Improved synchronous light scattering method for measuring baker’s yeast biomass using thickened suspensions

Measuring yeast biomass is important in the processes of microbial fermentations. It has been demonstrated that synchronous light scattering (SLS) signals could be applied for the quantification of model bioparticles such as Saccharomyces cerevisiae. In this study, an improved synchronous light scattering method was developed for yeast biomass estimation. The settlement of yeast cells during SLS signals measuring process was studied, and hydrolysis anionic polyacrylamide was added into yeast suspensions to increase the stability of the cells in liquid environment. By simultaneously scanning both the excitation and emission monochromators of a common spectrofluorometer with same starting excitation and emission wavelength (namely, ?λ = 0), the SLS intensity was found to be proportional to the yeast concentration in the range from 0 to 4.9 × 10⁶ cell/mL (R ² = 0.9907), the detection limit is 8.1 × 10³ cell/mL. The developed method exhibited good stability and sensitivity in the recovery test and growth curve drawing process, demonstrating the potential of the method in practical application of biomass estimation.     

Responses of soil microbial biomass and enzymatic activities to different forms of organic nitrogen deposition in the subtropical forests in East China

Numerous studies reported that inorganic nitrogen (N) deposition strongly affected forest ecosystems. However, organic N is also an important component of atmospheric N deposition. The influence of organic N deposition on soil microbial biomass and extracellular enzymatic activities (EEA) in subtropical forests remains unclear. Coniferous forest (CF) and broad-leaved forest (BF) were chosen from the Zijin Mountain in China. Five forms of organic N (urea, glycine, serine, nonylamine, and a mixture of all four) were used to fertilize the soils in CF and BF every month for 1 year. Soil samples were collected every 2 months. Subsequently, soil microbial biomass and EEA were assayed. Results showed that the microbial biomass and EEA of soils fertilized with urea and amino acids increased significantly, whereas those fertilized with nonylamine and mixed N decreased significantly. Urea and amino acid fertilizations had a more positive influence on EEA of BF than on those of CF. Nonylamine fertilization had a more negative influence on EEA of CF than on those of BF. Organic N fertilization shifted soil microbial biomass away from the excretion of N-degrading enzymes and toward the excretion of C-degrading enzymes. These results suggest that organic N type is an important factor that affects soil microbial biomass, EEA, and their relationship. Organic N deposition may seriously affect soil C and N cycling, as well as carbon dioxide releasing from the soils by influencing microbial activities and biomass. This study thereby provides evidence that soil microorganisms have strong feedback to different forms of organic N deposition.     

Comparative proteomic and physiological analysis of diurnal changes in Nostoc flagelliforme

Nostoc flagelliforme is a terrestrial cyanobacterium species whose metabolism follows an obvious diurnal pattern. Diurnal changes at physiological and proteomic levels of N. flagelliforme were obtained. In the morning (7:00 H), net photosynthesis, dark respiration, as well as the activities of total Rubisco, nitrogenase, glutamine synthetase, SOD and CAT were comparatively high. All these physiological activities significantly decreased in the afternoon (13:00 H), and then slowly increased in the evening (19:00 H). Thirty-one differentially expressed proteins with a variety of important functions were reproducibly detected and identified over a diurnal cycle. These proteins were categorized according to their predicted functions into secretion and regulation (15.79 %), antioxidative processes (21.05 %), nitrogen metabolism (10.53 %), carbohydrate and energy metabolism (10.53 %), as well as cell division (2.63 %). The remaining proteins had unclassified/unknown functions (21.05 %) or were unidentified (18.42 %). The results suggested a metabolic shift from active (7:00 H) to quiescent (13:00 H) and then to active (19:00 H) over the diurnal cycle. The differential expression level of ferritin, Mn-CAT, SOD and Fe-SOD may serve as molecular markers for the diurnal metabolism in N. flagelliforme.     

pEGFP-N1-mediated BmK CT expression suppresses the migration of glioma

Gliomas can diffuse into the normal brain and this invasion of glioma cells involves modification of receptor-mediated adhesive properties of tumor cells, degradation and remodeling of extracellular matrix by tumor-secreted metalloproteinase (MMPs) such as MMP-2, consequently creating an intercellular space for invasion of glioma cells. BmK CT, one of the key toxins in scorpion Buthus martensii Karsch venom, is a novel blocker of the chloride ion channel and MMP-2. In this report, a recombinant plasmid pEGFP-N1-BmK CT was constructed and characterized by in vitro studies. The results showed that pEGFP-N1 mediated BmK CT expression displayed a high activity in suppressing cell migration via MMP-2. The potential therapeutic effect of pEGFP-N1 mediated BmK CT against rat glioma C6 cells was assessed and its potential mechanism was elucidated. It represented an approach for developing a novel therapeutic agent—recombinant plasmid pEGFP-N1-BmK CT as an efficient and powerful adjuvant.     

Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization

Prorocentrum is a common dinoflagellate genus along the Chinese seacoast, which frequently causes harmful algal blooms. Efforts to understand and prevent blooms caused by these harmful species require the development of methods for rapid and precise identification and quantification so that an adequate early warning of harmful algal blooms may be given. Here, we report the development and application of rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection of Prorocentrum micans. The hypervariable D1–D2 regions of a large subunit rDNA of a strain isolated from East China Sea identified as P. micans were first sequenced to design species-specific probes. Analysis of sequences identified as P. micans and deposited in GenBank revealed significant base differences among them and phylogenetic analyses revealed multiple clades within the taxon P. micans. Thus, it is likely that more than one taxonomic and genetically distinct entity has been identified as P. micans, if not misidentified. A series of probes were identified to one of these clades and tested for their specificity. Second, whole cell in situ hybridization procedures were established and the optimal probes were screened among the candidate probes. Next, cross-reactivity was performed to test the specificity of the probes and the detection reliability under various culture conditions, including different nutrient levels, temperatures, and light intensities. Finally, an improved protocol for natural samples was applied to the field material. The designed rRNA-targeted probe was specific, showing no cross-reactivity with other microalgae. The optimized detection protocol could be completed within 1.5 h. All target cells were speculated to be identified during all stages of their whole growth cycle under different culture conditions because the difference in fluorescence intensities throughout the experiment was not significant (p?>?0.05). The cell densities determined by FISH and light microscopy (LM) were comparable, without any significant difference (p?>?0.05) between them. In general, the established FISH probe was promising for specific, rapid, precise detection of a selected set of P. micans in natural samples and served as a good detection model for other Prorocentrum in the future.     

Molecular cloning and sequence analysis of methionine adenosyltransferase from the economic seaweed Undaria pinnatifida

The methionine adenosyltransferase gene (MAT) had been isolated from an economic seaweed Undaria pinnatifida by PCR using degenerate primers. The cDNA was 1,491 bp in length with an open reading frame of 1,194 nucleotides, encoding a deduced protein of 397 amino acids. The protein had a predicted molecular weight of 43.2 kDa, and the isoelectric point was 5.244. The sequence contains a 92 bp 5′-untranslated region (UTR) and a 205 bp 3′-UTR. The methionine adenosyltransferase (MAT) sequence of U. pinnatifida (UpMAT) shared 68–92 % identities with the previous published MAT sequences of other species. Phylogenetic analysis indicated that the phylogenetic relationship of UpMAT with some other seaweeds was closer than with those of higher plants. Under different stress conditions, the relative mRNA expression levels of the MAT of U. pinnatifida (UpMAT) were measured by real-time quantitative PCR, and the results demonstrated that the UpMAT might help to protect the alga against various abiotic stresses.     

Pro-inflammatory cytokines modulate iron regulatory protein 1 expression and iron transportation through reactive oxygen/nitrogen species production in ventral mesencephalic neurons

Both inflammatory processes associated with microglia activation and abnormal iron deposit in dopaminergic neurons are involved in the pathogenesis of Parkinson's disease (PD). However, the relationship between neuroinflammation and iron accumulation was not fully elucidated. In the present study, we aimed to investigate whether the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) released by microglia, could affect cellular iron transportation in primary cultured ventral mesencephalic (VM) neurons. The results showed that IL-1β or TNF-α treatment led to increased ferrous iron influx and decreased iron efflux in these cells, due to the upregulation of divalent metal transporter 1 with the iron response element (DMT1 + IRE) and downregulation of ferroportin1 (FPN1). Increased levels of iron regulatory protein 1 (IRP1), transferrin receptor 1 (TfR1) and hepcidin were also observed in IL-1β or TNF-α treated VM neurons. IRP1 upregulation could be fully abolished by co-administration of radical scavenger N-acetyl-l-cysteine and inducible NO synthetase inhibitor N_ω-nitro-l-arginine methyl ester hydrochloride. Further experiments demonstrated that IL-1β and TNF-α release was remarkably enhanced by iron load in activated microglia triggered by lipopolysaccharide or 1-methyl-4-phenylpyridinium (MPP⁺). In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice, salicylate application could not block DMT1 + IRE upregulation in dopaminergic neurons of substantia nigra. These results suggested that IL-1β and TNF-α released by microglia, especially under the condition of iron load, might contribute to iron accumulation in VM neurons by upregulating IRP1 and hepcidin levels through reactive oxygen/nitrogen species production. This might provide a new insight into unraveling that microglia might aggravate this iron mediated neuropathologies in PD.     

Genome-wide analysis of Nilaparvata lugens nymphal responses to high-density and low-quality rice hosts

The brown planthopper （BPH） Nilaparvata lugens is an economically impor- tant pest on rice plants. In this study, the higher population density and yellow-ripe stage of rice plants were used to construct adverse survival conditions （ASC） against BPH nymphs. Simultaneously, the low population density and tillering stage of rice plants were used to establish a suitable survival condition （SSC） as a control. Solexa/Illumina sequencing was used to identify genes of BPH nymphs responding to ASC. Significantly longer duration development of BPH nymphs and significantly lower brachypterous ratio of BPH adults were observed by ASC compared with SSC. A total of 2 544 differentially expressed genes （DEGs） were obtained and analyzed by BLASTx, Gene Ontology and KEGG Orthology. Gene ontology analysis revealed that the DEGs were mainly involved in categories of cell, cell part, cellular process, binding, catalytic, organelle and metabolic processes. 1138 DEGs having enzyme commission numbers were assigned to different metabolic pathways. The largest clusters were neurodegenerative diseases （137, 12.0%）, followed by carbohy- drate metabolism （113, 9.9%）, amino acid metabolism （94, 8.3%）, nucleotide metabolism （76, 6.7%）, energy metabolism （64, 5.6%）, translation （60, 5.3%）, lipid metabolism （58, 5.1%）, and folding, sorting and degradation （52, 4.6%）. Expressing profile of 11 DEGs during eight nymphal developmental stages of BPH were analyzed by quantitative real- time polymerase chain reaction. The 11 genes exhibited differential expression between ASC and SSC during at least one developmental stage. The DEGs identified in this study provide molecular proof of how BPH reconfigures its gene expression profile to adapt to overcrowding and low-quality hosts.     

金荞麦果实中有效成分的分析

采用HPLC法对金荞麦果实中的有效成分进行了分析,结果表明:金荞麦果实中含有芦丁、槲皮素和表儿茶素等有效成分,其中主要以芦丁为主,槲皮素、表儿茶素等含量甚微.金荞麦果实中的有效成分主要集中在种子部分,果皮中则不合或含量甚微.