Production of high fructo-oligosaccharide syrup with two enzyme system of fructosyltransferase and glucose oxidase

Summary A novel two enzyme system of fructosyltransferase and glucose oxidase to enhance the content of the net fructo—oligosaccharide (FOS) fractions in the industrial production of FOS syrup from sucrose was devised. The net FOS content in the commercial FOS syrup has been limited only to 55–60 % due to the accumulation of glucose which acts as a feedback inhibitor of the fructosyltransferase. By supplementing glucose oxidase to the conventional FOS reaction system, we could convert the glucose to gluconic acid readily separable from neutral sugars by simple ion exchange operation in the next step. The simultaneous removal of glucose was proved effective in proceeding the reaction by fructosyltransferase further by relieving the product inhibition caused by glucose. By this way, we could raise the net FOS content as high as 90 %.     

Soluble Sema4D cleaved from osteoclast precursors by TACE suppresses osteoblastogenesis

Bone remodelling is mediated by orchestrated communication between osteoclasts and osteoblasts which, in part, is regulated by coupling and anti-coupling factors. Amongst formally known anti-coupling factors, Semaphorin 4D (Sema4D), produced by osteoclasts, plays a key role in downmodulating osteoblastogenesis. Sema4D is produced in both membrane-bound and soluble forms; however, the mechanism responsible for producing sSema4D from osteoclasts is unknown. Sema4D, TACE and MT1-MMP are all expressed on the surface of RANKL-primed osteoclast precursors. However, only Sema4D and TACE were colocalized, not Sema4D and MT1-MMP. When TACE and MT1-MMP were either chemically inhibited or suppressed by siRNA, TACE was found to be more engaged in shedding Sema4D. Anti-TACE-mAb inhibited sSema4D release from osteoclast precursors by ~90%. Supernatant collected from osteoclast precursors (OC-sup) suppressed osteoblastogenesis from MC3T3-E1 cells, as measured by alkaline phosphatase activity, but OC-sup harvested from the osteoclast precursors treated with anti-TACE-mAb restored osteoblastogenesis activity in a manner that compensates for diminished sSema4D. Finally, systemic administration of anti-TACE-mAb downregulated the generation of sSema4D in the mouse model of critical-sized bone defect, whereas local injection of recombinant sSema4D to anti-TACE-mAb-treated defect upregulated local osteoblastogenesis. Therefore, a novel pathway is proposed whereby TACE-mediated shedding of Sema4D expressed on the osteoclast precursors generates functionally active sSema4D to suppress osteoblastogenesis.     

Low genetic diversity and population connectivity fuel vulnerability to climate change for the Tertiary relict pine Pinus bungeana

Endemic species are important components of regional biodiversity and hold the key to understanding local adaptation and evolutionary processes that shape species distributions. This study investigated the biogeographic history of a relict conifer Pinus bungeana Zucc. ex Endl. confined to central China. We examined genetic diversity in P. bungeana using genotyping-by-sequencing and chloroplast and mitochondrial DNA markers. We performed spatial and temporal inference of recent genetic and demographic changes, and dissected the impacts of geography and environmental gradients on population differentiation. We then projected P. bungeana's risk of decline under future climates. We found extremely low nucleotide diversity (average π 0.0014), and strong population structure (global F_ST 0.234) even at regional scales, reflecting long-term isolation in small populations. The species experienced severe bottlenecks in the early Pliocene and continued to decline in the Pleistocene in the western distribution, whereas the east expanded recently. Local adaptation played a small (8%) but significant role in population diversity. Low genetic diversity in fragmented populations makes the species highly vulnerable to climate change, particularly in marginal and relict populations. We suggest that conservation efforts should focus on enhancing gene pool and population growth through assisted migration within each genetic cluster to reduce the risk of further genetic drift and extinction.     

Systematics of Mukdenia and Oresitrophe (Saxifragaceae): Insights from genome skimming data

Oresitrophe and Mukdenia (Saxifragaceae) are epilithic sister genera used in traditional Chinese medicine. The taxonomy of Mukdenia, especially of M. acanthifolia, has been controversial. To address this, we produced plastid and mitochondrial data using genome skimming for Mukdenia acanthifolia and Mukdenia rossii, including three individuals of each species. We assembled complete plastomes, mitochondrial CDS and nuclear ribosomal ETS/ITS sequences using these data. Comparative analysis shows that the plastomes of Mukdenia and Oresitrophe are relatively conservative in terms of genome size, structure, gene content, RNA editing sites and codon usage. Five plastid regions that represent hotspots of change (trnH-psbA, psbC-trnS, trnM-atpE, petA-psbJ and ccsA-ndhD) are identified within Mukdenia, and six regions (trnH-psbA, petN-psbM, trnM-atpE, rps16-trnQ, ycf1 and ndhF) contain a higher number of species-specific parsimony-informative sites that may serve as potential DNA barcodes for species identification. To infer phylogenetic relationships between Mukdenia and Oresitrophe, we combined our data with published data based on three different datasets. The monophyly of each species (Oresitrophe rupifraga, M. acanthifolia and M. rossii) and the inferred topology ((M. rossii, M. acanthifolia), O. rupifraga) are well supported in trees reconstructed using the complete plastome sequences, but M. acanthifolia and M. rossii did not form a separate clade in the trees based on ETS + ITS data, while the mitochondrial CDS trees are not well-resolved. We found low recovery of genes in the Angiosperms353 target enrichment panel from our unenriched genome skimming data. Hybridization or incomplete lineage sorting may be the cause of discordance between trees reconstructed from organellar and nuclear data. Considering its morphological distinctiveness and our molecular phylogenetic results, we strongly recommend that M. acanthifolia be treated as a distinct species.     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Molecular Cloning and Characterization of Transferrin from Wax Moth, Galleria mellonella

Complete mRNA sequence of transferrin from Galleria mellonella was obtained, and compared with those of other species. Until now, two types of insect transferrin were reported. Transferrins in cockroach and termite have two iron binding sites while those in most other insect groups, studied for the protein, have only one. It was suggested that the presence of two types of transferrin was related with transferrin evolution, because vertebrate transferrins have two iron binding sites, called N and C terminal lobe. It was shown that G. mellonella transferrin also has only one iron binding site (N terminal lobe), and the deduced amino acid sequence was most similar to those of Manduca sexta and Bombyx mori.     

Flow cytometric analysis of antibody producing cells using double immunofluorescent staining

Summary Double fluorescent labeling, with fluorescein isothiocyanate (FITC)-labeled F(ab)₂ specific for the heavy chain and R-phycoerythrin (R-PE)-labeled F(ab)₂ specific for the light chain, was demonstrated as a convenient means for the accurate evaluation of a heterogeneous non-antibody-producing population. Furthermore, it could be used for monitoring the changes in each immunoglobulin (Ig) chain content of the cells during the batch culture, which will facilitate the study on antibody synthesis, assembly and secretion.     

The Best Gastric Site for Obtaining a Positive Rapid Urease Test

Background Rapid urease tests (RUTs) provide a simple, sensitive method of detecting Helicobacter pylori infection.
Objectives. Our aim, therefore, was to determine whether the yield of detecting H. pylori infection by RUT varied depending on the site of gastric biopsy.
Materials and Methods. Gastric biopsies were obtained from 50 patients for RUT by use of hp fast (GI Supply, Camp Hill, PA). Biopsies were taken from the prepyloric greater curve antrum, from the gastric angle, and from the greater curve in mid-corpus. One biopsy specimen was placed in the RUT gel, and the biopsy from the adjacent mucosa was placed in formalin for subsequent histological evaluation by using the Genta stain. RUTs were examined and scored at intervals of 5, 10, 15, 30, and 45 minutes and after 1, 2, 4, and 24 hours.
Results. Fifty patients were entered in the test (150 RUTs), 32 having H. pylori infection. There were no false-positive RUTs (specificity, 100%). The gastric angle site was positive in 100%. The prepyloric site was positive in 87%, and the corpus site was positive in 84.4% ( p < .052 for angle or prepyloric antrum versus corpus). The most common pattern was for all to be positive (74%). The median time to positivity was similar with angle and prepyloric sites (37.5 and 60 minutes, respectively, p = not significant) and shorter than the corpus biopsy (180 minutes); ( p < .05 for angle or prepyloric antrum versus corpus).
Conclusion. The maximum probability for detecting H. pylori infection using a RUT is to obtain a biopsy from the gastric angle. To prevent missing a positive result when intestinal metaplasia is present, we recommend that (at a minimum) biopsies be taken from both the angle and the corpus.