Jian-Ning Liu

Glucagon‐like peptide‐1 (GLP‐1) plays an important role in the regulation of postprandial insulin release. Here, we used the split DnaB mini‐intein system to produce recombinant human GLP‐1/7‐36 (rhGLP‐1) in Escherichia coli. The C‐terminal domain of DnaB mini‐intein (IntC) was genetically fused at the N‐terminus of rhGLP‐1 to produce IntC–GLP‐1. IntC–GLP‐1 and N‐terminal domain of DnaB mini‐intein (IntN) protein were prepared in a denatured buffer of pH 8.0. IntC–GLP‐1 was diluted 1:8 into the phosphate buffer of pH 6.6. IntN was added into the diluted solution of IntC–GLP‐1 at the molar ratio of 1:2. Then, rhGLP‐1 was released from IntC–GLP‐1 via inducible C‐terminal peptide‐bond cleavage by shifting pH from 8.0 to 6.6 at 25 °C for 24‐H incubation. Then, the supernatant was applied to a Ni‐Sepharose column, and the pass through fraction was collected. About 5.34 mg of rhGLP‐1 with the purity of 97% was obtained from 1 L of culture medium. Mass spectrometry showed the molecular weight of 3,300.45 Da, which was equal to the theoretical value of GLP‐1/7‐36. The glucose‐lowering activity of rhGLP‐1 was confirmed by the glucose tolerance test in mice. In conclusion, the reported method was an efficient strategy to produce rhGLP‐1 without using enzyme or chemical reagents, which could also be used for other similar peptides.

The investigation of agents for the treatment of osteoporosis has been a long-standing effort. The Wnt pathway plays an important role in bone formation and regeneration, and expression of Wnt pathway inhibitors, Dickkopf-1 (DKK1), appears to be associated with changes in bone mass. Inactivation of DKK1 leads to substantially increased bone mass in genetically manipulated animals. DKK1-derived peptides (DDPs) were added to BMP2-stimulated MC3T3-E1 preosteoblastic cells in vitro to evaluate inhibitory activity of DDPs in MC3T3-E1 cell differentiation. Study was extended in vivo on old female mice to show whether or not inhibition of endogenous DKK1 biological activity using DDPs vaccination approach leads to increase of bone formation, bone density, and improvement of bone microstructure. We reported that synthetic DDPs were able to reduce alkaline phosphatase activity, prevent mineralization and inhibit the differentiation of MC3T3-E1 cells in vitro. Furthermore, vaccination with these DDPs in aged female mice 4 times for a total period of 22 weeks promoted bone mass and bone microstructure. 3D microCT and histomorphometric analysis showed that there were significant increase in bone mineral densities, improvement of bone microstructure and promotion of bone formation in the vaccinated mice, especially in the mice vaccinated with DDP-A and DDP-C. Histological and scanning electron microscopy image analysis also indicated that vaccination increased trabecular bone mass and significantly decreased fragmentation of bone fibers. Taken together, these preclinical results suggest that vaccination with DDPs represents a promising new therapeutic approach for the treatment of bone-related disorders, such as osteoporosis.

Summary: Objectives: To elucidate the efficacy and safety of pharmacoinvasive therapy by using prourokinase (prouk) in patients with ST‐segment elevation myocardial infarction (STEMI).Background: Patients with STEMI often have long percutaneous coronary intervention (PCI)‐related delays due to various reasons, which are associated with poor outcomes.Methods: A randomized study which enrolled patients from four centers in China was conducted. Patients were randomly assigned to accept routine primary PCI or prouk‐PCI. The primary end points were the angiographic parameters, including thrombolysis in myocardial infarction (TIMI) flow grade, TIMI frame count, and myocardial blush grade. Secondary endpoints were incidence of major adverse cardiac events (MACE, defined as death from all causes, reinfarction, revascularization, or rehospitalization due to new or worsening congestive heart failure) at 30 days and 1 year.Results: One hundred and ninety‐seven eligible patients were enrolled, of whom 100 were randomized to the prouk‐PCI group. Significantly more patients in the prouk‐PCI group than in the PCI group had an opened infarct‐related artery on arrival in the catheterization laboratory (48% vs. 21%, P = 0.0002) and better TIMI frame count after PCI (33 ± 6 vs. 40 ± 10, P < 0.001). At 1‐year follow‐up, there was a trend that patients in the prouk‐PCI group had less chances to have MACE (7.0% vs. 12.6%, P = 0.235) or be readmitted to hospital due to new or worsening congestive heart failure (1.0% vs. 4.1%, P = 0.209).Conclusion: A strategy of emergent PCI preceded by fibrinolysis with prouk results in a better myocardial perfusion in infarct‐related artery compared with primary PCI alone in patients with STEMI and long PCI‐related delay.

NUCB2^1–83 has been recently reported as an anorexigenic and anti-hyperglycemic peptide. Here we report that NUCB2^1–83 promotes osteogenesis. It was found after two months of once-a-day intravenous injection of NUCB2^1–83, bone mineral density of femora and lumbar vertebrae were increased in ovariectomized rats. NUCB2^1–83 also increased the alkaline phosphatase activity and promoted mineralization in mouse MC3T3-E1 preosteoblastic cell line. When either both Arg⁶⁰ and Arg⁶³ or Ser⁷² were mutated to Ala, the pro-osteogenic activity was completely lost, indicating that these residues are structurally important for its biological function. Furthermore, it encumbered osteoclastic differentiation of RAW 264.7 macrophage. It also excluded any possibility of the effect caused by contaminants or experimental faults, and demonstrated that the pro-osteogenic activity observed was a specific effect of NUCB2^1–83 itself. These findings warranted that further studies on NUCB2^1–83 would be valuable for the treatment of bone metabolic diseases especially for osteoporosis.

Angiogenesis is critical for tumor growth and metastasis. Tumor tissues induce the expression of angiogenesis-associated proteins on endothelial surface that can be targeted for tumor immunotherapy. In our study, the rat tumor endothelial proteins (EP) were isolated in situ via biotinylation of tumor vascular endothelial luminal surface followed by streptavidin affinity chromatography. The isolated tumor EP contained numerous up-regulated angiogenesis-associated endothelial proteins. The administration of these tumor EP as a vaccine to mice reduced the microvessel density in subcutaneous primary LLC tumors, delayed spontaneous LLC tumor metastasis and prolonged post-surgery life span. T lymphocytes from tumor EP-vaccinated mice lysed human umbilical vascular endothelial cells, but not tumor cells in vitro, in a dose-dependent manner. Furthermore, adoptive transfer of antitumor EP antibodies in vivo targeted to tumor endothelium and inhibited spontaneous LLC tumor metastasis. This study provides a successful preclinical exploration of the active immunotherapy for tumor by targeting tumor angiogenesis. © 2009 UICC

A proof-of-concept study demonstrated the feasibility of a novel gel-pad microarray on porous silicon chips, by initiation of an atom transfer radical propagation (ATRP) polymerisation of (polyethylene glycol) methacrylate (PEGMA) with surface Si–H species, stepwise chemical conversions of the gel membrane to an NTA-Ni²⁺/histidine-tagged protein system, and matrix-assisted laser desorption/ionisation mass spectroscopy (MALDI MS) and fluorescence detections.

To prevent in vivo degradation, small peptides are usually expressed in fusion proteins from which target peptides can be released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the production of recombinant human urodilatin, a hormone for the treatment of acute decompensated heart failure. The fusion protein, which was overexpressed mainly as inclusion bodies in Escherichia coli, constituted about 25% of the total cell proteins. After purification by Ni-sepharose affinity chromatography and renaturation in refolding buffer, the fusion protein was cleaved with SUMO protease 1. Urodilatin was separated from the fusion partner by the subtractive chromatography using Ni-sepharose once again, and then further purified with reverse-phase high performance liquid chromatography. In vitro activity assay demonstrated that the recombinant urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC₅₀ of 1.77 ± 0.53 μg/ml, which was similar to that of the synthetic urodilatin standard. The expression strategy presented in this study allows convenient high yield and easy purification of small recombinant peptides with native sequences.