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Objectives: To evaluate possible lipid catabolism and body fat regulation effects of 3-caffeoylquinic acid in Green coffee bean extract (GCBE) in high-fat diet (HFD)-induced obese mice., Methods: Obesity was induced in mice using a HFD for four weeks. Then, mice were fed only HFD or HFD with GCBE at 50, 100, and 200 mg/kg. Fatty acid synthesis mechanism regulation of body fat was investigated through real-time PCR and Western blot assay. Body fat reduction was measured through dual-energy X-ray absorptiometry., Results: In HFD-induced obese mice, GCBE treatment significantly decreased body weight gain, liver weight and white adipose tissue weights with regulation of adipose tissue lipolysis hormones, like adiponectin and leptin. GCBE treatment decreased mRNA expression levels of adipogenesis and adipocyte metabolism related genes in adipose tissues and the liver, and decreased the corresponding protein expression. Dual energy X-ray absorptiometry measurements were used to compare body fat between mice on high-fat and those treated with GCBE. GCBE treated mice had a lower fat mass compared to HFD alone fed mice and relative body weight and fat mass were markedly decreased., Conclusions: GCBE has a potential anti-obesity effect with lowering body fat accumulation by regulating adipogenesis and lipid metabolism-related genes and proteins in WAT and liver., (Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.)

Yam anthracnose caused by Colletotrichum gloeosporioides (C.g) is the most devastating disease of yam (Dioscorea sp.). In the present study, we evaluated the culture filtrate extract (CFE) of azalomycin-producing Streptomyces malaysiensis strain MJM1968 for the control of yam anthracnose. MJM1968 showed strong antagonistic activity against C.g in vitro. Furthermore, the MJM1968 CFE was tested for inhibition of spore germination in C.g, where it completely inhibited spore germination at a concentration of 50 μg/ml. To assess the in planta efficacy of the CFE and spores of MJM1968 against C.g, a detached leaf bioassay was conducted, which showed both the treatments suppressed anthracnose development on detached yam leaves. Furthermore, a greenhouse study was conducted to evaluate the CFE from MJM1968 as a fungicide for the control of yam anthracnose. The CFE non-treated plants showed a disease severity of >92% after 90 days of artificial inoculation with C.g, whereas the disease severity of CFE-treated and benomyl-treated yam plants was reduced to 26% and 15%, respectively, after 90 days. Analysis of the yam tubers from the CFE-treated and non-treated groups showed that tubers from the CFE-treated plants were larger than that of non-treated plants, which produced abnormal smaller tubers typical of anthracnose. This study demonstrated the utility of the CFE from S. malaysiensis strain MJM1968 as a biofungicide for the control of yam anthracnose.

Ecumicin is a novel anti-tuberculosis agent produced by Nonomuraea sp. MJM5123 as a new strain of actinomycetes. First, in order to increase the cell mass of Nonomuraea sp. MJM5123, we optimized the culture conditions with regard to carbon and nitrogen sources. The cell mass of Nonomuraea sp. MJM5123 increased by approximately twofold when glucose and soybean flour were used as carbon and nitrogen sources, respectively. For maximum production of ecumicin, we optimized the culture conditions by adding amino acids as building blocks for ecumicin, by adding vegetable oils and by controlling the temperature and pH. Ecumicin production was two times higher with the addition of valine as the building blocks for ecumicin compared with the production in the absence of valine. Interestingly, with the addition of 1% corn oil, the production of ecumicin increased by 4.6-fold compared with the production in the absence of corn oil. Finally, by controlling the pH and temperature, we established an optimized culture condition in which Nonomuraea sp. MJM5123 produced 576 mg ecumicin per litre of medium, which is about 50 times higher than in the control medium at 30 °C and pH 7.0.

In recent years, microalgae have attracted considerable interest as a biofuel resource owing to their rapid growth, tolerance to harsh conditions, and ability to accumulate a large amount of triacylglycerols (TAGs). However, the economic effectiveness of algal biofuel is still low. In this study, we attempted to increase oil production of the microalga Scenedesmus quadricauda by elevating intracellular malonyl-CoA and glycerol-3-phosphate (G3P) pools. To increase intracellular oil content, yeast-derived genes encoding acetyl-CoA carboxylase (ACC1), glycerol kinase (GPD1), and glycerol-3-phosphate dehydrogenase (GUT1) were overexpressed under the control of CaMV 35S and NOS promoters with SV40 large T antigen components. Fatty acid profiling, G3P content, and the number of cells with high oil content were analyzed by gas chromatography-mass spectrometry, G3P assay kit, and flow cytometry, respectively. Overexpression of ACC1 increased the total fatty acid content by 1.6-fold. Overexpression of GPD1 and GUT1 increased intracellular G3P content by 1.6- and 1.9-fold, respectively. Multi-gene expression of ACC1, GPD1, and GUT1 increased the number of cells with high oil content by 1.45-fold compared with that observed with the wild-type. This study is the first to report increased oil production by overexpression of the key genes (ACC1, GPD1, and GUT1) for TAG biosynthesis in microalgae.

Key Message: The loss-of-function mutants of the Arabidopsis orthologue of the wheat LRK10 gene shows ABA-insensitive and drought stress-sensitive phenotypes, suggesting that LRK10L1.2 is positively involved in ABA signaling. A subset of receptor-like kinases (RLKs) superfamily proteins play a key role in sensing internal and external signals. A gene encoding Arabidopsis thaliana Leaf rust 10 disease-resistance locus receptor-like protein kinase 1 (AtLRK10L1), most closely related to wheat LRK10, expresses two different transcripts, LRK10L1.1 and LRK10L1.2, using alternative promoters. The T-DNA insertion mutant, lrk10l1-2, that specifically shuts down LRK10L1.2 transcription displayed an abscisic acid (ABA)-insensitive phenotype in seed germination and seedling growth. However, the lrk10l1.2 mutant exhibited reduced tolerance to drought stress, compared with wild type, which is accompanied by alteration of stomatal apertures. The transgenic plants overexpressing full-length LRK10L1.2, which localizes to the plasma membrane (PM) complemented the phenotypes of lrk10l1-2 mutant background, while those expressing LRK10L1.2 Nu1, which switched its localization to the endoplasmic reticulum (ER) by skipping of a mini-exon, showed even higher ABA insensitivity and drought sensitivity than its mutant background. Our results suggest that ABA signaling involves the PM-localized LRK10L1.2.

Key Message: AtLRK10L1.2 produces a variety of alternatively spliced variants in the region a mini-exon and skipping of the mini-exon alters the subcellular localization of the protein. We have examined expression and alternative splicing in the gene encoding Arabidopsis LRK10-like 1 (AtLRK10L1) which is most closely related to wheat leaf rust 10 disease-resistance locus receptor-like protein kinase (LRK10). AtLRK10L1 produces two different transcripts, LRK10L1.1 and 1.2 through the use of two different promoters. We found no evidence of alternative splicing for the AtLRK10L1.1 transcript but identified numerous alternative splicing variants of AtLRK10L1.2 by sequencing of cloned cDNAs prepared from RNA isolated from whole cell, nucleolar and nucleoplasmic fractions. Many of these transcripts contained unspliced introns and accumulated differentially in the nucleolus and the nucleoplasm consistent with intron retention transcripts being retained in the nucleus (Göhring et al., Plant Cell 26:754-764, 2014). We examined the fate of different alternatively spliced transcripts by fusing variants to YFP and expressing them by agroinfiltration in Nicotiana benthamiana. AtLRK10L1 contains a 45 nt mini-exon which encodes part of a putative transmembrane domain. Full-length cDNA of LRK10L1.2 fused to YFP targeted the fusion protein to the plasma membrane while expression of transcripts where the mini-exon had been deleted, altered the localization of the fusion protein to the endoplasmic reticulum. Similarly, expression of full-length and mini-exon deleted versions of three other members of the LRK10 receptor-like kinase (RLK) gene family also showed the switch in localization. Thus, the mini-exons in Arabidopsis LRK10 genes are required for localization to the plasma membrane.

Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H2O2). Here we show that PknA phosphorylation of unprocessed proteasome β-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H2O2 and that H2O2 stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.

Bacteriophytochromes (BphP) are phytochrome-like light sensing proteins in bacteria, which use biliverdin as a chromophore. In order to study the biochemical properties of the DrBphP protein, five (2B8, 2C11, 3B2, 3D2, and 3H7) anti-DrBphP monoclonal antibodies were produced through the immunization of mice with purified full-length DrBphP and DrBphN (1-321 amino acid) proteins, and epitope mapping was then carried out. Among the five antibodies, 2B8 and 2C11 preferentially recognized the N-terminal region of BphP whereas 3B2, 3D2, and 3H7 showed preference for the C-terminal region. We performed further epitope mapping using recombinant truncated BphP proteins to narrow down their target sequences. The results demonstrated that each of the five monoclonal antibodies recognized different regions on the DrBphP protein. Additionally, epitopes of 2B8 and 3H7 antibodies were discovered to be shorter than 10 amino acids (2B8: RDPLPFFPP, 3H7: PGEIEEA). These two antibodies with such specific recognition epitopes could be especially valuable for developing new peptide tags for protein detection and purification., (© 2014 The Protein Society.)

Biological control and plant growth promotion by plant beneficial microbes has been viewed as an alternative to the use of chemical pesticides and fertilizers. Bacteria and fungi that are naturally associated with plants and have a beneficial effect on plant growth by the alleviation of biotic and abiotic stresses were isolated and developed into biocontrol (BCA) and plant growth-promoting agents (PGPA). Actinobacteria are a group of important plant-associated spore-forming bacteria, which have been studied for their biocontrol, plant growth promotion, and interaction with plants. This review summarizes the effects of actinobacteria as BCA, PGPA, and its beneficial associations with plants.

The regulatory function of extracellular ATP (exATP) in bacteria is unknown, but recent studies have demonstrated exATP induced enhanced secondary metabolite production and morphological differentiation in Streptomyces coelicolor. The growth of Streptomyces coelicolor, however, was unaffected by exATP, although changes in growth are common phenotypes. To identify bacteria whose growth is altered by exATP, we measured exATP-induced population changes in fast-growing microbes and actinomycetes in compost. Compared with the water-treated control, the addition of 10 ml 100 μM ATP to 10 g of compost enhanced the actinomycetes population by 30% and decreased fast-growing microbial numbers by 20%. Eight microbes from each group were selected from the most populated colony, based on appearance. Of the eight isolated fast-growing microbes, the 16S rRNA sequences of three isolates were similar to the plant pathogens Serratia proteamaculans and Sphingomonas melonis, and one was close to a human pathogen, Elizabethkingia meningoseptica. The growth of all fast-growing microbes was inhibited by ATP, which was confirmed in Pseudomonas syringae DC3000, a pathogenic plant bacterium. The growth of six of eight isolated actinomycetes strains, all of which were identified as close to Streptomyces neyagawaensis, was enhanced by ATP treatment. This study suggests that exATP regulates bacterial physiology and that the exATP response system is a target for the control of bacterial ecology.