OBJECTIVETo study the impact of complicated intra-abdominal infections on albumin synthesis rate.

METHODSEight patients with complicated intra-abdominal infections associated with intestinal fistula were admitted to the Research Institute of General Surgery at the Jinling Hospital between December 2009 and October 2010. Eight healthy volunteers matched for age, sex, and body mass index were enrolled as controls. All the subjects were given a primed, constant infusion of sterile L-[ring-(2)H(5)]-phenylalanine solution (priming dose: 4 μmol/kg, infusion rate: 6 μmol·kg(-1)·min(-1)) via peripheral venous lines in fast state. Arterial blood samples(3 ml) were drawn before and throughout the infusion at hourly intervals. The enrichment of L-[ring-(2)H(5)]-phenylalanine from the plasma free amino acid pool and from albumin were determined by gas chromatography/mass spectrometry analysis.

RESULTSBoth plasma total protein concentration(62.2±1.0) g/L and plasma albumin concentration (32.5±4.0) g/L in patients with complicated intra-abdominal infection were lower compared with controls[(74.2±1.7) g/L and (46.1±2.6) g/L, both P<0.05]. Body temperature, neutrophil count and plasma C-reactive protein concentration in patients with infection were significantly greater than the levels in control subjects(P<0.05). Albumin synthesis rate in patients with intra-abdominal infection was significantly lower than that in the control group [(5.3±1.6)%/d and (7.8±1.2)%/d respectively, P<0.05]. The measurement of plasma free amino acid concentration showed that plasma glutamic acid level was greater than that in control subjects, and that plasma phenylalanine and proline levels were lower than those in controls.

CONCLUSIONComplicated intra-abdominal infection inhibits albumin synthesis rate in patients with intestinal fistula, which may partially contribute to the decrease of plasma albumin concentration.

Adolescent ; Adult ; Case-Control Studies ; Female ; Humans ; Intraabdominal Infections ; blood ; Male ; Middle Aged ; Serum Albumin ; biosynthesis ; Young Adult

In order to make a large-scale preparation of(GLP-1A2G)2-HAS (GGH), the double-plamid pPICZalphaB and pPIC9K co-expression system was introduced into Pichia pastoris GS115. Firstly, the GGH fusion gene was amplified by PCR to create the recombinant expression plasmid pPICZalphaB-ggh, which was transformed into P. pastoris GS 115/F2 that was integrated by another recombinant expression plasmid pPIC9K-ggh. The immunology method combined with high concentration antibiotic was used to screen recombinant strain P. pastoris GS115/F3 capable of high-level expression of GGH protein. The GGH fusion protein expressed by GS115/F3 increased 49.7% compared with the GS 115/F2 in the expression conditions of 3% methanol inducing 80 h at 30 degrees C. At the same time, the quantitative PCR results showed that GGH gene dose in GS115/F3 increased 26.7% with respect to that of GS 115/F2. Furthermore, the Western blotting experiment indicated that the recombinant GGH possess the two antigenicities of GLP-1 and HSA.
Genetic Vectors ; genetics ; Glucagon-Like Peptide 1 ; biosynthesis ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Plasmids ; genetics ; Polymerase Chain Reaction ; methods ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics

It is generally believed that liposomes modified with polyethylene glycol (PEG) have no or lower immunogenicity. However, based on many recent literatures, when the PEGylated liposomes were repeatedly applied to the same animal, the immune responses occurred. The first injection of PEGylated liposomes resulted in a reduction in the circulation time and an increase in hepatic and splenic accumulation of the second dose of PEGylated liposomes in a time-interval, which was called "accelerated blood clearance (ABC)" phenomenon. Such immunogenicity of PEGylated liposomes presents a barrier in the research of liposomal formulations and their use in the clinics. This review focused on the definition, the method of verification, the development of the reason for ABC phenomenon, influencing factors of ABC phenomenon, and discussed if other PEGylated nanocarriers also induce ABC phenomenon.
Animals ; Antibiotics, Antineoplastic ; administration & dosage ; pharmacokinetics ; Doxorubicin ; administration & dosage ; pharmacokinetics ; Drug Carriers ; Immunoglobulin M ; biosynthesis ; blood ; Liposomes ; administration & dosage ; blood ; pharmacokinetics ; Liver ; metabolism ; Metabolic Clearance Rate ; Particle Size ; Polyethylene Glycols ; administration & dosage ; metabolism ; pharmacokinetics ; Serum Albumin, Bovine ; pharmacokinetics ; Spleen ; immunology ; metabolism

AIMTo obtain Clenbuterol monoclonal antibodies.

METHODSClenbuterol complete antigen was prepared with diazotization method. BALB/c mice was immunized with subtractive immunization, Clenbuterol monoclonal antibody was prepared with rule hybridoma technique.

RESULTSThe mice obtained tolerance to BSA by subtractive immunization. The rate of the hybridoma cell with positive reaction which had obtained was 8.2%, and the specific clenbuterol monoclonal antibody was obtained at last.

CONCLUSIONMonoclonal antibodies to micromolecule contaminant be prepared by subtractive immunization, could decrease the workload in the bolting of monoclonal antibodies, and increase the chance to obtain the antibody of expected.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Clenbuterol ; immunology ; Female ; Hybridomas ; metabolism ; Immunization ; methods ; Male ; Mice ; Mice, Inbred BALB C ; Serum Albumin, Bovine ; immunology

OBJECTIVETo purify the recombinant human serum albumin fusion protein with interleukin 1 (HAS/IL1ra) and to detect the bioactivity of the fusion protein.

METHODSThe recombinant HAS/IL1ra protein was purified by affinity chromatography and ion exchange chromatography, the bioactivity of the fusion protein was detected by IL1-induced A375 S2 cell killing.

RESULTThe purity of the fusion protein was at least 98 % as assessed by HPLC and the protective effect from the IL1-induced A375 S2 cell killing was similar to natural IL1ra.

CONCLUSIONThe purified recombinant HAS/IL1ra protein in this study has a satisfactory bioactivity.

Apoptosis ; drug effects ; Cell Line, Tumor ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; Melanoma ; pathology ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; pharmacology ; Serum Albumin ; biosynthesis ; genetics

Human serum albumin (HSA) is an important biological product in clinical pharmacy practice at present. Its main clinical use is in maintaining colloid osmotic pressure and increasing circulating plasma volume so as to cure hemorrhagic shock, burn, cancer, hypercytosis, hypoalbuminosis, etc. HSA is isolated by fractionating human plasma, which entails possible contamination by viruses or prions. Recombinant human serum albumin (rHSA) has been successfully produced by biological engineering. The structural and functional properties of rHSA are similar to those of plasma derived human serum albumin (pdHSA). Preclinical and clinical trials have confirmed the safety and efficacy of using this rHSA preparation in the treatment of certain diseases.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Saccharomyces cerevisiae ; genetics ; metabolism ; Serum Albumin ; biosynthesis ; genetics

In order to obtain enough fusion protein for developing preclinical studies of IFNbeta-HAS, we screened Pichia pastoris transformants expressing high-level protein by immunology method. The yield of IFNbeta-HSA was about 500 mg/L by fed-batch fermentation. The purity of IFNbeta-HSA reached 96% through the steps of ultrafiltration, Blue Sepharose FF, Ni2+-IMAC and DEAE Sepharose FF. Analysis of Western blotting showed that IFNbeta-HSA had the antigenicity of IFNbeta and HSA. The specific activity was about 1.96 x 10(7) IU/mg by standard survival activity test on WISH cells challenged with VSV virus. This study provided a method to produce IFNbeta-HSA.
Fermentation ; Humans ; Interferon-beta ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Serum Albumin ; biosynthesis ; genetics

OBJECTIVETo investigate the causes and influencing factors of heterogeneity of HSA/IL1ra fusion protein expression in Pichia pastoris.

METHODSThe heterogeneity of HSA/IL1ra fusion protein expressed in Pichia pastoris was studied by removing glycosylation and inhibiting glycosylation, as well as by different ways of fusion, different clones, and different expression host.

RESULTGlycosylation caused expression heterogeneity of fusion protein, but in SMD1168 and some GS115 clones there was no this phenomenon.

CONCLUSIONThe expression heterogeneity of HSA/IL1ra fusion protein in Pichia pastoris is due to the glycosylation, and different ways of fusion, different clones, different expression host also have some impact.

Escherichia coli ; genetics ; metabolism ; Genetic Heterogeneity ; Genetic Vectors ; genetics ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics

OBJECTIVETo obtain recombinant fusion protein HSA (human serum albumin)-PTH(1-34) in Pichia pastoris.

METHODSHSA and PTH(1-34) cDNA were obtained with PCR and the DNA segments were cloned into vector pPIC9 with linker. The linearized plasmids were transformed GS115 competent cells treated with LiCl, and mut+ transformants were screened on MD plate. With AOX promoter and alpha-MF signal sequences leading, fusion protein was expressed in GS115. PCR and SDS-PAGE were employed to confirm the integration and expression of HSA-PTH(1-34). The fusion protein was identified by Western blotting and classical adenylate cyclase assay.

RESULTThe PCR results showed that the gene of HSA-PTH(1-34) was integrated into GS115 genome. Western bolt approved the existence of two domains of HSA and PTH(1-34). The bioactivity assay in rabbit cortical membranes indicated that HSA-PTH (1-34) activated adenylate cyclase, but the activity was lower than that of the synthetic PTH(1-34).

CONCLUSIONActive fusion protein HSA-PTH (1-34) is successfully expressed in Pichia pastoris.

Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Peptide Fragments ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics ; Teriparatide ; analogs & derivatives

OBJECTIVETo observe the effect of bombesin noncytoskeleton form and intracellular free calcium ([Ca2+]i) concentration in PC-3 prostate cancer cell line.

METHODSImmunofluorescent histochemistry (IH) combined with laser scanning confocal microscopy (LSCM) was used to examine the expression of cytokeratin (CK) in PC-3 cells treated with definite concentrations of BBS and observe its effect on cytoskeleton form. Fluo-3/AM fluorescence technique and LSCM were adopted to measure the [Ca2+]i concentration after different concentrations (10(-9), 10(-7) and 10(-5) mol/L) of BBS were added in PC-3 cells.

RESULTSBBS (10(-5) mol/L) stimulated the expression of CK in PC-3 cells and the formation of lamellipodium, and increased the [Ca2+]i concentration, with concentration dependence.

CONCLUSIONDefinite concentrations of BBS could obviously enhance the [Ca2+] i concentration, CK expression and cytoskeleton morphology of PC-3 cells. The results provide a basis for further studies on the role of BBS in tumour researches as well as in intracellular signal transmission.

Bombesin ; pharmacology ; Calcium ; analysis ; Cytoskeleton ; drug effects ; metabolism ; Fluoroimmunoassay ; Humans ; Keratins ; biosynthesis ; Male ; Microscopy, Confocal ; Prostatic Neoplasms ; metabolism ; Serum Albumin, Bovine ; Tumor Cells, Cultured