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Biomedical subjects

An Hong

Publications and source records attributed to An Hong.

15 recordsLinked to original sources
PubMed2006

Cross-species pharmacokinetic comparison from mouse to man of a second-generation antisense oligonucleotide, ISIS 301012, targeting human apolipoprotein B-100.

The pharmacokinetics of a 2'-O-(2-methoxyethyl)-modified oligonucleotide, ISIS 301012 [targeting human apolipoprotein B-100 (apoB-100)], was characterized in mouse, rat, monkey, and human. Plasma pharmacokinetics following parental administration was similar across species, exhibiting a rapid distribution phase with t(1/2alpha) of several hours and a prolonged elimination phase with t(1/2beta) of days. The prolonged elimination phase represents equilibrium between tissues and circulating drug due to slow elimination from tissues. Absorption was nearly complete following s.c. injection, with bioavailability ranging from 80 to 100% in monkeys. Plasma clearance scaled well across species as a function of body weight alone, and this correlation was improved when corrected for plasma protein binding. In all of the animal models studied, the highest tissue concentrations of ISIS 301012 were observed in kidney and liver. Urinary excretion was less than 3% in monkeys and human in the first 24 h. ISIS 301012 is highly bound to plasma proteins, probably preventing rapid removal by renal filtration. However, following 25 mg/kg s.c. administration in mouse and 5-mg/kg i.v. bolus administration in rat, plasma concentrations of ISIS 301012 exceeded their respective protein binding capacity. Thus, urinary excretion increased to 16% or greater within the first 24 h. Albeit slow, urinary excretion of ISIS 301012 and its shortened metabolites is the ultimate elimination pathway of this compound, as demonstrated by 32% of dose recovered in total excreta by 14 days in a rat mass balance study. The pharmacokinetics of ISIS 301012 in human is predictable from the pharmacokinetics measured in animals. The pharmacokinetic properties of ISIS 301012 provide guidance for clinical development and support infrequent dose administration.

Animals↗
PubMed2006

Effects of IkappaBalpha and its mutants on NF-kappaB and p53 signaling pathways.

AIM: To study the effects of IkappaBalpha and its mutants (IkappaBalphaM, IkappaBalpha243N, IkappaBalphaM244C) on NF-kappaB, p53 and their downstream target genes. The relationship of NF-kappaB, p53, and IkappaBalpha was further discussed. METHODS: pECFP-IkappaBalpha, pECFP-IkappaBalphaM (amino acides 1-317, Ser32, 36A), pECFP-IkappaBalpha243N (amino acides 1-243), pECFP-IkappaBalpha244C (amino acides 244-317), pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-alpha-1 cells. Cells were transfected with pECFP-C1 as a control. 30 h after the transfection, location patterns of NF-kappaB, p53 and IkappaBalpha (IkappaBalphaM, IkappaBalpha243N, IkappaBalpha244C) were observed by a laser scanning microscope (LSM510/ConfoCor2, Zeiss). RNA extraction and reverse transcription were performed in cells transfected or co-transfected with different plasmids. Effects of IkappaBalpha and its mutants on the transprition level of NF-kappaB, NF-kappaB downstream target gene TNF-alpha, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments beta-actin was reference. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the control. Different transfected cells were incubated with CCK-8 for 2 h in the incubator. Then the absorbance at 450 nm was measured by using a microplate reader. RESULTS: Cells that were transfected with p53-DsRed revealed a predominant nuclear localization. YFP-p65 mainly existed in the cytoplasm. Cells were transfected with CFP-IkappaBalpha, CFP-IkappaBalphaM, and CFP-IkappaBalpha243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-IkappaBalpha244C revealed a predominant nuclear localization. The mRNA levels of p65, TNF-alpha, p53 and Bax in CFP-IkappaBalpha transfected cells did not change significantly, while in YFP-p65/CFP-IkappaBalpha co-transfected cells, IkappaBalpha decreased the transcription of p65 downstream gene TNF-alpha (2.24+/-0.503) compared with the YFP-p65/CFP-C1 co-transfected cells (5.08+/-0.891) (P<0.05). Phosphorylation defective IkappaBalpha (IkappaBalphaM) decreased the transcription levels of all the four genes compared with the control (P<0.05). The N terminus of IkappaBalpha (IkappaBalpha243N) increased the transcription of NF-kappaB (1.84+/-0.176) and TNF-alpha (1.51+/-0.203) a little bit. However, the C terminus of IkappaBalpha (IkappaBalpha244C) increased the transcription of NF-kappaB, TNF-alpha, p53 and Bax significantly (8.29+/-1.662, 14.16+/-2.121, 10.2+/-0.621, 3.72+/-0.346) (P<0.05). The CCK-8 experiment also showed that IkappaBalpha244C and p53 synergistically mediate apoptosis. CONCLUSIONS: IkappaBalpha and its mutants (IkappaBalphaM, IkappaBalpha243N, IkappaBalphaM244C) have different effects on NF-kappaB and p53 signaling pathways, according to their different structures. IkappaBalphaM bounds with NF-kappaB and p53 in cytoplasm steadily, and inhibits both of the two signaling pathways. p53 and IkappaBalpha244C may be co-factor in inducing apoptosis. The C terminal of IkappaBalpha enhanced cell death, which suggests that it may be a pro-apoptotic protein existed in cells.

Apoptosis↗
PubMed2006

Intein-mediated rapid purification and characterization of a novel recombinant agonist for VPAC2.

In order to obtain the recombinant VPAC2 agonist efficiently by intein-mediated single column purification, a gene encoding 32-amino acids peptide was designed, synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-ROM was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the rMROM-intein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by beta-mercaptoethanol and the rMROM with the homogeneity over 95% was released from the chitin-bound intein tag. The recombinant linear rMROM competitively displaced [125I] PACAP38 on VPAC2 with a half-maximal inhibitory concentration (IC50) of 60 +/- 5 nM, whereas the IC50 of rMROM at human VPAC1 was observed up to 10 microM and no binding was detected at PAC1. rMROM stimulated the cAMP accumulation in Chinese hamster ovary (CHO) cells expressing the human VPAC2 with a half-maximal stimulatory concentration (EC50) of 0.6 nM, which was 500-fold less potent at VPAC1and had no activity on PAC1. An efficient production procedure of a novel recombinant VPAC2-selective agonist was established.

Amino Acid Sequence↗
PubMed2006

[Expression and characterization of VPAC2 in CHO cells].

VPAC2 is a co-receptor of pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) and mediates multiple bio-functions. In order to construct the CHO line expressing VPAC2 stably, pcDNA-VPAC2 was used to transfect CHO cells. The positive clones were selected by G418 and the clone VPAC2-CHO with high sensitivity to PACAP38 was picked out by its ability to promoting the concentration of cAMP. RT-PCR, Western blot and Immunofluorescenece assay were used to identify the express of VPACS. Binding competition with VPAC2 agonist and the bioactivity of mediating the ligand to promote the concentration of cAMP showed that VPAC2 was expressed effectively in VPAC2-CHO. The results of Scatchard analysis revealed that VAPC2-CHO expressed a receptor density of (1.1 +/- 0.2) pmol/mg protein, respectively, with Kd values of (0.55 +/- 0.10) nmol/L for PACAP38 used as a tracer. The construction of CHO cells expressing VPAC2 specially and functionally lays a foundation not only for the further research on the characters and functions of VPAC2 but also for the screening and characterization of novel agonists of antagonists for VPAC2.

Animals↗
PubMed2005

Biochemical properties of C78SC96S rhFGF-2: a double point-mutated rhFGF-2 increases obviously its activity.

Fibroblast growth factor-2 (FGF-2) is a multifunctional polypeptide that affects many cellular functions and phenomena. The wild-type recombinant human fibroblast growth factor rhFGF-2(W) and the mutant C78SC96S rhFGF-2(M) were expressed in Escherichia coli and their products were purified. The results by the means of fluorescence spectroscopy and CD spectrums, suggested that due to its decreased hydrophobicity rhFGF-2 is not deposited as an inclusion body. The mitogenic activity of the expressed rhFGF-2(M) on 3T3 fibroblasts was shown to be 10-fold more than the expressed rhFGF-2(W) of which the biological activity was a little less than that of the standard rhbFGF(W), indicating that the increased biological activity was due to the change of its secondary structure, dimerization and affinity binding to FGF receptor (FGFR).

3T3 Cells↗
PubMed2005

Update on therapeutic neovascularization.

Therapeutic neovascularization for cardiovascular ischemia is a promising avenue in spite of disappointing early clinical trial results. The concept of three different mechanisms of neovascularization has served to define potential therapeutic targets such as vascular remodeling and stem cell recruitment, but it is anticipated that this will lose significance as the pleiotropic nature of angiogenic cytokines becomes fully understood. With the rapidly growing body of data on growth factors and pro-angiogenic strategies, approaches will emerge that are more effective than the ones that have been tested clinically thus far. Combinations of growth factors, for instance to stabilize vessels, or growth factors combined with cell transplants deserve more attention but will make the design of preclinical and clinical studies increasingly complex. Recent developments suggest that when using the appropriate dose and treatment regimens, even single growth factor therapy can result in stable and functional vessels. Whether gene therapy or protein therapy will be optimal for this purpose depends mainly on technical developments in vector design and production and on progress in the engineering of slow release matrix formulations for proteins. With the increasing complexity of therapeutic strategies, it remains imperative that these approaches are rationally based on fundamental and preclinical data.

Angiogenesis Inducing Agents↗
PubMed2005

Basic fibroblast growth factor alleviates brain injury following global ischemia reperfusion in rabbits.

The aim of this study was to explore the protective effect of basic fibroblast growth factor (bFGF) on brain injury following global ischemia reperfusion and its mechanisms. Brain injury following global ischemia was induced by four vessels occlusion and systemic hypotension. Twenty-four rabbits were randomized into three groups: group A, only dissection of vessels; group B, intravenous infusion of normal saline after reperfusion for 6 h; group C, 30 microg/kg bFGF injected intravenously at the onset of reperfusion, then infused with 10 microg/(kg.h) for 6 h. Serum neuron specific enolase (NSE), S-100B, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-8 (IL-8) were measured before ischemia, 30 min after ischemia, 0.5, 1, 3, 6 h after reperfusion. Brain water content was determined and cerebral histopathological damages were compared. NSE and S-100B were increased 1 h after reperfusion and reached their peaks 6 h after reperfusion, but were much higher in group B than those in group C 3, 6 h after reperfusion. In groups B and C, TNF-alpha was increased after ischemia and IL-1 and IL-8 were increased significantly 0.5 h after reperfusion, then reached their peaks 6 h, 3 h, 6 h after reperfusion respectively. TNF-alpha and IL-8 at the time points of 1 h and 3 h and IL-1 at 3 h and 6 h in group C were correspondingly lower than those in group B. These indices in group A were nearly unchanged. There were less severe cerebral histopathological damages in group C compared with group B, but no difference in brain water content. It could be concluded that bFGF alleviates brain injury following global ischemia and reperfusion by down-regulating expression of inflammatory factors and inhibiting their activities.

Animals↗
PubMed2005

[On-column refolding and purification of human EGF receptor L2 domain inclusion body overexpressed in Escherichia coli].

The human epidermal growth factor receptor (EGFR) extracellular region (residues 1-621) consists of four subdomains, i.e. L1, S1, L2, and S2. The L2 domain (EGFR-L2) is composed of residues 311-479 and plays a major role in ligand-binding. Due to the high content of cysteine residues (42 cysteines) in the S1 and S2 domains, it is quite difficulty to get a correctly refolded product of the complete EGFR extracellular domain. In contrast, only 4 cysteine residues are present in EGFR-L2 domain. The aim of the present study is to prepare a soluble EGFR-L2 domain from the recombinant protein inclusion body overexpressed in Escherichia coli (E. coli). DNA fragment encoding EGFR-L2 containing a polyhistidine-tag at the carboxyl terminus was amplified by PCR from the cDNA of EGFR extracellular region, and was inserted into pET-3c to construct the prokaryotic expression vector. The target protein was highly expressed in E. coli BL21 (DE3) strain and was only present in the inclusion body as revealed by immunoblotting analysis. No soluble product could be refolded through dilution or stepwise dialysis strategies. However, on-column refolding of denatured EGFR-L2 bound to Ni2+ -NTA produced a soluble one. Furthermore,the soluble EGFR-L2 was simultaneously purified to high purity (>95%) through eluting from the same Ni2+ -NTA column with a linear imidazole gradient. The refolded EGFR-L2 had specific binding activity with the cognate ligand EGF, although its affinity was low. These results suggest that a polyhistidine-tag fused with a recombinant protein facilitate not only the purification but also the renaturation of the target product through on-column refolding. Besides, this refolding strategy may be suitable for the preparation of those recombinant proteins which are hard to refold through conventional approaches.

ErbB Receptors↗
PubMed2004

Intein-mediated rapid purification of recombinant human pituitary adenylate cyclase activating polypeptide.

In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the PACAP-intein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by DTT and the rhPACAP was released from the chitin-bound intein tag. The activity of the rhPACAP to stimulate cyclic AMP accumulation was detected using the human pancreas carcinoma cells SW1990. Twenty-two milligrams of rhPACAP with the purity over 98% was obtained by single column purification from 1 liter of induced culture. The preliminary biological assay indicated that the rhPACAP, which has an extra Met at its N-terminus compared with the native human PACAP, had the similar activity of stimulating cAMP accumulation with the standard PACAP38 in the SW1990 cells. A new efficient production procedure of the active recombinant human PACAP was established.

Base Sequence↗
PubMed2004

High-ratio differentiation of embryonic stem cells into hepatocytes in vitro.

BACKGROUND/PURPOSE: To date, in differentiating system of embryonic stem (ES) cells into hepatocytes, hepatic differentiation ratio was still not shown. Here, after investigating hepatic differentiation from ES cells, we determined the differentiation ratios of hepatocytes and studied how to improve the ratios in ES cell differentiating system. METHODS: Embryonic bodies (EBs) formed from ES cells for 5 days were plated onto culture dishes and some growth factors were added into medium for hepatic differentiation. Expressions of hepatic genes and proteins were analysed using reverse transcriptase-polymerase chain reaction, immunocytochemistry (ICC) and radioimmunoassay. The relative counts of hepatocyte-like cells among all EBs cells were analysed by flow cytometry by which hepatic differentiation ratios were determined. Then, we observed the spatial distribution of ICC-positive cells in EB cells cluster and isolated the cells of positive areas in other EBs clusters without ICC examined. At last, isolated cells were re-cultured with previous condition and hepatic differentiation ratios were also determined. RESULTS: The hepatic genes and proteins were, respectively, expressed in cytoplasm. Hepatic differentiation ratio was first determined at day 11 to be 12.1% and the level reached maximum to be 33.4% at day 21. In isolated cells culture system, hepatic genes and proteins expressed stronger than that expressed in EBs cluster and hepatic differentiation ratio was got to 72.6% at day 21. CONCLUSIONS: Isolating hepatocyte-like cells from EBs cell cluster and re-culturing them could produce hepatocytes with high differentiation ratio. This culture system may produce a new source of cell types for hepatocytes replacement therapies in hepatic failure.

Albumins↗
PubMed2003

[Study on high-expression of soluble hbFGF in Escherichia coli].

The formation of inclusion body is correlated with the overexpression of foreign proteins in Escherichia coli. Controlling of the expression level seems to be critical to increase the soluble component. With the prerequisite of no alterations in amino acid sequence, the wobble bases of the first 3 codons downstream the initiation codon ATG of a hbFGF high-expression mutation, established previously, were changed into the bases of the native hbFGF sequences. Seven primers were synthesized for retro-mutations, after PCR were undertaken, the products were cloned into pET-3c, and recombinants were transformed into BL21 (DE3) plysS for expression. One strain with high solubility and bioactivity were identified, indicating that restriction of the expression level of recombinant protein is helpful for high-solubility.

Codon↗
PubMed2002

Bergenin is the antiarrhythmic principle of Fluggea virosa.

Bergenin was isolated from the aerial parts of Fluggea virosa (Euphorbiaceae). Its structure was elucidated on the basis of chemical and spectral data. Anti-arrhythmic effects of bergenin were investigated. At concentrations of 0.2 mg/kg, 0.4 mg/kg, and 0.8 mg/kg, bergenin showed distinct therapeutic effects on BaCl2-induced arrhythmias in rats. At concentrations of 0.4 mg/kg and 0.8 mg/kg bergenin significantly countered arrhythmias induced by ligation and reperfusion of the coronary artery. At 0.8 mg/kg, bergenin elevated the atria fibrillation threshold in rabbits from 1.34 mV to 1.92 mV. Our results suggest that bergenin has good potential to treat cardiac arrhythmias.

Animals↗
PubMed2002

Cloning and high expression of hbFGF with a new strategy.

Computer program DNASIS v2.5 was used to help designing the site-directed mutations for optimizing the expression of hbFGF in E. coli. The secondary structure of the translation initiation region (TIR) is a determinant factor for translation initiation rate, meanwhile, codon preference plays an important role, too. According to the two principles, 4 sites in 5' end of hbFGF cDNA were definitely changed, and another 4 sites randomly changed. These mutations will lead to potential variation in the secondary structure of TIR. Then computer program DNASIS v2.5 was utilized to analyse the total 32 TIR sequences resulted from the combination of the 4 randomly mutated sites. Ten sequences with highest free formation energy (delta G0) were chosen for subsequent cloning. By PCR using synthetic primers containing the 8 changed sites described above, ten hbFGF cDNA were amplified and cloned to pET-3c respectively. E. coli strain BL21 (DE3) was transformed and induced to express recombinant hbFGF. Two high-expression clones were obtained by SDS-PAGE and MTT assay, indicating that computer program-aided design for optimizing expression of foreign genes in E. coli is useful.

Cloning, Molecular↗
PubMed2002

Cloning and high level nonfusion expression of recombinant human basic fibroblast growth factor in Escherichia coli.

AIM: To obtain high-level expression of nonfusion recombinant human basic fibroblast growth factor (rhbFGF). METHODS: hbFGF cDNA was prepared from the total RNA of embryonic brain tissue. As a template, the obtained gene was used to clone nonfusion rhbFGF. New primers were employed to alter the translation initiation region (TIR) and reduce the G+C content through nucleotide change. Using pET-3C as vector, the cloned rhbFGF was expressed in BL21 (DE3). RESULTS: rhbFGF was expressed in E coli up to 30 % of the total cellular protein. Cation exchange and heparin affinity chromatography were employed to purify the target protein from the supernatant of bacteria lysate. The bioactivity of the purified rhbFGF was identical with the standard bFGF. CONCLUSION: Modification of TIR is an effective means to increase nonfusion expression rate of recombinant proteins, such as rhbFGF, in E coli.

Base Sequence↗