Factors affecting incidence and severity of leaf spot disease on lettuce caused by Septoria birgitae

In the 1990s during wet seasons a new disease causing brown leaf spots on lettuce (Lactuca sativa) was found for the first time in many lettuce‐growing areas of Austria and Germany. The causal agent, a new pathogenic species called Septoria birgitae, may be responsible for total crop loss. To study how temperature, inoculum density and leaf wetness period influence disease incidence and severity of leaf spot on lettuce caused by S. birgitae, we carried out in vivo experiments in growth chambers and in the field. Additionally, we evaluated the relevance of infected plant debris acting as a primary inoculum source in soil for subsequent crops. S. birgitae produces spores over a wide temperature range between 5°C and 30°C, and can infect plants at temperatures between 10°C and 30°C, with an optimum between 20°C and 30°C. Spores of S. birgitae at a density of at least 10³ conidia mL^–1 are essential for disease outbreak on lettuce. Because leaf wetness is crucial for releasing conidia from pycnidia, we studied the impact of leaf wetness duration on disease development under various temperature conditions. For relevant leaf spot disease development on lettuce in vivo, a leaf wetness duration of at least 24 h and temperatures higher than 10°C were necessary. Leaf spot disease development in the field required several leaf wetness periods longer than 20 h at approximately 15°C at the beginning of crop cultivation. Incorporating S. birgitae infected plant debris in soil as a primary inoculum was not relevant for leaf spot disease outbreak in the next year. However, in cases of continuous cropping of lettuce on the same field and in the same season, Septoria‐infected lettuce debris may become more relevant.     

Immunohistochemical Evaluation of Global DNA Methylation: Comparison with in Vitro Radiolabeled Methyl Incorporation Assay

The in vitro radiolabeled methyl incorporation assay, a commonly used technique to evaluate global methylation of DNA, has some disadvantages and limitations. The purpose of the present study was to compare the results of global DNA methylation evaluated by radiolabeled methyl incorporation (CPM/μg of DNA) with immunohistochemical staining of the same tissue sections with a monoclonal antibody developed against 5-methylcytosine (5-mc). We used archival specimens of squamous cell cancer (SCC) of the human lung with a matched uninvolved specimen (n = 18 pairs) and 18 lung specimens from subjects without lung cancer (noncancer specimens) to make this comparison. The immunostaining for 5-mc was reported as a percentage of cells positive for staining as well as a weighted average of the intensity score. The results suggested that both radiolabeled methyl incorporation assay and immunostaining for 5-mc can be used to demonstrate hypomethylation of DNA in SCC tissues compared to matched uninvolved tissues. An advantage of immunostaining, however, is its ability to demonstrate hypomethylation of SCC compared to adjacent bronchial mucosa on the same archival specimen, obviating the need to use sections from both SCC and matched uninvolved tissues. Only by using the immunostaining technique were we able to document a statistically significant difference in DNA methylation between SCC and noncancer tissues. We conclude that the immunostaining technique has advantages over the radiolabeled methyl incorporation assay and may be best suited for evaluation of global DNA methylation when the methylation status of cancer cannot be normalized by methyl incorporation of normal tissues or when the number of samples available for evaluation is small.     

The SBP2 protein central to selenoprotein synthesis contacts the human ribosome at expansion segment 7L of the 28S rRNA

SBP2 is a pivotal protein component in selenoprotein synthesis. It binds the SECIS stem–loop in the 3′ UTR of selenoprotein mRNA and interacts with both the specialized translation elongation factor and the ribosome at the 60S subunit. In this work, our goal was to identify the binding partners of SBP2 on the ribosome. Cross-linking experiments with bifunctional reagents demonstrated that the SBP2-binding site on the human ribosome is mainly formed by the 28S rRNA. Direct hydroxyl radical probing of the entire 28S rRNA revealed that SBP2 bound to 80S ribosomes or 60S subunits protects helix ES7L-E in expansion segment 7 of the 28S rRNA. Diepoxybutane cross-linking confirmed the interaction of SBP2 with helix ES7L-E. Additionally, binding of SBP2 to the ribosome led to increased reactivity toward chemical probes of a few bases in ES7L-E and in the universally conserved helix H89, indicative of conformational changes in the 28S rRNA in response to SBP2 binding. This study revealed for the first time that SBP2 makes direct contacts with a discrete region of the human 28S rRNA.     

Ovarian Cancer Cell Heparan Sulfate 6-O-Sulfotransferases Regulate an Angiogenic Program Induced by Heparin-binding Epidermal Growth Factor (EGF)-like Growth Factor/EGF Receptor Signaling

Heparan sulfate (HS) is a component of cell surface and extracellular matrix proteoglycans that regulates numerous signaling pathways by binding and activating multiple growth factors and chemokines. The amount and pattern of HS sulfation are key determinants for the assembly of the trimolecular, HS-growth factor-receptor, signaling complex. Here we demonstrate that HS 6-O-sulfotransferases 1 and 2 (HS6ST-1 and HS6ST-2), which perform sulfation at 6-O position in glucosamine in HS, impact ovarian cancer angiogenesis through the HS-dependent HB-EGF/EGFR axis that subsequently modulates the expression of multiple angiogenic cytokines. Down-regulation of HS6ST-1 or HS6ST-2 in human ovarian cancer cell lines results in 30–50% reduction in glucosamine 6-O-sulfate levels in HS, impairing HB-EGF-dependent EGFR signaling and diminishing FGF2, IL-6, and IL-8 mRNA and protein levels in cancer cells. These cancer cell-related changes reduce endothelial cell signaling and tubule formation in vitro. In vivo, the development of subcutaneous tumor nodules with reduced 6-O-sulfation is significantly delayed at the initial stages of tumor establishment with further reduction in angiogenesis occurring throughout tumor growth. Our results show that in addition to the critical role that 6-O-sulfate moieties play in angiogenic cytokine activation, HS 6-O-sulfation level, determined by the expression of HS6ST isoforms in ovarian cancer cells, is a major regulator of angiogenic program in ovarian cancer cells impacting HB-EGF signaling and subsequent expression of angiogenic cytokines by cancer cells.     

Sulfite formation by wine yeasts

Five different strains of wine yeasts were investigated with respect to active uptake of [³⁵S] sulfate and its regulation by methionine. Considerable differences exist between low and high sulfite-producing strains in the initial velocity of sulfate uptake. Further differences were established in repression of sulfate permease by l-methionine, most evident in a total lack of repression in one of the high sulfite producers. These findings explain in part variable sulfite and sulfide formation.List of Abbreviations CCMP carbonylcyanide-p-trifluormethoxyphenylhydrazone     

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding

Activated sulfate in the form of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is needed for all sulfation reactions in eukaryotes with implications for the build-up of extracellular matrices, retroviral infection, protein modification, and steroid metabolism. In metazoans, PAPS is produced by bifunctional PAPS synthases (PAPSS). A major question in the field is why two human protein isoforms, PAPSS1 and -S2, are required that cannot complement for each other. We provide evidence that these two proteins differ markedly in their stability as observed by unfolding monitored by intrinsic tryptophan fluorescence as well as circular dichroism spectroscopy. At 37 °C, the half-life for unfolding of PAPSS2 is in the range of minutes, whereas PAPSS1 remains structurally intact. In the presence of their natural ligand, the nucleotide adenosine 5'-phosphosulfate (APS), PAPS synthase proteins are stabilized. Invertebrates only possess one PAPS synthase enzyme that we classified as PAPSS2-type by sequence-based machine learning techniques. To test this prediction, we cloned and expressed the PPS-1 protein from the roundworm Caenorhabditis elegans and also subjected this protein to thermal unfolding. With respect to thermal unfolding and the stabilization by APS, PPS-1 behaved like the unstable human PAPSS2 protein suggesting that the less stable protein is evolutionarily older. Finally, APS binding more than doubled the half-life for unfolding of PAPSS2 at physiological temperatures and effectively prevented its aggregation on a time scale of days. We propose that protein stability is a major contributing factor for PAPS availability that has not as yet been considered. Moreover, naturally occurring changes in APS concentrations may be sensed by changes in the conformation of PAPSS2.     

汉阳陵文物表面硫酸盐形成原因微生物学证据

用仅含硫源的无机盐培养基对采自汉阳陵13号和15号遗址坑文物表面的土样进行细菌富集筛选实验,并对分离的菌株进行生理生化特性研究、16SrDNA序列分析及硫代谢能力测试。分离得到5株细菌:TD1、TD2、TD3、TD15及PL13。其中TD1属于Alcaligenes,TD3属于Bacillus;硫代谢能力测试结果显示TD2、TD15和PL13代谢产生硫酸盐的速率远远小于TD3和TD1,TD3氧化硫源生成硫酸盐的速率是TD1的1.4倍。实验证实这些菌株均能氧化S、S2O32-和S2-生成硫酸盐从而获得能量,这表明微生物氧化土壤中硫化物是汉阳陵文物表面硫酸盐增加的原因,从而为汉阳陵文物表面硫酸盐的形成原因提供了微生物学依据。     

G protein β subunit is closely associated with microtubules

Previously, we have identified the association of G protein β subunit (Gβ) with mitotic spindles in various mammalian cells. Since microtubules are the main component of mitotic spindles, here we have isolated bovine brain microtubules and purified Gβ subunit to identify the close association of Gβ subunit with purified brain microtubules and have shown the direct incorporation of Gβ subunit into the microtubules both in vitro and in vivo. It was found that: (1) microtubular fraction isolated from bovine brain contained Gβ subunit, (2) coimmunoprecipitation demonstrated that Gβ subunit could be coprecipitated with tubulin, (3) addition of purified Gβ subunit into cytosolic extract for microtubule assembly caused direct incorporation of Gβ subunit into assembled microtubules and increased the association of microtubule-associated proteins with microtubules, and (4) incubation of exogenous Gβ subunit with detergent-permeabilized cells resulted in direct incorporation of Gβ subunit into microtubule fibers and depolymerized tubulin molecules. We conclude that G protein β subunit is closely associated with microtubules and may play an important role in the regulation of microtubule formation in addition to its regulatory role in cellular signal transduction. J. Cell. Biochem. 70:553–562, 1998. © 1998 Wiley-Liss, Inc.     

α-1 acid glycoprotein inhibits insulin responses by glucose oxidation,protein synthesis and protein breakdown in mouse C2C12 myotubes

Increased plasma α-1 acid glycoprotein (AGP) is correlated with reduced growth rates in neonatal swine. The specific physiological mechanisms contributing to this relationship are unknown. This study was performed to determine if AGP can modify muscle metabolism by examining glucose oxidation and protein synthesis in the C2C12 muscle cell line. Cells were used for experiments 4 days post-fusion as myotubes. Myotubes were exposed to AGP for 24 h, with the last 4 h used to monitor ¹⁴C-glucose oxidation or to measure protein synthesis by incorporation of ³H-tyrosine. Treatment of C2C12 myotubes with mouse AGP (100 µg/ml) reduced glucose oxidation (P<0.01, n=3 trials), whereas bovine insulin (1 µM) stimulated glucose oxidation (P<0.05, n=3 trials). Treatment with AGP in combination with insulin reduced ¹⁴C-glucose oxidation (P<0.05, n=3 trials), similar to the effect of AGP alone. Glucose transport, as measured by ³H-deoxyglucose uptake, was increased by 38% with 1 µM insulin (P<0.05, n=3 trials), whereas AGP alone increased glucose uptake by 36% (P<0.05, n=3 trials). The combination of insulin and AGP in the medium resulted in an 88% increase in glucose uptake (P<0.01, n=3 trials). Protein synthesis was measured by ³H-tyrosine incorporation into C2C12 myotubes. Insulin stimulated a 18% increase in ³H-tyrosine incorporation (P<0.05, n=6 trials). The incorporation of ³H-tyrosine into myotubes was reduced by 20% with AGP incubation (P<0.01, n=6 trials), like the 20% decrease in ³H-tyrosine incorporation in response to the combination of AGP and insulin (P<0.01, n=6 trials). Protein breakdown, as measured by the release of ³H-tyrosine from C2C12 myotubes, was reduced 27% by insulin (P<0.01, n=6 trials). Treatment with AGP had no effect on protein breakdown (P>0.05, n=6 trials), whereas incubation with both AGP and insulin reduced ³H-tyrosine release by 15% (P<0.01, n=6 trials). First, these data indicate that the acute phase protein AGP can interact with the skeletal muscle to reduce glucose oxidation, but this is not the result of an effect on glucose transport. Second, AGP can specifically reduce protein synthesis. Lastly, AGP can inhibit insulin-stimulated glucose oxidation, protein synthesis and breakdown.