Pathogenicity of various species of Candida in an experimental murine system

The systemic infection induced by Candida albicans, Candida krusei and Candida viswanathii was studied in an experimental murine system. Candida albicans is able to kill outbred CD1 mice within a few days and at a very low concentration; C. krusei is not pathogenic not even when inoculated at a higher concentration; C. viswanathii is able to kill animals only a a higher concentration. The different resistances do not seem to be under genic control, in as much as the different strains of mice used (hybrid CD2F1 and B6C3HF1, inbred Balb/c) show the same degree of resistance as the CD1 mice to the three species of Candida. The colony forming units (CFU) in the kidneys of CD1 mice inoculated intravenously with 10(5) cells of the three species of Candida, collected at various intervals showed a good correlation with the median survival times: a rapid moltiplication of the C. albicans is evident in the kidneys of the animals 24 hours after the inoculation, while the C. krusei and the C. viswanathii do not moltiply.     

8-Oxoguanine DNA-glycosylase repair activity and expression: a comparison between cryopreserved isolated lymphocytes and EBV-derived lymphoblastoid cell lines

Several lines of evidence suggest an association between oxidative DNA-damage repair capacity and cancer risk. In particular, a DNA-glycosylase assay for removal of 8-oxoguanine (8-oxoG) in peripheral blood mononuclear cells (PBMC) has been successfully applied to identify populations with increased risk for lung cancer and squamous cell carcinomas of head and neck. In order to verify whether EBV-transformed lymphoblastoid cell lines (LCL) are a suitable surrogate for PBMC in specific DNA-repair phenotypic assays, a validation trial was conducted. PBMC from 20 healthy subjects were collected and an aliquot was transformed with EBV to obtain LCL. The ability of cell-free extracts from both cell types to incise a 3'-fluorescently labelled duplex oligonucleotide containing a single 8-oxoG (OGG assay) was evaluated. Since this activity is mediated predominantly by OGG1, the OGG1 gene expression was also measured. 8-oxoG DNA-glycosylase activity and OGG1 expression were significantly higher (p<0.0001) in LCL than in PBMC. However, while this assay was shown to be robust and reproducible when used on PBMC (intra-assay CV=8%), a high intra-culture variability was observed with LCL (intra-culture CV=16.8%). Neither differences on OGG1 gene expression nor the cell-cycle distribution seemed to account for this variability. Inter-individual variability of OGG activity in PBMC and LCL was not associated with OGG1 gene expression. We have therefore established a non-radioactive cleavage assay that can be easily applied to measure OGG activity in human PBMC. The use of LCL for DNA-repair genotype-phenotype correlation studies seems to be inappropriate, at least with cell-free based functional assays.     

Determination of metabolic equivalents during low- and high-intensity resistance exercise in healthy young subjects and patients with type 2 diabetes

The purpose of this study was to quantify the metabolic equivalents (METs) of resistance exercise in obese patients with type 2 diabetes (T2DM) and healthy young subjects and to evaluate whether there were differences between sessions executed at low- versus high-intensity resistance exercise. Twenty obese patients with T2DM (62.9±6.1 years) and 22 young subjects (22.6±1.9 years) performed two training sessions: one at vigorous intensity (80% of 1-repetition maximum (1RM)) and one at moderate intensity (60% of 1RM). Both groups carried out three strength exercises with a 2-day recovery between sessions. Oxygen consumption was continuously measured 15 min before, during and after each training session. Obese T2DM patients showed lower METs values compared with young healthy participants at the baseline phase (F= 2043.86; P<0.01), during training (F=1140.59; P<0.01) and in the post-exercise phase (F=1012.71; P<0.01). No effects were detected in the group x intensity analysis of covariance. In this study, at both light-moderate and vigorous resistance exercise intensities, the METs value that best represented both sessions was 3 METs for the obese elderly T2DM patients and 5 METs for young subjects.     

Identification and characterization of a novel bacterial sulfite oxidase with no heme binding domain from Deinococcus radiodurans

An open reading frame (draSO) encoding a putative sulfite oxidase (SO) was identified in the sequence of chromosome II of Deinococcus radiodurans; the predicted gene product showed significant amino acid sequence homology to several bacterial and eukaryotic SOs, such as the biochemically and structurally characterized enzyme from Arabidopsis thaliana. Cloning of the Deinococcus SO gene was performed by PCR amplification from the bacterial genomic DNA, and heterologous gene expression of a histidine-tagged polypeptide was obtained in a molybdopterin-overproducing strain of Escherichia coli. The recombinant protein was purified to homogeneity by nickel chelating affinity chromatography, and its main kinetic and chemical physical parameters were determined. Northern blot and enzyme activity analyses indicated that draSO gene expression is constitutive in D. radiodurans and that there is no increase upon exposure to thiosulfate and/or molybdenum(II).     

Membrane charge dependent states of the β-amyloid fragment Aβ (16-35) with differently charged micelle aggregates

Aβ (16-35) is the hydrophobic central core of β-amyloid peptide, the main component of plaques found in the brain tissue of Alzheimer's disease patients. Depending on the conditions present, β-amyloid peptides undergo a conformational transition from random coil or α-helical monomers, to highly toxic β-sheet oligomers and aggregate fibrils. The behavior of β-amyloid peptide at plasma membrane level has been extensively investigated, and membrane charge has been proved to be a key factor modulating its conformational properties. In the present work we probed the conformational behavior of Aβ (16-35) in response to negative charge modifications of the micelle surface. CD and NMR conformational analyses were performed in negatively charged pure SDS micelles and in zwitterionic DPC micelles “doped” with small amounts of SDS. To analyze the tendency of Aβ (16-35) to interact with these micellar systems, we performed EPR experiments on three spin-labeled analogues of Aβ (16-35), bearing the methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl) methanethiolsulfonate spin label at the N-terminus, in the middle of the sequence and at the C-terminus, respectively. Our conformational data show that, by varying the negative charge of the membrane, Aβ (16-35) undergoes a conformational transition from a soluble helical-kink-helical structure, to a U-turn shaped conformation that resembles protofibril models.     

Exploring interaction of beta-amyloid segment (25-35) with membrane models through paramagnetic probes.

The accumulation of beta-amyloid peptides into senile plaques is one of the hallmarks of Alzheimer's disease (AD). There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the beta-sheet oligomerization process of beta-amyloid. Abeta(25-35), the sequence of which is GSNKGAIIGLM, is a highly toxic segment of amyloid beta (Abeta)-peptides, which forms fibrillary aggregates. In the present work, two spin-labelled Abeta(25-35) analogues containing the nitroxide group of the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe at the N- or the C-terminus of the peptide sequence, respectively, were synthesized in order to investigate the peptide-membrane interaction. The orientation and associated changes of the peptide conformation in the presence of different artificial membrane models (micelles, liposomes) were evaluated by electron paramagnetic resonance and circular dichroism techniques. The results of this study allowed us to propose a model in which the C-terminal portion of the peptide is highly associated to the membrane, while the N-terminal part extends into the aqueous phase with occasional contacts with the lipid head-group region. Interestingly, the interaction of the C-terminal portion of the peptide is particularly enhanced in the presence of sodium dodecyl sulfate (SDS) molecules.     

New functions of XPC in the protection of human skin cells from oxidative damage

Xeroderma pigmentosum (XP) C is involved in the recognition of a variety of bulky DNA-distorting lesions in nucleotide excision repair. Here, we show that XPC plays an unexpected and multifaceted role in cell protection from oxidative DNA damage. XP-C primary keratinocytes and fibroblasts are hypersensitive to the killing effects of DNA-oxidizing agents and this effect is reverted by expression of wild-type XPC. Upon oxidant exposure, XP-C primary keratinocytes and fibroblasts accumulate 8,5'-cyclopurine 2'-deoxynucleosides in their DNA, indicating that XPC is involved in their removal. In the absence of XPC, a decrease in the repair rate of 8-hydroxyguanine (8-OH-Gua) is also observed. We demonstrate that XPC-HR23B complex acts as cofactor in base excision repair of 8-OH-Gua, by stimulating the activity of its specific DNA glycosylase OGG1. In vitro experiments suggest that the mechanism involved is a combination of increased loading and turnover of OGG1 by XPC-HR23B complex. The accumulation of endogenous oxidative DNA damage might contribute to increased skin cancer risk and account for internal cancers reported for XP-C patients.