Evaluation of peroxidative stress of cancer cells in vitro by real time quantification of volatile aldehydes in culture headspace

Rationale Peroxidation of lipids in cellular membranes results in the release of volatile organic compounds (VOCs), including saturated aldehydes. The real‐time quantification of trace VOCs produced by cancer cells during peroxidative stress presents a new challenge to non‐invasive clinical diagnostics, which as described here, we have met with some success. Methods A combination of selected ion flow tube mass spectrometry (SIFT‐MS), a technique that allows rapid, reliable quantification of VOCs in humid air and liquid headspace, and electrochemistry to generate reactive oxygen species (ROS) in vitro has been used. Thus, VOCs present in the headspace of CALU‐1 cancer cell line cultures exposed to ROS have been monitored and quantified in real time using SIFT‐MS. Results The CALU‐1 lung cancer cells were cultured in 3D collagen to mimic in vivo tissue. Real‐time SIFT‐MS analyses focused on the volatile aldehydes: propanal, butanal, pentanal, hexanal, heptanal and malondialdehyde (propanedial), that are expected to be products of cellular membrane peroxidation. All six aldehydes were identified in the culture headspace, each reaching peak concentrations during the time of exposure to ROS and eventually reducing as the reactants were depleted in the culture. Pentanal and hexanal were the most abundant, reaching concentrations of a few hundred parts‐per‐billion by volume, ppbv, in the culture headspace. Conclusions The results of these experiments demonstrate that peroxidation of cancer cells in vitro can be monitored and evaluated by direct real‐time analysis of the volatile aldehydes produced. The combination of adopted methodology potentially has value for the study of other types of VOCs that may be produced by cellular damage

Biogeneze a degradace prenasece bazickych aminokyselin CAN 1 kvasinky Candida albicans experinovaneho v Saccharomyces cerevisiae

Available from STL Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi

Parkin (PARK 2) mutations are rare in Czech patients with early-onset Parkinson's disease.

The aim of the study is to determine the frequency of parkin allelic variants in Czech early-onset Parkinson's disease patients and healthy controls.A total of 70 early-onset Parkinson's disease patients (age at onset ≤40 years) and 75 controls were screened for the sequence variants and exon rearrangements in the parkin gene.Parkin mutations were identified in five patients (7.1%): the p.R334C point mutation was present in one patient, four patients had exon deletions. The detected mutations were observed in the heterozygous state except one homozygous deletion of the exon 4. No mutations were obtained in control subjects. A novel sequence variant p.V380I (c.1138G>A) was identified in one control. Non-pathogenic polymorphisms p.S167N and p.D394N were seen in similar percentage in patients and controls, polymorphism p.V380L was almost twice as frequent in controls as in patients.Our study contributes to the growing body of evidence on the low frequency of the parkin mutations in the early-onset Parkinson's disease suggesting the potential role of other genes in the pathogenesis of the disease

Genotypic characteristics of patients and controls.

<p>het - heterozygous, hom- homozygous.</p><p>Genotypic characteristics of patients and controls.</p

Frequency of allelic variants of the parkin gene in patients (n = 70) and controls (n = 75).

<p>CI - confidence interval, DM - disease causing mutation, DP - disease-associated polymorphism, EVS - Exome variant server, het - heterozygous, HGMD - Human gene mutation database, hom - homozygous, NA - not available, OR - odds ratio.</p><p>Frequency of allelic variants of the parkin gene in patients (n = 70) and controls (n = 75).</p

Clinical characteristics of patients (n = 70).

<p>Clinical characteristics of patients (n = 70).</p

Glucose-Induced Monoubiquitination of the Saccharomyces cerevisiae Galactose Transporter Is Sufficient To Signal Its Internalization

In Saccharomyces cerevisiae, the addition of glucose to cells growing on galactose induces internalization of the galactose transporter Gal2p and its subsequent proteolysis in the vacuole. Here we report that the essential step in Gal2p down-regulation is its ubiquitination through the Ubc1p-Ubc4p-Ubc5p triad of ubiquitin-conjugating enzymes and Npi1/Rsp5p ubiquitin-protein ligase. Moreover, Gal2p appears to be stabilized in mutant cells defective in the ubiquitin-hydrolase Npi2p/Doa4p, and the mutant phenotype can be reversed by overexpression of ubiquitin. An analysis of the fate of Gal2p in cells overexpressing wild-type ubiquitin as well as its variants incompetent to form polyubiquitin chains showed that monoubiquitination of Gal2p is sufficient to signal internalization of the protein into the endocytic pathway

The Candida albicans GAP Gene Family Encodes Permeases Involved in General and Specific Amino Acid Uptake and Sensing▿†

The Saccharomyces cerevisiae general amino acid permease Gap1 (ScGap1) not only mediates the uptake of most amino acids but also functions as a receptor for the activation of protein kinase A (PKA). Fungal pathogens can colonize different niches in the host, each containing various levels of different amino acids and sugars. The Candida albicans genome contains six genes homologous to the S. cerevisiae GAP1. The expression of these six genes in S. cerevisiae showed that the products of all six C. albicans genes differ in their transport capacities. C. albicans Gap2 (CaGap2) is the true orthologue of ScGap1 as it transports all tested amino acids. The other CaGap proteins have narrower substrate specificities though CaGap1 and CaGap6 transport several structurally unrelated amino acids. CaGap1, CaGap2, and CaGap6 also function as sensors. Upon detecting some amino acids, e.g., methionine, they are involved in a rapid activation of trehalase, a downstream target of PKA. Our data show that CaGAP genes can be functionally expressed in S. cerevisiae and that CaGap permeases communicate to the intracellular signal transduction pathway similarly to ScGap1