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Human proinsulin C-peptide with C-terminal glutamine amide could be prepared through solid phase method by combining the gamma-carboxyl group of glutamic acid with the amino group of MBHA resin. The protecting groups were cleaved by HF. MBHA resin is relatively inexpensive. The new method is another way for the preparation of human proinsulin C-peptide. The preparation human proinsulin C-peptide of analogue using of PAM resin was also reported.

Erythromycin may accelerate gastric emptying in animals and human probably as an motilin agonist, but its prokinetic effects show obvious individual disparity. This study was to find the mechanism of this phenomenon. Microarray analysis was used to screen genes that might be involved in the response of diabetic gastroparesis rats to erythromycin. It was found that erythromycin accelerated gastric emptying of diabetic rats with great individual disparity. Through microarray analysis we screened differential expression genes that might be involved in the effect of erythromycin. Among 10 genes screened out, dopamine D3 receptor (DRD3) and neuropeptide Y Y5 receptor (NPYY5) genes were submitted to RT-PCR quantification and showed consistent results with microarray. It can be concluded that erythromycin promote gastric emptying of gastroparetic rats; DRD3 and NPYY5 may be involved in prokinetic action of erythromycin; and targets other than motilin receptor of erythromycin might exist as prokinetics.

In this paper, we report the semisynthesis of B27 Lys destripeptide insulin (B27 Lys DTrI), i.e. destetrapeptide insulin with an additional Lys residue at the C-terminus of B-chain. B27 Lys DTrI is also monomeric as shown by gel filtration. Its in vivo biological activity is 80% in comparison with that of native insulin. The addition of a Lys residue at the C-terminus of B-chain makes it possible to obtain monomeric B27 Lys DTrI from a precursor expressed in Saccharomyces cerevesiae by tryptic hydrolysis instead of the less efficient tryptic transpeptidation.

Osteogenic growth peptide (OGP) has been synthesized through Fmoc solid phase synthesis procedure. The purity of synthetic OGP (sOGP) is over 98.6% identified by HPLC, the amino acid sequence and electro-spray mass spectroscopy are consistent with theoretical values. The synergetic effect of sOGP with recombinant human granulocyte-colony stimulating factor (rhG-CSF) on the hematopoiesis was investigated in normal mice. To assess the synergy of sOGP with rhG-CSF, two schemes were designed. In one scheme rhG-CSF was used at the last 8 days of a 13-day treatment with sOGP, while in the other one both cytokines were given concurrently for 10 days [sOGP, 0.5 nmol/day (mouse); rhG-CSF, 2 microg/day (mouse)]. Both schemes showed that sOGP remarkably synergized with rhG-CSF on increment of white blood cell number and lymphocyte number in peripheral blood without any change of red blood cell and platelet counts. Quantitative differential analysis of bone marrow and histological examination of the spleen and sternum showed that sOGP plus rhG-CSF did not cause abnormal hyperplasia, so sOGP is a very hopeful new drug to improve the effectiveness of clinical used rhG-CSF.

Western blot result showed that T(8)C(12), an anti-trichosanthin (TCS) monoclonal antibody, could bind to a CNBr-cleaved TCS fragment with MW of 8 kD. The epitope was located in 1-72 of the N terminal of TCS as shown by amino acid analysis. A random 6-aa peptide library cloned in pIII of phage M13 was screened by T(8)C(12). After two cycles screening, 15 positive clones were randomly selected and six kinds of 6-aa sequences were determined, which showed to be highly homologous with a Ser/Thr-(X)-(X)-Arg motif, the X denoting hydrophobic amino acids. The motif was found to be similar to the first 3-5 amino acid sequence of the TCS N-terminal. The synthesized 1-8 peptide of TCS showed competitive binding to T(8)C(12) with native TCS. It suggested that 3-5 amino acid residues of TCS N-terminal was the core of epitope recognized by T(8)C(12).

By growing the mouse mammary tumor-derived cell line GR2H6 in 96-well plates, we have developed an in vitro bioassay for the growth promoting activities of insulin. This bioassay system offers several advantages over currently used alternatives, such as higher sensitivity, better reproducibility and the processing of many samples simultaneously. Using this method, the mitogenic activities of insulin and its analogues were studied. The analogue with elongated C-tenminus of insulin B chain ( B31Lys Ins-NH(2)) had a higher mitogenic activity than insulin (130% of insulin). The mitogenic activities of analogues with B29Lys amino group blocked was one third of those with B29Lys amino group free, indicating that the C-terminal part of B chain and the amino group of B29Lys were important for the growth promoting activities of insulin.

The porcine insulin precursor (PIP) gene and its derivative form sp-PIP gene, which had a nona-peptide (called spacer peptide, sp) added at the 5' terminus of PIP gene, were inserted into the plasmid pPIC9 of Pichia pastoris to obtain secretory plasmid pPIC9/PIP and pPIC9/sp-PIP, respectively. P.pastoris GS115 was transformed by pPIC9/PIP or pPIC9/sp-PIP and the high-copy strains, P39(-sp) and S51(+sp), were selected by dot-blotting. The expression levels of PIP and sp-PIP were 10 mg/L and 40 mg/L in 1 L shake flask, respectively, indicating that the spacer peptide could increase the expression. The expression level of PIP (sp-PIP) in P.pastoris was higher than that of PIP in S.cerevisiae and K.lactis reported in this laboratory. The expression level of sp-PIP was 250 mg/L in 10 L fermentor. Recombinant human insulin was obtained by means of transpeptidation of PIP or sp-PIP. The receptor binding capacity is identical with that of porcine insulin. In vivo biological activity of the recombinant human insulin is 27 IU/mg.

The potency of salmon calcitonin (sCT) is higher but the structural homology between sCT and human calcition (hCT) is only 50%. Based on the comparison of the structure between sCT and hCT, we have designed and synthesized a hCT analogue (mhCT-2) by solid phase method, using air oxidation in diluted solution to obtain a peptide with intramolecular disulfide bond. Through HPLC purification, we obtained a capillary electrophoresis-homogeneous mhCT, results of analysis of its mass spectrum and N-terminal sequencing were in accordance with the theoretical values. The results of calcitonin bioassay by estimating the blood calcium levels in rats showed that the potency of mhCT-2 was around 2 000 IU/mg, one order of magnitude higher than that of hCT. In RIA assay, we have found that the immunoactivity of mhCT-2 and hCT was much different because of their different binding abilities to anti-hCT antibody. This indicated that the conformation of mhCT-2 was changed as compared with hCT. In rat osteoporosis model, the results showed that pharmacologic effects of mhCT-2 was the same as that of sCT. The synthetic mhCT-2 seems promising to be a clinically useful peptide with high potential in osteoporosis therapy, because it is similar in biological properties to, but less immunogenic than sCT.

A HPLC and CE pure human proinsulin C-peptide was synthesized by solid-phase method and TSK column purification. Its amino acid sequence and MS were consistent with theoretical values. In comparison with the formly reported chemical synthesis of C-peptide, this method has the advantage of simplicity and higher overall yield (41%). To improve the immunogenicity and specificity of oligopeptide antibody, the acrylyl-C-peptides were transformed into a polymer the product had a poly-propionyl-core matrix with C-peptide branches. This treatment gave a macromolecule with a M(r) about 25 kD. By using the polymer to immunize New Zealand rabbits for 30 days, specific antiserum was obtained with titer of 2.5x10(4) (by ELISA), which did not cross react with BSA. Thus, the poly-propionyl-peptide system provided a new approach for preparing synthetic peptide antibody and therefore is promising for the preparation of synthetic peptide-based vaccine.

sOGP was synthesized by standard solid phase method, with the purity of 99.2% shown by HPLC and CE. Its amino acid sequence and MS were consistent with theoretical values. In New Zealand white rabbits, sOGP could promote the for mation of new bone and up-regulate the serum level of ALP and BGP as observed by means of biochemical analysis, X-ray, bone mineral density, callus tissue histological analysis and biomechanical tests, demonstrating that sOGP might play a significant role in the tibia fracture healing. Particularly the bone density at 4 weeks and the amounts of trabecular bone in the callus at 2 and 4 weeks of the sOGP-treated group were significantly different from those of control group (P0.05). Effects of sOGP in different medium in vitro has been studied. sOGP could accelerate osteogenic cell proliferation in 1%BSA-10%NCS-MEM in a dose dependent manner, with a peak at 10(-11) mol/L. But interestingly, this prolife ration activity was not observed in 10%NCS-MEM or 4%BSA-serum free MEM. It suggests that sOGP's osteogenic cell proliferation effects may depend on the involvement of BSA and some regulatory molecules in serum.

Human calcitonin (hCT) is a 32 amino acid peptide hormone that requires C-terminal amidation for full biological activity. Calcitonin has important physiological function in vivo. We describe the couple expression of a synthesized modified human calcitonin(hmCT) gene fused with glutathione-S-transferase and rat peptidylglycine alpha-amidation monooxygenase (PAM) in insect cells infected by recombinant baculovirus GSTCT/PAM. Using Western blotting against hmCT or rat PAM, the GSThmCT fusion protein had been identified as well as the PAM. Following affinity chromatography with glutathione agarose column, the GSThmCT fusion protein produced by insect cells was purified. The purified fusion protein was also interacted with antibody against hmCT. The couple expression of a modification enzyme and its substrate in eucaryotic expression system may be used for producing other biological activity peptides.

In this article, we introduce a kind of capillary electrophoresis microchip in PDMS. This paper describes the procedure to design and fabricate channels in an elastomeric material - PDMS> The format of the capillary electrophoresis microchip is converted into a transparency, which serves as the photomask in contact photolithography to produce a SU-8 positive relief photoresist on silicon wafer. PDMS cast against the master yield s a replica contain ga network of channels, which are 200(Mu) m wide and 200micrometers deep. After the surface of this replica and a glass substrate being fabricated with the gold down-leads are oxidized in oxygen plasma, the two cured surfaces are brought into conformal contact and the capillary electrophoresis microchip is gotten.

DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase. However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitro reactivation and suppressing aggregation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding. Carboxymethylation of DsbC at Cys98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation. Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH. The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled and S-carboxymethylated RNases, but not by shorter peptides, including synthetic 10- and 14-mer peptides and S-carboxymethylated insulin A chain. In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI. DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer. Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI.

Two amphiphilic peptides, Amp1 and Amp2, were synthesized according to the sequence of the lipid-binding domain in apolipoprotein. Amp2 has a Val residue substituted for the Lys at the 4th position of Amp1. Intrinsic fluorescence spectra, peptide-induced leakage of calcein-laoded liposomes, quenching of tryptophan fluorescene by iodide and acrylamide, circular dichroism spectra, and measurement of the membrane penetration depth of tryptophan residue with spin-labeled phospholipids indicate unexceptionally that Amp1 interacted more strongly with the lipid bilayer than Amp2. It is proposed that class A amphiphilic alpha-helix is buried in the membrane in such a way that its long axis is oriented parallel to the membrane plane, and the electrostatic interaction between the positively charged residues located at the polar-nonpolar interface of the amphiphilic helix with the negatively charged head groups of phospholipids is important to the lipid affinity of the amphiphilic peptide.

Two amphiphilic peptides, Ampl and Amp2, were synthesized according to the sequence of the lipid-binding domain in apoliporotein. Amp2 has a Val residue substituted for the Lys at the 4th position of Ampl. Interaction between Ampl / Amp2 and liposomes with different lipid compositions were compared by studying the blue shifts of the intrinsic fluorescence emission maxima, the peptide-induced lipsome leakage and the quenching of tryptophan fluorescence by acrylamide. The influence of temperature on interactions was studied as well. Ampo1 interacted stronger with acidic lipids while Amp2 interacted stronger with zwitterionic lipids. The interaction was reinforced with the increasing extent of lipid unsaturation as well as with the increase of temperature. The lipid unsaturation had amore prominent effect.