Functional studies of *Ras and Bcl-2 oncoproteins in keratinocyte homeostasis and multistep skin carcinogenesis

To assess the effect of deregulated Ha-ras and bcl-2, individually and in combination on epidermal keratinocyte homeostasis and during multistep skin carcinogenesis, we generated skin-specific transgenic mice and keratinocyte transfectants constitutively expressing oncogenic Ha-ras and bcl-2 proteins. The deregulated Ha-ras and bcl-2 expression contributing to homeostatic imbalances in the skin had an additive effect on the probability of tumor development. They were also cooperative in incidence, growth, and latency of tumor formation, and they exhibited synergistic cooperation in malignant transformation of benign papillomas. To explain the homeostatic imbalances by Ha-ras and bcl-2 overexpression in the skin, we investigated the three major cellular processes of proliferation, cell death, and differentiation. Epidermal expression of Bcl-2 retarded keratinocyte proliferation in the epidermis of neonatal mice compared with results for control littermates. Constitutive expression of Ha-ras increased keratinocyte proliferation, and co-expression of bcl-2 modestly suppressed the ras-mediated abnormal proliferation of neonatal keratinocytes. Bcl-2 proteins in keratinocytes protected UV-treated cells from apoptotic cell death regardless of oncogenic ras expression in both non-neoplastic neonatal epidermis and human keratinocyte cell lines. The spontaneous apoptotic index (AI) was also lower in papillomas constitutively expressing bcl-2 compared with the ones that developed in control mice. Ras-overexpressing epidermis, including that in ras/bcl-2 double transgenic mice, had abnormal differentiation patterns compared with controls. The oncogenic ras protein had alterations in both epidermal distribution and the extent of cytokeratin 14 and involucrin expression. Abnormal expression of the hyperproliferation marker cytokeratin 6 and modest down regulation of cytokeratin 1 were also detected. Late appearance of filaggrin was another abnormal phenotype of the ras-expressing epidermis. Overexpression of bcl-2 had no effect on epidermal differentiation. Together, these findings suggest that constitutive expression of oncogenic Ha-ras and bcl-2 are important determinants of epidermal proliferation, viability and differentiation. In summary, our results demonstrated that the disruption of epidermal homeostasis by overexpressed ras and bcl-2 predisposes to hyperplastic growth of the epidermis and to papilloma development and that these proteins with distinct mechanisms for oncogenesis are functionally synergistic for malignant transformation of chemically induced skin carcinogenesis. ^

第6章 取引額行列の推計におけるRAS 法の特徴

取引額表においてすべての要素は知られておらず、行および列の合計がベクトルとして既知であるとき、適当な行列を初期値にすることにより取引額の要素を推計することができる。比例反復法あるいはRAS 法は、繰り返し計算によって解を求めるそのような推計方法である。本章は正数を要素とする行列X (0) に対して任意の正数を要素とするベクトルa とb、D(a) をa を要素とする対角行列とするとき、初期値をX (0) と(0) ( ) (0) ( )X1 = D a X D b としたRAS 法による解は一致することを示す。RAS 法の解はその初期値においてD(a) とD(b) に対して無関係である。

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Impact on agronomic parameters in Vines and wine quality of foliar treatments with specific fractions of yeast derivatives

In hot Spanish climate, Toledo, Syrah and Sauvignon blanc Vineyard were treated in pre veraison with yeast derivatives RD-LM and RD- LA to stimulate phenolic and aromatic maturity respectively (application of yeast derivatives specifically designed to be used with the patent foliar application technology WO/2014/024039, Lallemand Inc. Canada). For studied effects in berry and wine composition three harvest time had been done. Experimented yeast derivatives had no significant effects on yield components and vegetative growth in both varieties. The Syrah RD-LM variety presented higher total and extractable anthocyanins and also more amount of tannins, although this last ones are not evident in the sensory analysis. The sensory analysis of wine has given very similar results in both varieties but with significant results in favored by phenols and tannins derived RD- LM and RD-LA respectively.

Quantitative analysis of morphological changes in yeast colonies growing on solid medium: the eccentricity and Fourier indices

The colony shape of four yeast species growing on agar medium wasmeasured for 116 days by image analysis. Initially, all the colonies are circular, with regular edges. The loss of circularity can be quantitatively estimated by the eccentricity index, Ei, calculated as the ratio between their orthogonal vertical and horizontal diameters. Ei can increase from 1 (complete circularity) to a maximum of 1.17–1.30, depending on the species. One colony inhibits its neighbour only when it has reached a threshold area. Then, Ei of the inhibited colony increases proportionally to the area of the inhibitory colony. The initial distance between colonies affects those threshold values but not the proportionality, Ei/area; this inhibition affects the shape but not the total surface of the colony. The appearance of irregularities in the edges is associated, in all the species, not with age but with nutrient exhaustion. The edge irregularity can be quantified by the Fourier index, Fi, calculated by the minimum number of Fourier coefficients that are needed to describe the colony contour with 99% fitness. An ad hoc function has been developed in Matlab v. 7.0 to automate the computation of the Fourier coefficients. In young colonies, Fi has a value between 2 (circumference) and 3 (ellipse). These values are maintained in mature colonies of Debaryomyces, but can reach values up to 14 in Saccharomyces.All the species studied showed the inhibition of growth in facing colony edges, but only three species showed edge irregularities associated with substrate exhaustion. Copyright © 2014 John Wiley & Sons, Ltd.

A simple mathematical model that describes the growth of the area and the number of total and viable cells in yeast colonies

We propose a model, based on the Gompertz equation, to describe the growth of yeasts colonies on agar medium. This model presents several advantages: (i) one equation describes the colony growth, which previously needed two separate ones (linear increase of radius and of the squared radius); (ii) a similar equation can be applied to total and viable cells, colony area or colony radius, because the number of total cells in mature colonies is proportional to their area; and (iii) its parameters estimate the cell yield, the cell concentration that triggers growth limitation and the effect of this limitation on the specific growth rate. To elaborate the model, area, total and viable cells of 600 colonies of Saccharomyces cerevisiae, Debaryomyces fabryi, Zygosaccharomyces rouxii and Rhodotorula glutinis have been measured. With low inocula, viable cells showed an initial short exponential phase when colonies were not visible. This phase was shortened with higher inocula. In visible or mature colonies, cell growth displayed Gompertz-type kinetics. It was concluded that the cells growth in colonies is similar to liquid cultures only during the first hours, the rest of the time they grow, with near-zero specific growth rates, at least for 3 weeks.

Combine use of Selected Schizosaccharomyces pombe and Lachancea thermotolerans Yeast Strains as an Alternative to the Traditional Malolactic Fermentation in Red Wine Production

Most red wines commercialized in the market use the malolactic fermentationprocess in order to ensure stability from a microbiological point of view. In this secondfermentation, malic acid is converted into L-lactic acid under controlled setups. Howeverthis process is not free from possible collateral effects that on some occasions produceoff-flavors, wine quality loss and human health problems. In warm viticulture regions suchas the south of Spain, the risk of suffering a deviation during the malolactic fermentationprocess increases due to the high must pH. This contributes to produce wines with highvolatile acidity and biogenic amine values. This manuscript develops a new red winemakingmethodology that consists of combining the use of two non-Saccharomyces yeast strains asan alternative to the traditional malolactic fermentation. In this method, malic acid is totallyconsumed by Schizosaccharomyces pombe, thus achieving the microbiological stabilizationobjective, while Lachancea thermotolerans produces lactic acid in order not to reduce andeven increase the acidity of wines produced from low acidity musts. This technique reducesthe risks inherent to the malolactic fermentation process when performed in warm regions.The result is more fruity wines that contain less acetic acid and biogenic amines than thetraditional controls that have undergone the classical malolactic fermentation.

Impact of using new commercial glutathione enriched inactive dry yeast oenological preparations on the aroma and sensory properties of wines

The effect of the addition of a commercial enriched glutathione inactive dry yeast oenological preparation in the volatile and sensory properties of industrially manufactured rosé Grenache wines was evaluated during their shelf-life. In addition, triangle tests were performed at different times during wine aging (among 1 and 9 months) to determine the sensory differences between wines with and without glutathione inactive dry yeast preparations. Descriptive sensory analysis with a trained panel was carried out when sensory differences in the triangle test were noticed. In addition, consumer tests were performed in order to investigate consumers’ acceptability of wines. Results revealed significant sensory differences between control and glutathione inactive dry yeast wines after 9 months of aging. At that time, glutathione inactive dry yeast wines were more intense in fruity aromas (strawberry, banana) and less intense in yeast notes than control wine. The impact of the glutathione inactive dry yeast in the aroma might be the consequence of different effects that these preparations could induce in wine composition: modification of yeast byproducts during fermentation, release of volatile compounds from inactive dry yeast, interaction of wine volatile compounds with yeast macromolecules from inactive dry yeast and a possible antioxidant effect of the glutathione released by the inactive dry yeast preparation on some specific volatile compounds.

Influence of the training system on the agronomic behavior and grape composition of Albariño in Rías Baixas apellation

The aim of this study was to evaluate if early defoliation can be an alternative to bunch thinning in limiting yield and improving quality in grapes of the white cultivar Loureiro (Vitis vinifera L.), grafted onto 1103P. The field trial had been set up in a commercial vineyard in Vinhos Verdes Region (Northwest of Portugal, 41º 48? 53? N, 8º 24? 42? W). Treatments studied, performed five days before full bloom were: LR5 ? Leaf removal of the first five basal leaves, performed manually, LR8 ? Leave removal of the first eight basal leaves, LRM ? mechanical leaf removal and C ? the control, without defoliation. This paper reports the results of four years (2010-2013). The results presented a significant removal of main leaf area after defoliation principally in the most intensive treatment (LR8) but at harvest, the total leaf area had been compensated by lateral regrowth and no statistical differences between the treatments and the control were found. Early defoliation caused a decrease in fruit set and also a significant reduction in the diameter of the berry within the more severe defoliation treatments (LR5 and LR8). Yield factors were also significantly affected by the defoliation, causing a reduction of bunch weight and in 2013 a yield reduction in LR8 and LRM, and in 2010 in LR8. Conversely, LR5 presented a yield always similar to the control C. The reduction of cluster compactness and the substantial improvement of the microclimate at the cluster level significantly reduced bunch rot incidence in the defoliated modalities compared to control. No carry-over effects, along the four years trial were observed Early defoliation proved to be a canopy management technique that can have a strong impact in the final quality of grapes, reducing the compactness and lower the incidence and intensity of bunch rot, even if the reduction of yield observed in other papers had not been observed in all modalities.

Specific interaction of the yeast cis-Golgi syntaxin Sed5p and the coat protein complex II component Sec24p of endoplasmic reticulum-derived transport vesicles

The generation of transport vesicles at the endoplasmic reticulum (ER) depends on cytosolic proteins, which, in the form of subcomplexes (Sec23p/Sec24p; Sec13p/Sec31p) are recruited to the ER membrane by GTP-bound Sar1p and form the coat protein complex II (COPII). Using affinity chromatography and two-hybrid analyses, we found that the essential COPII component Sec24p, but not Sec23p, binds to the cis-Golgi syntaxin Sed5p. Sec24p/Sed5p interaction in vitro was not dependent on the presence of [Sar1p⋅GTP]. The binding of Sec24p to Sed5p is specific; none of the other seven yeast syntaxins bound to this COPII component. Whereas the interaction site of Sec23p is within the N-terminal half of the 926-aa-long Sec24p (amino acid residues 56–549), Sed5p binds to the N- and C-terminal halves of the protein. Destruction by mutagenesis of a potential zinc finger within the N-terminal half of Sec24p led to a nonfunctional protein that was still able to bind Sec23p and Sed5p. Sec24p/Sed5p binding might be relevant for cargo selection during transport-vesicle formation and/or for vesicle targeting to the cis-Golgi.