The mechanism of oxidative refolding of proteins was elucidated in more detail from the intensive and extensive studies in the past decades. 1. Most of the proteins examined so far proceed oxidative refolding via multiple pathways rather than a single and specific pathway. This is consistent with the folding energy landscape theory. 2. It is the native interactions rather than the non-native interactions that direct the folding process. This is not necessarily incompatible with the importance of the non-native disulfide intermediates in the bovine pancreatic trypsin inhibitor (BPTI) pathway, which are just a chemical necessity in the intramolecular arrangement to facilitate native disulfide formation. 3. Based on the BPTI refolding it was suggested that disulfide bonds have a stabilizing effect on the native state without determining either the folding pathway or the final three-dimensional structure of the protein. This point of view is not applicable to other proteins. Studies on the refolding of prochymosin unequivocally demonstrated that the formation of native disulfides is the prerequisite to the recovery of the native conformation. It is more likely that the interdependence between the native disulfide formation and the formation of native structure is a general rule. 4. At the early stage of oxidative refolding disulfide formation is essentially a random process, with the progress of refolding further disulfide formation is increasingly dependent on the conformations of the intermediates. Enhancing the renaturation yield of recombinant proteins is a major challenge in biotechnology. In addition to aggregation, the formation of species with mispaired disulfide bonds is a leading cause of decreased yield. Progress in understanding the mechanism of oxidative refolding has provided insight into how to solve this problem. As described above, at the later stage of refolding disulfide formation depends on the conformations of intermediates. The intermediates with native-like and flexible structure favourable for native disulfide formation and correct refolding are productive intermediates, while the unproductive intermediates tend to adopt stable conformations, which render the thiol groups and disulfide bond(s) inaccessible and further folding unfavourable energetically. Therefore, the principle to enhance the renaturation yield of disulfide-containing proteins is to cause the productive intermediates to predominate by destabilizing the unproductive intermediates. To approach this, alkaline pH, low temperature, labilizing agents, protein disulfide isomerase and its analogues and alteration of primary structure have been proved useful to adjusting the structure of the unproductive intermediates so as to facilitate thiol/disulfide interchange and in turn the native disulfide formation. The prospects for the oxidative refolding of proteins both in basic and applied researches are discussed in this review article.
Animals ; Biotechnology ; Disulfides ; chemistry ; Humans ; Oxidation-Reduction ; Protein Folding ; Proteins ; chemistry ; genetics ; metabolism

Algorithms ; Crystallography, X-Ray ; Disulfides ; chemistry ; Models, Molecular ; Protein Conformation ; Proteins ; chemistry

This study is (1) to improve the stabilization of human scFv to rabies virus; (2) to prepare active human dsFv fragment; and (3) to evaluate the biological activities of dsFv. The dsFv V(H) and VL were separately expressed in PET22b(+)/BL21 (DE3), solublized and combined in appropriate molar ratio in refolding solution. The resultant dsFv fragments were evaluated for its protection against rabies virus, its affinity and stability, in reference to the cognate scFv. The dsFv was found to bind specifically to Vero vaccine of rabies virus. Compared to the scFv, the dsFv was more stable, had higher affinity, and was able to inhibit the infection of Rabies virus to Vero cell. This established a solid basis for the clinical application of dsFv to rabies virus.
Antibodies, Viral ; immunology ; Disulfides ; chemistry ; Humans ; Immunoglobulin Fragments ; immunology ; metabolism ; Immunoglobulin Variable Region ; immunology ; metabolism ; Rabies virus ; immunology

OBJECTIVETo investigate the effects of garlicin on fibroblasts proliferation and type I collagen synthesis and explore its anti-fibrosis mechanism.

METHODSGarlicin was added into the culture fluid of NIH3T3 cell, taking Radix Salviae miltiorrhizae as the control medicine. The spiking of H3-thymidine DNA was detected, also the hydroxyproline (HOP) concentration in the culture fluid by alkali digestion method and the protein expression of type I collagen in NIH3T3 cells by immunofluorescent staining.

RESULTSThe NIH3T3 cell growth and proliferation rate were obviously reduced after garlicin treatment concentration-dependently in range of 0.2 - 5 microg/mL; HOP level and protein expression of type I collagen also lowered.

CONCLUSIONGarlicin could inhibit NIH3T3 cell proliferation, reduce the synthesis and protein expression of type I collagen so as to exert the anti-fibrosis effect.

Allyl Compounds ; pharmacology ; Animals ; Cell Proliferation ; drug effects ; Collagen Type I ; biosynthesis ; Disulfides ; pharmacology ; Dose-Response Relationship, Drug ; Garlic ; chemistry ; Hydroxyproline ; analysis ; Mice ; NIH 3T3 Cells

OBJECTIVETo evaluate the influence of preoperatively selected gut decontamination (SGD) on intestinally derived endotoxemia(ETM) in patients with rheumatic heart disease undergoing valve replacement operation with cardiopulmonary bypass(CPB).

METHODSThirty patients were randomly divided into control group and SGD group. The patients in control group underwent preoperative bowel preparation, i.e, diet preparation and enema. The patients in SGD group were administrated 100 mg Tobramycin, 40 mg garlicin and 20% Lactulose for 10 ml three times per day for 3 days besides routinely preoperative bowel preparation. Bacteria cultivation and identification and Gram staining of feces in both groups were used to evaluate species of intestinal flora and their ratios. The levels of endotoxin, D-lactate, TNF-alpha and complement 3 were determined at four time points of anesthetic induction, CPB end, 2 h after CPB, 24 h after CPB. And the related clinical biochemical and clinical markers were recorded.

RESULTSAerobic gram-negative bacilli (AGNB) ratio in post-SGD group decreased significantly as compared with that in control group and pre-SGD group (P less than 0.05). The level of D-lactate reduced significantly at time points of anesthetic induction and 2 h after CPB (P less than 0.05). Endotoxin levels of patients in both groups elevated significantly after CPB (P less than 0.05), and endotoxin levels of the patients in SGD group decreased significantly at points of CPB end (P less than 0.01) and 24 h after CPB (P less than 0.05) compared with those in control group. The levels of TNF-alpha and complement 3 were similar in both groups as well as clinical and biochemical markers.

CONCLUSIONSCPB induces endotoxemia, while the regime of SGD is an effective way to prevent endotoxemia but may not affect activation of inflammatory media and clinical outcomes.

Adult ; Allyl Compounds ; therapeutic use ; Cardiopulmonary Bypass ; adverse effects ; Decontamination ; Disulfides ; therapeutic use ; Endotoxemia ; prevention & control ; Humans ; Intestines ; microbiology ; Preoperative Care ; Rheumatic Heart Disease ; surgery ; Tobramycin ; therapeutic use

OBJECTIVETo investigate the molecular mechanisms of Z-ajoene mitosis blocking and telomerase inhibitory effects on HL-60 cells.

METHODSProliferation inhibition of HL-60 cell line was evaluated by MTT assay. Z-ajoene-induced mitotic blocking effect was investigated by flow cytometry. Immunoblotting analysis was used to determine cell cycle regulatory proteins. The telomerase activity of HL-60 cells was detected by TRAP-silver stain assay. Telomerase hTRT and TP1 mRNA level were determined by RT-PCR.

RESULTSZ-ajoene displayed great proliferation inhibiting effect on HL-60 cells. Progressive increase in the percentage of mitotic block at G(2)/M phase was observed from 4 h to 12 h after treatment with 10 micromol/L Z-ajoene, with a peak at 10 h, which was 1.95 times higher than that in control. Z-ajoene also caused an increase in cyclin B1 accumulation and a decrease of p34(cdc2) expression. But Z-ajoene did not change the level of cyclin A. After treating with 10 micromol/L Z-ajoene for 24 h, the telomerase activity of HL-60 cells was also decreased in a dose-independent manner. Furthermore, telomerase hTRT and TP1 mRNA levels decreased after 10 micromol/L Z-ajoene treatment for 24 h.

CONCLUSIONThe results suggest that Z-ajoene has potent anti-cancer activity, and that its inhibitory effect on telomerase activity and on cell growth might be the result of G(2)/M phase blocking.

Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Cycle ; drug effects ; Disulfides ; pharmacology ; Garlic ; chemistry ; HL-60 Cells ; Humans ; Mitosis ; drug effects ; Telomerase ; metabolism

The molecular characterization and phylogenetic analysis of hemagglutinin (HA) genes of human influenza H3N2 viruses in Guangdong, China from 2007 to 2010 were studied in this study. By space-time sampling of strains, the HA genes of H3N2 strains from Guangdong were sequenced and searched from Internet, and then the variation and evolution of HA genes were conducted by Lasergene 7.1 and Mega 5.05 and evolutionary rates were analyzed by epidemiological data. The phylogenetic tree was established by alignment of 17 Guangdong strains and 26 global reference strains. Ks rates and Ka rates of HA genes were 2.06 x 10(-3)-2.23 x 10(-3) Nt/Year and 1.05 x 10(-3)-1.21 x 10(3) Nt/Year during 2007-2010, while the velocity of HA1 evolution of Ka was 3. 13 times than that of HA2 evolution. Compared with HA of vaccine strain A/Perth/16/2009, the genetic homologies of Guangdong strains in 2009 reached to 98.8%-99.7% and of Guangdong strains in 2010 reached to 98.0%-98.4%. There were some amino acid substitutions in five epitope regions of HA1 during 2007-2010, especially in B region (N160K) and D region (K174R/N); the K189E/N/Q and T228A in RBS (receptor-binding site) occurred in 2010 as two glycoproteins sites substituted impacted on the HA1 antigenicity. The antigenicity of epidemic H3N2 strains in 2010 was to some degree different that of the vaccine strain A/ Perth/16/2009. According to that there were variations of B and D epitopes and two sites of RBS and two glycoprotein in Guangdong H3N2 HA1 genes, WHO/ CDC should recommend new representative strains during 2011-2012 influenza seasons if H3N2 HA genes further evolve in the near future.
Amino Acid Substitution ; China ; Disulfides ; chemistry ; Epitopes ; genetics ; Evolution, Molecular ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; Mutation ; Phylogeny

To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.
Animals ; COS Cells ; Cercopithecus aethiops ; Cysteine ; genetics ; metabolism ; Disulfides ; metabolism ; Factor VIII ; genetics ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; Mutation ; Peptide Fragments ; genetics ; metabolism ; Protein Splicing ; Transfection

We study the techniques of synthesis of disulfide bond-bearing cyclopeptides FIK. This experimentation with the material of Fmoc-aa use Solid-Phase synthesis after condensation by HBTU/HOBt/DIEA to synthesize linear peptide, then cyclopeptide was synthesized by creation of intramolecular disulfide bond by means of 12 oxidation of bis cysteine sulfhydryl of the linear peptide. The crude production was cleaved from the resin together with all protecting group and identified and separated by MALDI-MS and RP-HPLC. The peptide yield was 18%, after purification the purity was more than 97%. It was identified on MALDI-MS and Ellman reagent detection. This method is effective, simple, rapid and obtained good yield, and it's fit for the large-scale production.
Amino Acids ; chemistry ; Combinatorial Chemistry Techniques ; methods ; Disulfides ; chemistry ; Fluorenes ; chemistry ; Molecular Structure ; Peptides, Cyclic ; chemical synthesis ; chemistry ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The study was aimed to investigate the expression of VEGF mRNA and VEGF protein in HL-60 cells treated with diallyl disulfide (DADS), and to explore the antileukemic mechanism of DADS in respect of VEGF production. Semi-quantitative RT-PCR and ELISA were used to detect the expression of VEGF mRNA and secretion of VEGF protein in HL-60 cell lines treated by DADS respectively. The results showed that the expression of VEGF mRNA and secretion of VEGF protein were found in HL-60 cells. The expression of VEGF mRNA and secretion of VEGF protein in HL-60 cells could be down regulated by treatment with 0.625, 1.25, and 2.5 microg/mL DADS for 48 and 72 hours and the effects had a dose dependent relationship (r > 0.9, P < 0.01). The differences between DADS treated HL-60 cell groups and the control group were statistically significant (P < 0.01), there were also statistically significant differences among three DADS-treated HL-60 cell groups (P < 0.05). It is concluded that DADS effectively inhibits the proliferation of human leukemia cell line HL-60 cells; DADS exerts its antileukemic effects by reduction of the expression of VEGF mRNA and VEGF protein secretion.
Allyl Compounds ; pharmacology ; Antineoplastic Agents ; pharmacology ; Cell Proliferation ; drug effects ; Disulfides ; pharmacology ; HL-60 Cells ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics