfolobus solfataricus MCM protein N-terminal domain†

The Mini-Chromosome Maintenance (MCM) proteins are candidates of replicative DNA helicase in eukarya and archaea. Here we report a 2.8 Å crystal structure of the N-terminal domain (residues 1–268) of the Sulfolobus solfataricus MCM (Sso MCM) protein. The structure reveals single-hexameric ring-like architecture, at variance from the protein of Methanothermobacter thermoautotrophicus (Mth). Moreover, the central channel in Sso MCM seems significantly narrower than the Mth counterpart, which appears to more favorably accommodate single-stranded DNA than double-stranded DNA, as supported by DNA-binding assays. Structural analysis also highlights the essential role played by the zinc-binding domain in the interaction with nucleic acids and allows us to speculate that the Sso MCM N-ter domain may function as a molecular clamp to grasp the single-stranded DNA passing through the central channel. On this basis possible DNA unwinding mechanisms are discussed

Amino acids of the Sulfolobus solfataricus mini-chromosome maintenance-like DNA helicase involved in DNA binding/remodeling.

Herein we report the identification of amino acids of the Sulfolobus solfataricus mini-chromosome maintenance (MCM)-like DNA helicase (SsoMCM), which are critical for DNA binding/remodeling. The crystallographic structure of the N-terminal portion (residues 2–286) of the Methanothermobacter thermoautotrophicum MCM protein revealed a dodecameric assembly with two hexameric rings in a head-to-head configuration and a positively charged central channel proposed to encircle DNA molecules. A structure-guided alignment of the M. thermoautotrophicum and S. solfataricus MCM sequences identified positively charged amino acids in SsoMCM that could point to the center of the channel. These residues (Lys-129, Lys-134, His-146, and Lys-194) were changed to alanine. The purified mutant proteins were all found to form homo-hexamers in solution and to retain full ATPase activity. K129A, H146A, and K194A SsoMCMs are unable to bind DNA either in single- or double-stranded form in band shift assays and do not display helicase activity. In contrast, the substitution of lysine 134 to alanine affects only binding to duplex DNA molecules, whereas it has no effect on binding to single-stranded DNA and on the DNA unwinding activity. These results have important implications for the understanding of the molecular mechanism of the MCM DNA helicase action

Dermal regeneration template: reconstruction in oral cancer defects

BackgroundPost ablative oral mucosal defect resulting from the removal of tumors can be treated with various techniques.PurposeIn this paper, we are showing what, in our experience, are the advantages and disadvantages given using biosynthetic skin substitutes when dealing with this kind of lesions.Materials and methodsPatients included in the sample came to our attention with both neoplastic lesions (11 subjects) and important scar retraction after previous oncologic surgery (1 subject). All patients underwent trans-oral resection surgery following the same surgical protocol and post ablative oral mucosal defect were treated using the dermal regeneration template. The surgical defect location, size, and time of removal of the silicone layer varied from one subject to the other.ResultsMost patients showed good healing with reduced scarring and adequate remucosalisation of the defect. The main complications were shown in a palatal lesion treated with concomitant osteal resection, which developed an oroantral fistula at follow up, and tongue lesions which showed some scarring.ConclusionsGiven our experience, we would advise using dermal substitutes when reconstructing oral defects only after a cautious evaluation of the area of the lesion, the gap size, the possible adherence of the membrane to the gap, and the presence of tissue supporting the overlying membrane

CD4+CD25+Foxp3+ T regulatory cells are not involved in oral desensitization.

Oral tolerance has been related to generation of T regulatory cells (Treg) or clonal anergy/deletion, respectively by administering low and high doses of fed antigens. CD4+CD25+ regulatory T cell clones can be induced by the antigen in Peyer's patches of animal models. We selected ten subjects (mean age: 89.4 ± 36.21 months; group A) with severe cow's milk allergy. They underwent oral desensitization (OD) according to the current protocols. In six months they reached a tolerance of 50 ml of cow's milk. CD4+CD25+Foxp3+ T(reg) blood levels were measured at the beginning of OD (A) and after 6 months (A'), but almost the same values were obtained: A = 0.36 ± 0.11%; A'= 0.59 ± 0.15%. These results were compared with a control group (C) of non-atopic children. Naturally outgrowing cow's milk allergy can be related to high blood levels of CD4+CD25+Foxp3+ T(reg), as previously reported in children. On the other hand, a forced oral desensitization through a progressive intake of the antigenic food seems not to be related to an enhancement of CD4+CD25+Foxp3+ T(reg) levels in peripheral blood, making the role of long-lasting systemic immunologic changes unlikely

Epoxy resin doped with Coumarin 6: Example of accessible luminescent collectors

We report on the preparation of luminescent collectors based on epoxy resins containing Coumarin 6 as fluorescent dye. Fluorescent epoxy slabs were obtained by carefully mixing from 60 to 150 ppm of the fluorophore with bisphenol A diglycidyl ether and 4,4′-methylenebis(2-methylcyclohexylamine) as curing agent. Spectroscopic (FT-IR, solid-state NMR, Raman) investigations and calorimetric analysis evidence the success of the preparation procedure in terms of slab homogeneity, fluorophore dispersibility and its role in promoting the crosslinking extent. The concentrating ability and the derived optical efficiencies of the epoxy-based collectors are determined with a properly designed set-up and result greater (∼10%) than that of poly(methyl methacrylate) concentrators with the same fluorophore and geometry. Optical efficiencies as high as 7.4% are obtained and enable the potential use of epoxy resins as bulk thermosetting materials for solar collectors

A CDC6-like factor from the archaea Sulfolobus solfataricus promotes binding of the mini-chromosome maintenance complex to DNA

The archaeal replication apparatus appears to be a simplified version of the eukaryotic one with fewer polypeptides and simpler protein complexes. Herein, we report evidence that a Cdc6-like factor from the hyperthermophilic crenarchaea Sulfolobus solfataricus stimulates binding of the homohexameric MCM-like complex to bubble- and fork-containing DNA oligonucleotides that mimic early replication intermediates. This function does not require the Cdc6 ATP and DNA binding activities. These findings may provide important clues to understanding how the DNA replication initiation process has evolved in the more complex eukaryotic organisms

Thermochromic polyethylene films doped with perylene chromophores: experimental evidence and methods for characterization of their phase behaviour

We report on a thermochromic system suitable for sensing temperature changes in the 30-70 degrees C regime based on linear low density polyethylene (LLDPE) films doped with N,N'-bis-(1'-phenylethyl)-perylene-3,4,9,10-tetracarboxydiimide (PE-Pery), a fluorescent aggregachromic dye. At low PE-Pery concentration (0.01-0.02 wt%), the dye monomers were well dispersed in the polymer matrix showing their maximum fluorescence intensity at 525 nm. As the dye content was increased, monomers emission quenched whereas dye aggregates prevailed above 0.05 wt% as well as their red fluorescence band at 620-680 nm. Upon heating from 30 to 70 degrees C, all films displayed a thermochromic response, more evident for the less concentrated samples (<0.05 wt%) in which the emission of the dye as a monomer continuously increased with increasing temperature. This phenomenon promoted effective color changes from a dull red-violet at 30 degrees C to a bright yellow-green at 70 degrees C. Combined DSC and variable-temperature Solid State NMR (SSNMR) measurements addressed the thermochromic behavior to the increased amount of the available amorphous phase and to the increased mobility of both the interphase and amorphous components with temperature, which favored PE-Pery dispersion and diffusion, thus recovering their fluorescence. Overall, the present results support the use of PE-Pery-enriched LLDPE films as a chromogenic material suitable for the detection of temperature changes close to the physiological regime

Biochemical characterization of a CDC6-like protein from the crenarchaeon sulfolobus solfataricus

Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya. Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far. Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa). We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function