Dissecting the telomere region of barley chromosome 5HL using rice genomic sequences as references: new markers for tracking a complex region in breeding

The terminal region of barley chromosome 5HL controls malt extract, diastatic power, free amino acid nitrogen, alpha-amylase activity, seed dormancy and pre-harvest sprouting. Comparative analysis of the barley and rice maps has established that the terminal region of barley chromosome 5HL is syntenic to rice chromosome 3L near the telomere end. The rice BAC (Bacterial Artificial Chromosome) sequences covering the region of chromosome 3L were used to search barley expressed sequenced tags database. Thirty-three genes were amplified by PCR (polymerase chain reaction) with the primers designed from barley ESTs (expressed sequence tag). Comparison of the sequences of the PCR generated DNA fragments revealed polymorphisms including single nucleotide polymorphism (SNP), insertions or deletions between the barley varieties. Seven new PCR based molecular markers were developed and mapped within 10 cM in three doubled haploid barley populations (Stirling × Harrington, Baudin × AC Metcalfe and Chebec × Harrington). The mapped genes maintain the micro-syntenic relationship between barley and rice. These gene specific markers provide simple and efficient tools for germplasm characterization and marker-assisted selection for barley malting quality, and ultimately lead to isolation and identification of the major gene(s) controlling multiple quality traits on barley chromosome 5HL.     

Mutant telomerase RNAs induce DNA damage and apoptosis via the TRF2-ATM pathway in telomerase-overexpressing primary fibroblasts

Mutant template human telomerase RNAs (MT-hTers) have been shown to induce apoptosis in various cancer cells with high telomerase activity. However, the mechanism by which MT-hTers inhibit the growth of cancer cells and their effects on normal cells remain unknown. To determine the effects of MT-hTers on normal cells, MT-hTer-47A and -AU5 were introduced into IMR90 lung fibroblasts, which have low telomerase levels. Growth of IMR90 cells after MT-hTers infection was not significantly impaired; however, similar treatments in telomerase-overexpressing IMR90 [IMR90 wild-type (WT)hTERT] cells inhibited cell proliferation and induced apoptosis. Confocal microscopy showed that MT-hTers induced DNA damage foci (i.e. 53BP1 and γ-H2AX) in IMR90 WThTERT cells. Microarray analysis revealed that GADD45γ was significantly elevated in MT-hTer-treated IMR90 WThTERT cells. MT-hTers also induced ATM phosphorylation at Ser1981 in IMR90 WThTERT cells, and western blot analysis revealed high levels of phosphorylated p53 after the down-regulation of cellular TRF2 expression in MT-hTer-treated IMR90 WThTERT cells. Taken together, we have shown that MT-hTers induce double-stranded DNA break-like damages in telomerase positive IMR90 WThTERT cells after phosphorylation of ATM and p53 via suppression of TRF2, which may eventually lead to apoptosis via elevation of GADD45γ.     

High throughput screening of hydrolytic enzymes from termites using a natural substrate derived from sugarcane bagasse

Background

Short rotation coppice willow is a potential lignocellulosic feedstock in the United Kingdom and elsewhere; however, research on optimising willow specifically for bioethanol production has started developing only recently. We have used the feedstock Salix viminalis × Salix schwerinii cultivar 'Olof' in a three-month pot experiment with the aim of modifying cell wall composition and structure within the stem to the benefit of bioethanol production. Trees were treated for 26 or 43 days with tension wood induction and/or with an application of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile that is specific to secondary cell walls. Reaction wood (tension and opposite wood) was isolated from material that had received the 43-day tension wood induction treatment.

Results

Glucan content, lignin content and enzymatically released glucose were assayed. All measured parameters were altered without loss of total stem biomass yield, indicating that enzymatic saccharification yield can be enhanced by both alterations to cell wall structure and alterations to absolute contents of either glucan or lignin.

Conclusions

Final glucose yields can be improved by the induction of tension wood without a detrimental impact on biomass yield. The increase in glucan accessibility to cell wall degrading enzymes could help contribute to reducing the energy and environmental impacts of the lignocellulosic bioethanol production process.     

Rapid cultivation of stable aerobic phenol-degrading granules using acetate-fed granules as microbial seed

cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 °C in amount corresponding to 8% of the cell soluble protein (0.6 U mg⁻¹). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 °C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.     

The use of sewage sludge and horticultural waste to develop artificial soil for plant cultivation in Singapore

Greenhouse pot experiments were performed with Ipomoea aquatica (Kang Kong) to evaluate artificial soil produced from poor fertility subsoil, horticultural compost, and sewage sludge. The addition of horticultural compost and sewage sludge to subsoil substantially improved plant growth, improved the physical properties of subsoil and enriched subsoil by essential nutrients for plants. The effect was enhanced when the two ingredients were added to subsoil together. The highest yield of biomass of I. aquatica was observed in artificial soil prepared by mixing subsoil with 4% (wet weight/wet weight) of horticultural compost and 2% (dry weight/wet weight) of sewage sludge. The contents of heavy metals in plants, grown in the artificial soil, were significantly lower than toxic levels. The artificial soil could be recommended for urban landscaping and gardening in Singapore.     

G protein-gated inhibitory module of N-type (ca(v)2.2) ca2+ channels

Voltage-dependent G protein (Gbetagamma) inhibition of N-type (CaV2.2) channels supports presynaptic inhibition and represents a central paradigm of channel modulation. Still controversial are the proposed determinants for such modulation, which reside on the principal alpha1B channel subunit. These include the interdomain I-II loop (I-II), the carboxy tail (CT), and the amino terminus (NT). Here, we probed these determinants and related mechanisms, utilizing compound-state analysis with yeast two-hybrid and mammalian cell FRET assays of binding among channel segments and G proteins. Chimeric channels confirmed the unique importance of NT. Binding assays revealed selective interaction between NT and I-II elements. Coexpressing NT peptide with Gbetagamma induced constitutive channel inhibition, suggesting that the NT domain constitutes a G protein-gated inhibitory module. Such inhibition was limited to NT regions interacting with I-II, and G-protein inhibition was abolished within alpha1B channels lacking these NT regions. Thus, an NT module, acting via interactions with the I-II loop, appears fundamental to such modulation.     

The antimicrobial activity of extracts of the lichen Hypogymnia tubulosa and its 3-hydroxyphysodic acid constituent

The antimicrobial activity and the MIC values of the diethyl ether, acetone, chloroform, petroleum ether, and ethanol extracts of the lichen Hypogymnia tubulosa and its 3-hydroxyphysodic acid constituent have been investigated against some microorganisms. At least one of the extracts or 3-hydroxyphysodic acid showed antimicrobial activity against Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Listeria monocytogenes, Proteus vulgaris, Salmonella typhimurium, Staphylococcus aureus, Streptococcus faecalis, and Candida albicans. No antifungal activity of the extracts has been observed against ten filamentous fungi.     

实验动物皮肤病原真菌检测方法的改良

目的对实验动物皮肤病原真菌2种培养方法进行了比较。方法将采集到的3只皮肤真菌感染病兔样品经由沙氏平皿法和沙氏试管斜面培养法分别进行培养。结果在3只真菌感染病兔中应用试管斜面法我们只检测到1例皮肤病原真菌阳性,而采用沙氏平皿法3例阳性全部检出。结论结合临床检测经验,我们认为本研究的沙氏平皿法优于沙氏试管斜面法,在实验动物皮肤病原真菌常规检测中具有推广应用价值。