REV1 protein interacts with PCNA: significance of the REV1 BRCT domain in vitro and in vivo

REV1 protein, a eukaryotic member of the Y family of DNA polymerases, is involved in the tolerance of DNA damage by translesion DNA synthesis. It is unclear how REV1 is recruited to replication foci in cells. Here, we report that mouse REV1 can bind directly to PCNA and that monoubiquitylation of PCNA enhances this interaction. The interaction between REV1 protein and PCNA requires a functional BRCT domain located near the N terminus of the former protein. Deletion or mutational inactivation of the BRCT domain abolishes the targeting of REV1 to replication foci in unirradiated cells, but not in UV-irradiated cells. In vivo studies in both chicken DT40 cells and yeast directly support the requirement of the BRCT domain of REV1 for cell survival and DNA damage-induced mutagenesis.     

MicroRNA Profiling in Prostate Cancer - The Diagnostic Potential of Urinary miR-205 and miR-214

Prostate cancer (PCa) is the most common type of cancer in men in the United States, which disproportionately affects African American descents. While metastasis is the most common cause of death among PCa patients, no specific markers have been assigned to severity and ethnic biasness of the disease. MicroRNAs represent a promising new class of biomarkers owing to their inherent stability and resilience. In the present study, we investigated potential miRNAs that can be used as biomarkers and/or therapeutic targets and can provide insight into the severity and ethnic biasness of PCa. PCR array was performed in FFPE PCa tissues (5 Caucasian American and 5 African American) and selected differentially expressed miRNAs were validated by qRT-PCR, in 40 (15 CA and 25 AA) paired PCa and adjacent normal tissues. Significantly deregulated miRNAs were also analyzed in urine samples to explore their potential as non-invasive biomarker for PCa. Out of 8 miRNAs selected for validation from PCR array data, miR-205 (p<0.0001), mir-214 (p<0.0001), miR-221(p<0.001) and miR-99b (p<0.0001) were significantly downregulated in PCa tissues. ROC curve shows that all four miRNAs successfully discriminated between PCa and adjacent normal tissues. MiR-99b showed significant down regulation (p<0.01) in AA PCa tissues as compared to CA PCa tissues and might be related to the aggressiveness associated with AA population. In urine, miR-205 (p<0.05) and miR-214 (p<0.05) were significantly downregulated in PCa patients and can discriminate PCa patients from healthy individuals with 89% sensitivity and 80% specificity. In conclusion, present study showed that miR-205 and miR-214 are downregulated in PCa and may serve as potential non-invasive molecular biomarker for PCa.     

A study on sex-determination and karyotypic evolution inTetranychidae

The chromosome complements of 45 species of spider mites (Tetranychidae) were studied, making the total number of species now examined in this family 57, approximately 10% of all species known. The chromosome numbers range fromn=2 ton=7. The modal number of the family is 3 (found in 44% of the species). It is argued that the ancestral number isn=2 (21% of the species).In the more primitive subfamily of theBryobiinae both thelytokous and arrhenotokous species occur, whereas the subfamily of theTetranychinae exclusively exhibits arrhenotoky. The karyotype evolution is discussed in connection with arrhenotoky. It is stated that karyotype information is a useful tool for the spider mite taxonomist.This study was financially supported by the Netherlands Foundation for the Advancement of Tropical Research (W.O.T.R.O.) and the Uyttenboogaart-Eliasen Foundation.     

The fluorescent cationic dye rhodamine 6G as a probe for membrane potential in bovine aortic endothelial cells.

The membrane potential of cultured bovine aortic endothelial cells was assessed by a fluorescent probe as an alternative to direct methods. We used the fluorescent cationic dye rhodamine 6G, a lipophilic probe with high permeability in cell membranes. A linear relationship was obtained between fluorescence intensity (F.I.) and membrane potential (Em) as a function of the extracellular Na(+) concentration in the presence of the ionophore gramicidin. From the equation derived from the linear relationship F.I. = -0.004 Em + 0. 03 (P < 0.001), the fluorescence measurements could be converted to membrane potential. The resting plasma membrane potential obtained was -65 +/- 7 mV. Nigericin (27 microM), ouabain (1 mM), and bradykinin (20 nM) induced a decrease in F.I. (depolarization), while ATP (25-100 microM) induced an increase in F.I. (hyperpolarization). Mitochondrial membrane potential inhibitors myxothiazol (3 microM) and oligomycin (4 microM) did not influence F. I. measured in the cultured bovine aortic endothelial cells. The results indicate that rhodamine 6G can be used as a sensitive and specific dye in studies of substances that affect the membrane potential of endothelial cells.     

The GABA Paradox

GABA, which is present in the brain in large amounts, is distributed among distinctly different cellular pools, possibly reflecting its multiple functions as metabolite, neurotransmitter, and neurotrophin. Its metabolic enzymes also exhibit heterogeneity, because glutamate decarboxylase exists in two isoforms with different subcellular distribution and regulatory properties. Moreover, recent evidence points to a more pronounced regulatory role of the tricarboxylic acid cycle than hitherto anticipated in the biosynthetic machinery responsible for formation of GABA from glutamine. Additionally, GABAergic neurons may contain distinct populations of mitochondria having different turnover rates of the tricarboxylic acid cycle with different levels of association with GABA synthesis from 2-oxoglutarate via glutamate. These aspects are discussed in relation to the different functional roles of GABA and its prominent involvement in epileptogenic activity.     

Genetic affinities between adjacent populations of spider mites (Tetranychus urticae Koch)

The interbreeding potentials of nine populations of spider mites were examined. The populations were gathered from horticultural crops in different glasshouses in a limited area of Aalsmeer. A great variety of genetic incompatibilities between these populations was found. In most crossing a partial sterility of the hybrids, as measured by egg hatchability, occurred. Some crosses showed reciprocal differences. It was suggested that genetic divergence of these adjacent populations arises especially from the haplo-diploid nature of spider mites.

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Zusammenfassung Die Kreuzbarkeit von neun Spinnmilben-Populationen wurde untersucht. Die Populationen wurden von Gartenpflanzen verschiedener Gewächshäuser in einem bestimmten Gebiet von Aalsmeer gesammelt. Es zeigte sich eine große Variabilität in der genetischen Verträglichkeit zwischen diesen Populationen. Bei den meisten Kreuzungen trat, gemessen an der Schlüpffähigkeit der Eier, eine partielle Sterilität der Hybriden auf. Einige Kreuzungen wiesen reziproke Unterschiede auf. Es wird vermutet, daß die genetische Divergenz dieser aneinandergrenzenden Populationen speziell auf der haplo-diploiden Natur der Spinnmilben beruht.

     

Xylose fermentation by genetically modified Saccharomyces cerevisiae 259ST in spent sulfite liquor

Spent sulfite pulping liquor (SSL) is a high-organic content byproduct of acid bisulfite pulp manufacture which is fermented to make industrial ethanol. SSL is typically concentrated to 240 g/l (22% w/w) total solids prior to fermentation, and contains up to 24 g/l xylose and 30 g/l hexose sugars, depending upon the wood species used. The xylose present in SSL is difficult to ferment using natural xylose-fermenting yeast strains due to the presence of inhibitory compounds, such as organic acids. Using sequential batch shake flask experiments, Saccharomyces cerevisiae 259ST, which had been genetically modified to ferment xylose, was compared with the parent strain, 259A, and an SSL adapted strain, T2, for ethanol production during SSL fermentation. With an initial SSL pH of 6, without nutrient addition or SSL pretreatment, the ethanol yield ranged from 0.32 to 0.42 g ethanol/g total sugar for 259ST, compared to 0.15-0.32 g ethanol/g total sugar for non-xylose fermenting strains. For most fermentations, minimal amounts of xylitol (<1 g/l) were produced, and glycerol yields were approximately 0.12 g glycerol/g sugar consumed. By using 259ST for SSL fermentation up to 130% more ethanol can be produced compared to fermentations using non-xylose fermenting yeast.     

Demonstration of Neuron-Glia Transfer of Precursors for Gaba Biosynthesis in a Co-Culture System of Dissociated Mouse Cerebral Cortex

Co-cultures of neurons and astrocytes were prepared from dissociated embryonic mouse cerebral cortex and cultured for 7 days. To investigate if these cultures may serve as a functional model system to study neuron-glia interaction with regard to GABA biosynthesis, the cells were incubated either in media containing [U-¹³C]glutamine (0.1, 0.3 and 0.5 mM) or 1 mM acetate plus 2.5 mM glucose plus 1 mM lactate. In the latter case one of the 3 substrates was uniformly ¹³C labeled. Cellular contents and ¹³C labeling of glutamate, GABA, aspartate and glutamine were determined in the cells after an incubation period of 2.5 h. The GABA biosynthetic machinery exhibited the expected complexity with regard to metabolic compartmentation and involvement of TCA cycle activity as seen in other culture systems containing GABAergic neurons. Metabolism of acetate clearly demonstrated glial synthesis of glutamine and its transfer to the neuronal compartment. It is concluded that this co-culture system serves as a reliable model in which functional and pharmacological aspects of GABA biosynthesis can be investigated. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella. An erratum to this article can be found at