cDNA isolation and functional characterization of UDP-glucose 4-epimerase from Davallia divaricate.

UDP-glucose 4-epimerase (UGE) is a universal enzyme responsible for interconversion of UDP-glucose and UDP-galactose. However, the gene encoding UGE from Davallia divaricate is elusive. In this study, two UGE genes, ddUGE1 and ddUGE2, were isolated and cloned from D. divaricate using a transcriptome-guided search strategy. Two unigenes sharing high sequence identity with UGE homologous genes were selected from transcriptome assembly. The enzymes, further functionally expressed in Escherichia coli, exhibit narrow substrate specificity. The biochemical characterization assays of DdUGE1 and DdUGE2 showed good thermal and pH stability, and metal ion independence, which provides a meaningful feature for biotechnological applications.[Formula: see text].

Berberine combined with stachyose induces better glycometabolism than berberine alone through modulating gut microbiota and fecal metabolomics in diabetic mice.

Berberine (BBR), a small alkaloid, is used as a hypoglycemic agent in China. Stachyose (Sta), a Rehmannia glutinosa oligosaccharide, acts as a prebiotic. This study aimed to evaluate whether BBR combined with Sta produced better glycometabolism than BBR alone, and explored the effects on gut microbiota and metabolomics. Type-2 diabetic db/db mice were administered BBR (100 mg/kg), Sta (200 mg/kg), or both by gavage once daily. Glucose metabolism, the balance of α- and ß-cells, and mucin-2 expression were ameliorated by combined treatment of BBR and Sta, with stronger effects than upon treatment with BBR alone. The microbial diversity and richness were altered after combined treatment and after treatment with BBR alone. The abundance of Akkermansia muciniphila was increased by combined treatment compared to treatment with BBR alone, while the levels of the metabolite all-trans-heptaprenyl diphosphate were decreased and the levels of fumaric acid were increased, which both showed a strong correlation with A. muciniphila. In summary, BBR combined with Sta produced better glycometabolism than BBR alone through modulating gut microbiota and fecal metabolomics, and may aid in the development of a novel pharmaceutical strategy for treating Type 2 diabetes mellitus.

The PI3K/Akt1-FoxO1 Translocation Pathway Mediates EXf Effects on NIT-1 Cell Survival.

EXf, a glucagon-like peptide 1 (GLP-1) receptor agonist, stimulates ß-cell proliferation and reduces apoptosis in diabetic animal models, but the underlying mechanisms are not fully understood. We constructed a FoxO1-GFP fusion protein expression plasmid and transiently transfected it into NIT-1 cells to investigate whether FoxO1 mediates EXf effects on NIT-1 cell survival. Our results showed that EXf could increase cell viability by inhibiting apoptosis and stimulating proliferation, and it could also promote the translocation of the FoxO1-GFP fusion protein from the nucleus to the cytoplasm in NIT-1 cells. However, the above effects of EXf were suppressed by the inhibitor of PI3K. Comparative transcription analysis showed up-regulation of igf-1r, irs-2, pI3k, akt1 and pdx-1 in NIT-1 cells after EXf treatment. Moreover, the up-regulation of PI3K and phosphorylation of Akt1 upon EXf treatment was confirmed by Western blot, both phenomena were abrogated by wortmannin, an inhibitor of PI3K. In summary, FoxO1 may mediate the effects of EXf on NIT-1 cell survival by activating the PI3K/Akt1 pathway.

[Enzymatic cyclization of peptides using immobilized sortase A].

Peptide cyclization, a pivotal approach to modifying linear precursors of proteins and pepticles, has been used to enhance their biological activities and serum stabilities. Recently, sortase A (SrtA) from Staphyloccus aureus becomes a promising new technology for efficiently incorporating site specific modifications into proteins, conjugating the cell surface and cyclizing the linear peptides. In this study, we constructed two recombinant expression systems, one with chitin binding domain and the other with six-histidine tag and chitin binding domain on the N-terminal of SrtA, separately. The results of enzymatic kinetics indicate that the two recombinant tags do not impair the transpeptidase activity of SrtA compared with the standard reaction reported under the same reaction condition. The two synthesized peptides with N-ternimal three glycines and C-terminal penta-amino acid motif, LPETG, were cyclized using immobilized and recycled SrtA. The SrtA-based cyclization promises to represent a simple method for easy and efficient enzymatic synthesis of large cyclic peptides.

[A cyclotide against influenza A H1N1 virus from Viola yedoensis].

Three cyclotides were isolated from the whole plant of Viola yedoensis in this study. The two, vary peptide E and cycloviolacin Y5, were previously reported, and a novel cycloviolacin VY1 was characterized according to the interpretation of MS/MS fragmentation of peptides which were produced from the reduced and alkylated parent peptide with the digestion of Endo Lys-C, trypsin and chymotrypsin, separately. The stability of remarkable resistance to proteolytic degradation by trypsin and chymotrypsin, and that of thermal denaturation was confirmed again. Besides, the IC50 value of cycloviolacin VY1 against influenza A H1N1 virus was (2.27 +/- 0.20) microg x mL(-1). It is the first cyclotide reported with anti-influenza A H1N1 virus activity in vitro assay.

[Pamidronate in treatment of pain caused by bone metastasis].

BACKGROUND & OBJECTIVE: Clinical studies showed that pamidronate has curative effects on pain induced by bone metastasis. This study was to observe curative effects, and side effects of different dosages of pamidronate on pain induced by bone metastasis. METHODS: Ninety patients with metastatic bone tumor were divided into 2 groups randomly. In group A, 120 mg pamidronate was given by intravenous infusion in 3 days (60 mg day(1), 30 mg day(2-3)); in group B, 90 mg pamidronate was given by intravenous infusion in 3 days (30 mg day(1-3)), repeated every 4 weeks. RESULTS: The notable effect, effect, and no effect were observed in 20, 23, and 2 patients of group A, and 16, 20, and 9 patients of group B, respectively. Total effective rate in group A was 95.6%(43/45) , in group B was 80.0% (36/45) (P< 0.05). Curative rate within 1 course of treatment in group A was 88.9% (40/45), in group B was 57.8% (26/45) (P< 0.01). Curative rate within 1 week in group A was 80% (36/45) , in group B was 57.8% (26/45) (P< 0.05). Side effects were observed in 3 patients (6.7%). CONCLUSIONS: Pamidronate has good curative effects in treating pain induced by bone metastasis. Efficacy of 120 mg pamidronate is better than 90 mg pamidronate.