A novel and effective human hepatocyte growth factor kringle 1 domain inhibits ocular neovascularization.

Neovascularization is the critical pathological process and the leading cause of blindness in a variety of clinical conditions. This angiogenesis process is still uncertain. Human hepatocyte growth factor 1 (HGFK1) is derived from the mature form of hepatocyte growth factor, which contains four kringle domains in its α-chain. This study aimed to investigate the antiangiogenic activity of HGFK1 using in vitro and in vivo assays. HGFK1 was added into the DMEM to test the proliferation and migration of human umbilical vascular endothelial cells (HUVEC), and it was intravitreously injected in laser photocoagulation-induced choroidal neovascularization (NV) model, oxygen-induced retinopathy model and rho/VEGF transgenic mice to test its antiangiogenic effect. The results showed that HGFK1 effectively inhibited VEGF-stimulated HUVEC proliferation and migration, and also had anti-NV activity in choroidal NV and retinal NV. It is suggested that HGFK1 has antiangiogenic activity in vitro and in vivo. It may lead to new potential drug discoveries and the development in addition to anti-VEGF therapy in the future clinical anti-angiogenesis treatment.

A potential therapeutic strategy for inhibition of ocular neovascularization with a new endogenous protein: rhEDI-8t.

BACKGROUND: Endogenous angiogenesis inhibitors act as natural negative feedback in the focal area during the neovascularization process, and have less interference on physiological angiogenesis, and thus fewer negative side-effects. These inhibitors are potential candidates to combine with or substitutes for current popular anti-angiogenesis treatments to have synergistic effect. In this study, the effects of recombinant endothelial growth inhibitor protein (rhEDI-8t), a novel endogenous protein originated from collagen VIII, was investigated on ocular neovascularization (NV). Endostatin, a well-identified endogenous angiogenesis inhibitor, was compared in parallel and served as a positive control. METHODS: The inhibitory effect of rhEDI-8t on vascular endothelial cells was evaluated by a human umbilical vascular endothelial cells (HUVEC) proliferation test and a bovine aortic endothelial cells (BAEC) migration experiment. The effect of rhEDI-8t on ocular NV was further investigated in mice with choroidal neovascularization (choroidal NV) induced by laser, ischemic retinopathy and transgenic mice with expression of VEGF in photoreceptors (rho/VEGF) respectively. RESULTS: RhEDI-8t inhibited the growth of HUVECs and migration of BAECs stimulated by basic fibroblast growth factor (bFGF). Mice intravitreally treated with rhEDI-8t showed a significant reduction of choroidal NV, retinal NV and subretinal NV. CONCLUSION: Endogenous angiogenesis inhibitor rhEDI-8t showed a potent anti-angiogenesis effect in both in vitro and in vivo experiments. It contributed to the suppression of ocular NV. The study suggested that rhEDI-8t could be a subsidiary potent therapeutic medicine in addition to anti-VEGF therapy in future clinical anti-angiogenesis treatment.

[Secretory Expression of the Superactive [Lys(17,18),Glu(21)]-Glucagon in E. coli].

One of the most important findings in structure-function studies on glucagon by means of chemical synthesis is the discovery that [Lys(17,18),Glu(21)]-glucagon had higher biological activity than native glucagon. This mutant of glucagon was called superactive glucagon (SA-glucagon). In the present work, the possibility to obtain SA-glucagon by means of genetic engineering was studied. The gene of SA-glucagon (SAG) was obtained by PCR from a constructed recombinant glucagon plasmid, pAGluT. A secretory expression vector harboring SAG, pBLSG7, containing P(L) promoter and the gene of phoA signal peptide was constructed. In expression studies after transformation of pBLSG7 into E. coli BL21, it was found that the expression yield of SA-glucagon reached 3.65 mg/L(A(600)=1), about 19.5% of total proteins in the culture medium under shaken flask conditions. In addition, the influence of induction temperature and of E. coli strain on the expression yield of SA-glucagon was also studied.

Cloning of Human Granulocyte-macrophage Colony Stimulating Factor (GM-CSF) cDNA and Its Expression in E. coli.

Human GM-CSF cDNA fragment encoding the mature GM-CSF was obtained by using RT-PCR method with total RNA extracted from induced human fetal lung cells HFL. The sequence of the h GM-CSF cDNA thus obtained was the same as those reported. In order to get expression of a high level in, the 5' terminal nucleotide sequence of hGM-CSF cDNA was modified by using PCR. The modified h GM-CSF cDNA was inserted into plasmid pET-11d containing T7 promoter, with the resulted expression of plasmid pETC-5. E. coli BL21(DE3) was transformed with pETC-5 and an expressed strain BLEC4 was selected. SDS-PAGE analysis revealed the rhGM-CSF was produced and accumulated up to 16% of the total cellular protein in the form of inclusion body in BLEC$ cells after induced by 0.5 mM IPTG for 2 h. ELISA and TF-1 cell culture assay showed that the biological activity of the partially purified and renatured rhGM-CSF was similar to that of the natural human GM-CSF.

On the Mechanism of Growth Inhibition of Epiregulin in A431 Epidermal Carcinoma Cells.

Preliminary investigation on the mechanism of the growth inhibition by recombinant epiregulin(EPI)of epidermal carcinoma cell A431 is reported. Northern blotting indicated that the mRNA level of cyclin dependent kinase(CDK)inhibitor, p21(WAF1/CIP1), was increased significantly after stimulation of the recombinant epiregulin protein. Luc reporter revealed that STAT1 could bind the promoter region of p21 in response to the EPI signal. Flow cytometry assay showed that the EPI-induced growth inhibition was not related to the apoptosis. The above results indicate that the EPI-induced cell growth inhibition might result from the STAT1-stimulated expression of p21, leading to the G1 arrest.

A Hepatitis B Virus Variant with an Ile to Ser Mutation at aa126 of HBsAg.

For the detection of HBV variants in patients vaccinated with HBV vaccine but failed to be protected, 16 children patients were studied by using the polymerase chain reaction (PCR) to amplify the HBV S gene fragment. To increase the sensitivity, a nested PCR method was used. These 10 HBV S gene fragments amplified from patients were cloned into M13mp18 phage vector and then sequenced respectively. One of them, No.19, was found to have a point mutation within a determinant coding region (nt524-nt595) of the HBsAg. There was a G at nt 531 instead of T, leading to a change of Ile to Ser at aa126 of the major HBsAg. As aa126 is located in the first loop of the two-looped conformational structure of the determinant, and the Ile to Ser at aa 126 is a drastic change, it is suggested that the antigenicity of the HBsAg might be altered and the immune failure in patient No.19 was probably related to the mutation.

The Effect of Partially Conserved Residues of hTGF-alpha C Domain in Its Structure and Function.

There is a highly homologous region in the C domain of the EGF family, some of its residues are semi-conserved. We constructed three hTGF-alpha mutants, hTGF-alphaV35, hTGF-alphaQ44, hTGF-alphaY45R46, by site-directed mutagenesis to replace the semi-conserved residues in the C domain of hTGF-alpha with the corresponding residues of hEGF. We observed that although the binding affinity of hEGF to hEGF receptor was about two fold that of hTGF-alpha, but the receptor binding affinity of the three mutants was respectively decreased to about 22 %, 13.4 % and 25 % compared of that of hTGF-alpha. On the other hand, the stimulating action of hEGF on NRK-49F cell proliferation was only 10 % that of hTGF-alpha, but those of the threemutants was about 4 fold, 10 fold and 5 fold more active than hTGF-alpha. Thus, the three mutants did not become more similar to hEGF in function. The functional difference between hEGF and hTGF-alpha was not simply determined by any single semi-conserved residue, but substitution at those sites in the C domain have altered the characters of hTGF-alpha sharply.

Potential GM-CSF Antagonists Selected from a Peptide Phage Display Library.

Peptide phage display libraries have been successfully applied in areas of mapping antibody epitope, finding ligands for enzymes, receptors, and many other molecules. But it has been demonstrated to be very difficult to select cytokine-binders from peptide phage display libraries probably because cytokine is not so sticky as antibody that there are rare chances of capturing peptide phages during biopanning. A pVIII-based peptide phage display library was panned with the cytokine GM-CSF and some GM-CSF binding clones were selected based on high throughput screening (HTS) method and confirmed by ELISA and micropanning assays. These cytokine-binders may be utilized in affinity chromatography in cytokine downstream processing and even act as potential antagonists of GM-CSF if their affinity are further improved through secondary library strategy.

[Expression and characterization of Kringle 1-5 domains of human plasminogen].

The cDNA encoding Kringle 1-5 domains of human plasminogen (designated as K1-5), obtained from HepG2 by RT-PCR, was cloned into expression vector pHIL-S1. The recombinant plasmid pHIL-K1-5 was transformed into Pichia pastoris GS115 and the recombinant yeast was induced by methanol to express the recombinant protein. The expressed protein was purified by lysine affinity chromatography. The recombinant K1-5 inhibited the growth of bovine aortic endothelial cells (BAEC) stimulated by the basic fibroblast growth factor (bFGF), in a dosage-dependent manner, with a half maximal concentration of 14 mg/L. And rhK1-5 inhibited 47% of the BAEC migration stimulated by bFGF at the concentration of 50 mg/L. rhK1-5 also affected the cell cycle of BAEC and caused G(0)-G(1) arrest at the concentration of 14 mg/L.

Expression of Human Angiostatin in Pichia pastoris and the Detection of Its Anti-angiogenic Activity.

Human angiostatin cDNA was amplified from human hepatoma cell line HepG2 using RT-PCR and was cloned into pPIC9K vector. Recombinant Pichia pastoris strain with 4 copies of angiostatin gene was obtained. Recombinant protein was purified by lysine-affinity Sepharose column and the finally purified angiostatin was 25 mg/L, higher than previously reported 17 mg/L. Amino acid sequence analysis revealed the identity of our protein the same with that previously reported. Recombinant angiostatin inhibited specifically the proliferation of bovine aortic endothelial cell stimulated by bFGF, with ED(50) being about 3 mg/L.

[Restin expressed in vivo suppresses the growth of tumors in nude mice].

Restin, a homologous protein of endostatin (62% homology), is the NC domain of collagen XV at C-terminal. The recombinant restin expressed in E. coli had the ability to suppress the proliferation of bovin aortic endothelial cells and cause apoptosis. In this report, mouse restin gene was fused with a sequence of human plasminogen signal peptide by PCR and cloned into eukaryotic expression vector pCDNA3. The plasmid containing restin gene was named pCDNAXV and was transfected into human hepatoma cell line Bel7404. Stable transfected clones were screened and expression of restin was confirmed by RT-PCR and Western blot. The proliferated cells were injected subcutaneusly into nude mice. The growth of tumors formed by cells transfected with restin gene was much slower than that of control group. These results indicated that the expressed restin in vivo could suppress the growth of tumor, and this suppression might be achieved by restraining angiogenesis since the restin had no effect on the proliferation of tumor cells. At the same time, this report provided a new method to investigate the effect of anti-angiogenetic proteins on the tumor growth.

Mouse restin inhibits bovine aortic endothelial cell proliferation and causes cell apoptosis.

Restin, a homologous protein of endostatin, was found by Ramchandran et al. It was the C-terminal fragment of type XV collagen. To analysis the inhibition activity of mouse restin on the proliferation of endothelial cells, the cDNA of restin was amplified from the total RNA of the mouse muscle and cloned into the prokaryotic expression plasmid pQE32. The recombinant protein was expressed in inclusion body with a yield about 60%--70% of total protein. After refolding, the purified recombinant protein specifically inhibits bovine aortic endothelial (BAE) cell proliferation stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent manner, but the activity of restin was weaker than that of endostatin. Treatment of BAE cell with recombinant restin caused G(1) arrest and apoptosis in BAE cells.

Expression of Human Epiregulin in E.coli.

Human epiregulin cDNA was amplified from the lung cancer cell line A549 using RT-PCR. After adding 6 His codon to its 3' end, it was cloned into a high efficient secretive Escherichia coli system with alkaline phosphatase promoter(phoA promoter)constructed in our lab and induced for expression. The product was purified one-step by Ni-NTA column. Amino acid sequence analysis revealed the identity of our product with that previously reported. The product showed strong proliferative effect on fibroblast cell line Balb/c3T3 and growth inhibitory effect on epithelial carcinoma cell line A431.

[Expression and characterization of Kringle 1-4.5 domains of human plasminogen].

The cDNA encoding Kringle 1-4 and part of Kringle 5 domains of human plasminogen (K1-4.5), obtained from HepG2 by RT-PCR, was cloned into expression vector pHIL-S1. The recombinant plasmid pHIL-K1-4.5 was transformed into Pichi pastoris GS115 and the recombinant yeast was induced to express the recombinant proteins by methanol. The expressed proteins were purified by lysine affinity chromatography to a purity of 95%. The recombinant K1-4.5 inhibited the growth of bovine capillary endothelial cells (BAEC) stimulated by the basic fibroblast growth factor (bFGF), in a dosage-dependent manner with a half maximal concentration of 2 mg/L. rhK1-4.5 also inhibited 40% of the BAEC migration stimulated by bFGF in the concentration of 1 mg/L.

Functional Difference between N Domain and C Domain of hEGF and hTGF-alpha.

By exchanging the N domain and C domain of hEGF and hTGF-alpha genes by PCR, two chimeras E-TGF(EGF(1-32)-TGF-alpha(34-50))and T-EGF(TGF-alpha(1-33)-EGF(33-53))were constructed. The wild and chimeric molecules were expressed in E.coli under phoA system. The expressed hEGF, hTGF-alpha and two chimeras were purified. The EGF receptor competitive binding affinity of the four molecules was hEGF > hTGF-alpha and E-TGF > T-EGF and the cell proliferation stimulating activity of them was hTGF-alpha and E-TGF > T-EGF > hEGF. The result suggests that the N domain of hEGF and hTGF-alpha may play a major role in receptor binding activity and C domain of them may be responsible for stimulating cell proliferation.

Cloning and Characterization of fup1, A Gene Highly Expressed in Hepatocellular Carcinoma.

Primary hepatocellular carcinoma(HCC) is one of the common malignant tumors in China. In our previous work, a gene named fup1(function-unknown protein 1) was isolated that was expressed differently in HCC and in normal liver. We assumed that it might be a candidate oncogene for the HCC. The fup1 gene had a ORF of 1 233 bp, encoding a protein with M(r) of 46 kD and isoelectric point of 5.48. The sequence characteristics showed its possible localization in nuclei. Northern blots showed that this gene was weakly expressed in many types of human tissues, except in the heart, implying its tissue-specific expression pattern. MTT assay of the NIH 3T3 cells transfected with this gene in the form of recombinant eukaryotic expression plasmid showed its enhancing role to cellular proliferation.

Expression and subcellular localization of P9-ZFD protein in patients with myasthenia gravis.

OBJECTIVE: To express and purify the protein coded by the TRAF-type zinc finger domain of myasthenia gravis (MG)-related gene P9 (P9-ZFD) and to prepare P9-ZFD antiserum for detecting expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of patient with MG. METHODS: The cDNA encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD cDNA was ligated into pET24a, and the P9-ZFD recombinant protein was induced via E. coli. BL21 (DE3) and purified by histidine affinity chromatography. P9-ZFD antiserum was prepared and its titer and specificity were determined by ELISA and Western blot. Expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of MG and control were studied. RESULTS: The molecular weight of purified P9-ZFD protein was about 30 kD. Its purity was more than 95%. Antiserum specific for P9-ZFD was excellent. P9-ZFD protein is fully confined to the cytoplasm membrane of skeletal muscle cell of MG, obvious immunostaining was absent in the A, I, and Z bands of cytoplasm and no immunoreactivity was observed in the skeletal muscle cell of control. CONCLUSION: P9-ZFD protein is expressed as a cytoplasm membrane-bound protein and has obvious distribution difference in the skeletal muscle cells of patient with MG and normal control.

[Endothelial genesis inhibitor-8t (EDI-8t) against tumor growth].

On the basis of the origin comparison of known endothelial genesis inhibitors, a 417-bp cDNA fragment was amplified from umbilical cord by RT-PCR and cloned into the expression vector pPIC9, followed by transformation into Pichia pastoris GS115. The resulted yeast was induced with methanol to express recombinant protein. The resulted protein was purified from culture broth and designated as EDI-8t. The in vitro study showed that EDI-8t, originated from collagen VIII, could specifically inhibit the growth and migration of bovine aortic endothelial cells (BAEC) stimulated by basic fibroblast growth factor (bFGF). The protein also exhibited the activity to cause cell apoptosis. In vivo EDI-8t showed the identical activity comparing with endostatin to inhibit the growth of liver tumor transplanted into nude mice. Interestingly, EDI-8t showed higher activity than endostatin to inhibit tumor growth in metastatic model of melanoma mice.

Construction of secretory expression system suitable to express glucagon under the control of PL promoter.

It was reported that PL promoter and alkaline phosphatase (phoA) signal peptide were used to construct secretory expression plasmid suitable to express glucagon and [Des-His1] glucagon in E. coli BL21 herein. Expression studies showed these two peptides could be expressed and secreted into the culture medium. The expression yield of recombinant glucagon reached 3.46 mg/L/OD600 unit of cells in shake flask. The yield of [Des-His1] glucagon was found to be higher than that of glucagon. In addition, some factors involved in secretion were studied too.

[Study on the changes of IL-3 and its receptor in mice with immune-mediated aplastic anemia].

The aim of this study was to find new idea for clinical treatment of aplastic anemia. Immune-mediated aplastic anemia mice were developed, IL-3 in the supernatant with PHA stimulating splenic cells was detected by ELISA, semi-quantiting analysis of IL-3R was performed by point hybridization. The results showed that the IL-3 level in the supernatant with PHA stimulating splenic cells of immune-mediated aplastic anemia mice was higher than controls, difference between them was significant (P <0.001), while amount of IL-3 receptor by semi-quantiting analysis was lower than control significantly. In conclusion, the IL-3 receptor expression level is important for pathogenesis and treatment strategy of aplastic anemia.