Peng Geng

Development, Characterization, and Evaluation of a Fusion Protein of a Novel Glucagon-Like Peptide-1 (GLP-1) Analog and Human Serum Albumin in Pichia pastoris

Glucagon-like peptide-1 (GLP-1) has considerable potential as a possible therapeutic agent for type-2 diabetes. Unfortunately, this glucoincretin is short lived due to degradation by dipeptidyl-peptidase IV and rapid clearance by renal filtration. In this study, we attempted to extend GLP-1 action through the attachment of a lysine residue at the N-terminal of GLP-1 (named KGLP-1), and to make a fusion protein with human serum albumin (HSA) in Pichia pastoris. The protein, designated KGLP-1/HSA, was purified by an immunomagnetic separation technique. High performance liquid chromatography (HPLC) showed that the purified protein had an overall purity of 92.0%, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) confirmed the expected molecular mass of 70,297.8 Da. Additionally, the N-terminal sequence of KGLP-1/HSA was confirmed by N-terminal sequencing. The stability and biological activity of KGLP-1/HSA were then evaluated in vitro and in vivo. The findings indicated that fusion KGLP-1/HSA preserved the action of native GLP-1, and the active duration was greatly prolonged.

Four acarviosin-containing oligosaccharides identified from Streptomyces coelicoflavus ZG0656 are potent inhibitors of α-amylase

Four aminooligosaccharides were isolated and purified from the culture filtrate of Streptomyces coelicoflavus ZG0656. Their chemical structures were determined by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The names acarviostatins I03, II03, III03, and IV03 were given to the oligomers due to their acarviosin core structures. Acarviostatins III03 and IV03, which contain three and four acarviosin-glucose moieties, respectively, were identified as novel compounds. The four acarviostatins were all mixed noncompetitive inhibitors of porcine pancreatic alpha-amylase (PPA). The inhibition constants (K(i)) for acarviostatins III03 and IV03 were 0.008 and 0.033muM, respectively. Acarviostatin III03 is the most effective alpha-amylase inhibitor known to date, with a K(i) value 260 times more potent than acarbose.

Identification of anti-asthmatic compounds in Pericarpium citri reticulatae and evaluation of their synergistic effects

AbstractAim:To investigate the anti-asthmatic mechanisms of the traditional Chinese medicine Pericarpium citri reticulatae (PCR).Methods:The alkaloid section (AS) of PCR was extracted using an ion exchange resin, separated, and purified into different fractions by semi-preparative HPLC. These fractions were screened for beta2-adrenergic receptor (β2AR) agonistic activity using rat β2AR-transfected CHO-CRE-EGFP cells. AS and its isolated components were characterized by ultra-performance liquid chromatography/quadrupole time-of-flight MS (UPLC/Q-Tof MS) and were evaluated for their spasmolytic and antitussive activities both in vitro and in vivo in a guinea pig model.Results:We demonstrated that the AS component responsible for activating β2AR signaling was synephrine. Both AS and synephrine showed significant spasmolytic effects on acetylcholine chloride (ACh)-induced contractions in isolated guinea pig trachea, and they protected against histamine-induced experimental asthma by prolonging the latent period. We further identified stachydrine as the antitussive component that could significantly reduce citric acid–induced coughing. The combination of these two bioactive compounds had a more potent spasmolytic activity in comparison with the single use of synephrine or stachydrine.Conclusion:We conclude that synephrine and stachydrine are the key components of AS that mediate asthma relief due to their synergism when used in combination.

Taxonomy of the Streptomyces strain ZG0656 that produces acarviostatin α‐amylase inhibitors and analysis of their effects on blood glucose levels in mammalian systems

Aims:u2002 To clarify the taxonomic status of strain ZG0656 and analyse the effects of its acarviostatin products on blood glucose levels in mammalian systems.

Combined effect of total alkaloids from Feculae Bombycis and natural flavonoids on diabetes

Both total alkaloids from Feculae Bombycis (TAFB) and natural flavonoids can inhibit α‐glucosidase activity to depress the glucose level in blood. To investigate the cooperative effect of TAFB and flavonoids on blood glucose, we have studied their combined function compared with individual ingredients on enzymology, in‐vitro and in‐vivo. In the enzymological assay, the combination of TAFB and flavonoids showed more effective inhibition, compared with either TAFB or flavonoids alone, to α‐glucosidase activity. In the everted intestine model in‐vitro, the combined inhibition of starch hydrolysation and glucose transference to blood was much stronger than with separate components. In short‐term studies with normal and experimentally‐induced diabetic mice in‐vivo, the combination of TAFB and flavonoids also had a stronger suppressive effect on the postprandial elevation in blood glucose after oral administration. In long‐term treatment to diabetic mice in‐vivo, the compound prescription could depress not only the fasting blood glucose, but also the fasting blood total cholesterol. These results demonstrated that TAFB and flavonoids could inhibit α‐glucosidase activity cooperatively, which would successfully depress blood glucose level in the therapy of diabetes.

Identifying glucagon-like peptide-1 mimetics using a novel functional reporter gene high-throughput screening assay.

Glucagon-like peptide-1 (GLP-1) stimulates insulin and inhibits glucagon secretion and therefore could potentially be used to treat diabetes type II. However, its therapeutic use is limited by its short half-life in vivo, due mainly to enzymatic degradation by dipeptidyl peptidase IV (DPP-IV). Developing GLP-1 analogs with greater bioactivity is therefore an important step toward using them therapeutically. Accordingly, we aimed to identify GLP-1 mimetic peptides by creating a high-throughput screening (HTS) assay of a phage displayed (PhD) peptide library. This assay was functionally based using the GLP-1 receptor (GLP-1R) gene. Rat GLP-1R cDNA was transfected into CHO/enhanced green fluorescent protein (EGFP) cells by lipofection. The resulting stable, recombinant cell line functionally expressed the GLP-1R and a cAMP-responsive EGFP reporter gene, to monitor receptor activation, and was used to screen a PhD dodecapeptide library. After four rounds of selection, 10 positive clones were selected based on functional evaluation and sequenced. Three sequences were obtained, corresponding to three different domains of GLP-1 (Group 1: 22-34; Group 2: 18-29; and Group 3: 6-17). The Group 3 peptide had the highest bioactivity, was synthesized, and designated KS-12. Importantly, KS-12 activated GLP-1R in vitro and reduced blood glucose levels in a dose-dependent manner when administered to Chinese Kunming mice. Although KS-12 was not as effective as GLP-1, it was significantly resistant to DPP-IV both in vitro and in vivo. Thus, this study provides a novel way to screen DPP-IV resistant agonist peptides of GLP-1 from a PhD peptide library using the functional reporter gene HTS assay.