Proteomics Identification and Validation of Desmocollin‐1 and Catechol‐O‐Methyltransferase as Proteins Associated with Breast Cancer Cell Migration and Metastasis

Biological treatment of many cancers currently targets membrane bound receptors located on a cell surface. To identify novel membrane proteins associated with migration and metastasis of breast cancer cells, a more migrating subpopulation of MDA‐MB‐231 breast cancer cell line is selected and characterized. A high‐resolution quantitative mass spectrometry with SILAC labeling is applied to analyze their surfaceome and it is compared with that of parental MDA‐MB‐231 cells. Among 824 identified proteins (FDR < 0.01), 128 differentially abundant cell surface proteins with at least one transmembrane domain are found. Of these, i) desmocollin‐1 (DSC1) is validated as a protein connected with lymph node status of luminal A breast cancer, tumor grade, and Her‐2 status by immunohistochemistry in the set of 96 primary breast tumors, and ii) catechol‐O‐methyltransferase is successfully verified as a protein associated with lymph node metastasis of triple negative breast cancer as well as with tumor grade by targeted data extraction from the SWATH‐MS data of the same set of tissues. The findings indicate importance of both proteins for breast cancer development and metastasis and highlight the potential of biomarker validation strategy via targeted data extraction from SWATH‐MS datasets.     

Targeted proteomics of solid cancers: from quantification of known biomarkers towards reading the digital proteome maps

The concept of personalized medicine includes novel protein biomarkers that are expected to improve the early detection, diagnosis and therapy monitoring of malignant diseases. Tissues, biofluids, cell lines and xenograft models are the common sources of biomarker candidates that require verification of clinical value in independent patient cohorts. Targeted proteomics – based on selected reaction monitoring, or data extraction from data-independent acquisition based digital maps – now represents a promising mass spectrometry alternative to immunochemical methods. To date, it has been successfully used in a high number of studies answering clinical questions on solid malignancies: breast, colorectal, prostate, ovarian, endometrial, pancreatic, hepatocellular, lung, bladder and others. It plays an important role in functional proteomic experiments that include studying the role of post-translational modifications in cancer progression. This review summarizes verified biomarker candidates successfully quantified by targeted proteomics in this field and directs the readers who plan to design their own hypothesis-driven experiments to appropriate sources of methods and knowledge.     

Sub-milliMorgan map of the proximal part of mouse chromosome 17 including the hybrid sterility 1 gene

We have generated a high-resolution genetic map, 0.071 cM per backcross animal, of the 13 cM T–H2 region of the mouse Chromosome (Chr) 17. The map contains two phenotypic loci, T and Hst1, 12 RFLP markers, and 24 microsatellite loci. The Hst1 gene was mapped to a chromosomal interval contained within a single 580-kb YAC clone. The FFEH11 YAC is 0.44 cM long and carries, besides the Hst1 gene, five polymorphic DNA markers and recombination breakpoints of six backcross animals. Two candidate genes for Hst1 were identified based on their location and testicular expression. These are Tbp and D17Ph4e. The sub-milliMorgan map of the T–H2 region revealed significant clustering of (CA)_n loci. The clustering, if shown to be a common feature in the mouse genome, may cause gaps in the physical map of the mouse genome. Received: 11 September 1995 / Accepted: 9 October 1995     

Dysfunctional protection against advanced glycation due to thiamine metabolism abnormalities in gestational diabetes

While the pathogenic role of dicarbonyl stress and accelerated formation of advanced glycation end products (AGEs) to glucose intolerance and to the development of diabetic complications is well established, little is known about these processes in gestational diabetes mellitus (GDM), a condition pathogenically quite similar to type 2 diabetes. The aims of the present study were (i) to determine plasma thiamine and erythrocyte thiamine diphosphate (TDP) and transketolase (TKT) activity in pregnant women with and without GDM, (ii) to assess relationships between thiamine metabolism parameters and selected clinical, biochemical and anthropometric characteristics and, finally, (iii) to analyse relationship between variability in the genes involved in the regulation of transmembrane thiamine transport (i.e. SLC19A2 and SLC19A3) and relevant parameters of thiamine metabolism. We found significantly lower plasma BMI adjusted thiamine in women with GDM (P = 0.002, Mann-Whitney) while levels of erythrocyte TDP (an active TKT cofactor) in mid-trimester were significantly higher in GDM compared to controls (P = 0.04, Mann-Whitney). However, mid-gestational TKT activity - reflecting pentose phosphate pathway activity - did not differ between the two groups (P > 0.05, Mann-Whitney). Furthermore, we ascertained significant associations of postpartum TKT activity with SNPs SLC19A2 rs6656822 and SLC19A3 rs7567984 (P = 0.03 and P = 0.007, resp., Kruskal-Wallis). Our findings of increased thiamine delivery to the cells without concomitant increase of TKT activity in women with GDM therefore indicate possible pathogenic role of thiamine mishandling in GDM. Further studies are needed to determine its contribution to maternal and/or neonatal morbidity.     

Small‐Scale Distribution of Aquatic Macroinvertebrates in Two Spring Fens with Different Groundwater Chemistry

We examined responses of macroinvertebrate assemblages to environmental and temporal variations along spring source‐spring brook transects in two fen habitats, sharply differing in groundwater chemistry, and compared the patterns among individual taxonomical groups. We hypothesised a different importance of environmental heterogeneity and seasonal changes primarily linked to strong tufa precipitation, which causes stronger environmental filtering in the calcareous fen. In concordance, we observed that assemblages of the more homogenous calcareous fen primarily changed over time, due to seasonal shifts in source availability and favourable conditions. Their spatial distribution was determined by the amount of CPOM, tufa crusts and temperature variation, but a substantial part of the assemblage exhibited spatial uniformity (Plecoptera, Clitellata, and especially Trichoptera and Diptera). The assemblages of the more heterogeneous Sphagnum ‐fen were primarily driven by water pH and substrate and the season was a notably weaker predictor. We found that different macroinvertebrate groups can display various responses to the measured variables shaping the overall pattern obtained based on the whole community. Further, greater environmental heterogeneity can result in temporally stable species distribution patterns even at very small spatial scales within a single site. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Saprotrophic basidiomycete mycelia and their interspecific interactions affect the spatial distribution of extracellular enzymes in soil

Saprotrophic cord-forming basidiomycetes are important decomposers of lignocellulosic substrates in soil. The production of extracellular hydrolytic enzymes was studied during the growth of two saprotrophic basidiomycetes, Hypholoma fasciculare and Phanerochaete velutina, across the surface of nonsterile soil microcosms, along with the effects of these basidiomycetes on fungi and bacteria within the soil. Higher activities of α-glucosidase, β-glucosidase, cellobiohydrolase, β-xylosidase, phosphomonoesterase and phosphodiesterase, but not of arylsulphatase, were recorded beneath the mycelia. Despite the fact that H. fasciculare, with exploitative hyphal growth, produced much denser hyphal cover on the soil surface than P. velutina, with explorative growth, both fungi produced similar amounts of extracellular enzymes. In the areas where the mycelia of H. fasciculare and P. velutina interacted, the activities of N-acetylglucosaminidase, α-glucosidase and phosphomonoesterase, the enzymes potentially involved in hyphal cell wall damage, and the utilization of compounds released from damaged hyphae of interacting fungi, were particularly increased. No significant differences in fungal biomass were observed between basidiomycete-colonized and noncolonized soil, but bacterial biomass was reduced in soil with H. fasciculare. The increases in the activities of β-xylosidase, β-glucosidase, phosphomonoesterase and cellobiohydrolase with increasing fungal:bacterial biomass ratio indicate the positive effects of fungal enzymes on nutrient release and bacterial abundance, which is reflected in the positive correlation of bacterial and fungal biomass content.     

Production of extracellular enzymes and degradation of biopolymers by saprotrophic microfungi from the upper layers of forest soil

Production of extracellular enzymes participating in the degradation of biopolymers was studied in 29 strains of nonbasidiomycetous microfungi isolated from Quercus petraea forest soil based on the frequency of occurrence. Most of the isolates were ascomycetes and belonged to the genera Acremonium, Alternaria, Cladosporium, Geomyces, Hypocrea, Myrothecium, Ochrocladosporium, and Penicillium (18 isolates), and two isolates were zygomycetes. Only six isolates showed phenol oxidation activity which was low and none of the strains were able to degrade humic acids. Approximately half of the strains were able to degrade cellulose and all but six degraded chitin. Most strains produced significant amounts of the cellulolytic enzymes cellobiohydrolase and ??-glucosidase and the chitinolytic enzymes chitinase, chitobiosidase, and N-acetylglucosaminidase. The highest cellulase activities were found in Penicillium strains, and the highest activity of chitinolytic enzymes was found in Acremonium sp. The production of the hemicellulose-degrading enzymes ??-galactosidase, ??-galactosidase, and ??-mannosidase was mostly low. The microfungal strains were able to produce significant growth on a range of 41?C87, out of 95 simple C-containing substrates tested in a Biolog? assay, monosaccharides being for all strains the most rapidly metabolized C-sources. Comparison with saprotrophic basidiomycetes from the same environment showed that microfungi have similar cellulolytic capabilities and higher chitinase activities which testifies for their active role in the decomposition of both lignocellulose and dead fungal biomass, important pools of soil carbon.