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 tissue distribution of the diallyl disulfide (DADS) and diallyl trisulfide (DATS) in solid lipid nanoparticles loaded garlic oil (GO-SLN) in rats.

METHODThe gas chromatography-electron capture detection (GC-ECD) method was established to determined the DADS and DATS simultaneously in the biological samples of rats after administration of 0.5 mL garlic oil injection or GO-SLN (containing about 10 mg garlic oil) via jugular vein cannula. The conditions for gas chromatographic separation were as follows. The oven temperature was set at 110 degrees C and maintained for 15 min. Temperatures at the injection port and detector were 180 degrees C and 300 degrees C, respectively. Ultra-pure nitrogen (purity > 99.999%, Shenyang Kerui Special Gases Co. Ltd., China) was used as a carrier gas and made-up gas at flow-rates of 1 mL x min(-1) and 60 mL x min(-1), respectively. All injections were carried out in the split injection mode with a split ratio of 1:10.

RESULTThe GC-ECD method was fit for determing the concentration of DADS and DATS in garlic oil. The distribution character of GO-SLN in rats had changed to some extent and the concentration of GO-SLN in tissues was higher than that of GO-Injection.

CONCLUSIONThe SLN can elevate the passive targeting of drugs and lengthen their action time in tissues.

Allyl Compounds ; analysis ; pharmacokinetics ; Animals ; Disulfides ; analysis ; pharmacokinetics ; Female ; Garlic ; chemistry ; Male ; Nanoparticles ; administration & dosage ; chemistry ; Plant Oils ; administration & dosage ; chemistry ; pharmacokinetics ; Rats ; Rats, Wistar ; Sulfides ; analysis ; pharmacokinetics

Recent years, the incidence and mortality of prostate cancer have increased dramatically in China. At earlier stages, most diagnosed prostate cancers are responsive to androgen depletion treatment, yet, nearly all patients will eventually progress to metastatic androgen-independent prostate cancer (AIPC), which still has no effective therapeutic method or drug to deal with. 11'-Deoxyverticillin A (C42) belongs to the family of epipolythiodioxopiperazines (ETPs), an interesting class of fungal toxins that inhibit farnesyl transferase. Compounds holding such a property have been explored as putative anticancer agents. In this study, using PC3M cells, an AIPC cell line, we investigated the effect of the compound on apoptosis and explored the underlying mechanism. It revealed that C42 markedly enhanced the activity of caspase-3/7 and increased the accumulation of the cleaved PARP, all of which are the markers of apoptosis. It also revealed that C42 either decreased cell viability or inhibited the growth of PC3M cells. Moreover, we observed that the loss of cell viability and cell growth inhibition induced by C42 were both time- and dosage dependent. Taken together, we indicated that C42 can induce caspase-dependent apoptosis in AIPC cells, and the results presented here will broaden our knowledge about the molecular mechanisms by which C42 exerts its anticancer activity, and future work in this direction may provide valuable information in the development of these compounds into effective cancer therapeutic strategies against androgen-independent prostate cancer.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 7 ; metabolism ; Cell Line, Tumor ; Disulfides ; pharmacology ; Farnesyltranstransferase ; antagonists & inhibitors ; Humans ; Male ; Mycotoxins ; pharmacology ; Piperazines ; pharmacology ; Prostatic Neoplasms ; pathology

Transmembrane protein p185 (the product of Her2/c-erbB-2 gene) is a member of the epidermal growth factor receptor (EGFR) family. Its overexpression was found in about 30% of breast cancer. It is essential to obtain soluble extracellular domain (ECD) of p185, especially disulfide bond rich domains, for identifying the epitopes of anti-p185 antibodies and researching the interrelationship between the antigen and antibody. The disulfide bond rich domain I-II and domain IV of p185 ECD were amplified from plasmid pBabe/erbB-2 by PCR respectively. These two fragments were inserted into pGEX/4T-1 vector, transfected into E. coli Origami B (DE3) pLysS and expressed inductively by low concentration of IPTG and low temperature overnight. After the pressure lysis of cells, the supernatants were analyzed by SDS-PAGE and the result demonstrated that this GST-fusion protein was expressed solubly in the amount of 10-15 mg/L. By the ELISA, Western blot and other immunological assays, the fusion proteins and their GST cut-off derivates both showed binding activities with several anti-p185 antibodies respectively. These results indicated that it was a feasible and effectual method to express disulfide bond rich domain I-II and domain IV of p185 ECD and this method may also be used to express other disulfide bond rich proteins.
Antibodies, Monoclonal ; immunology ; Disulfides ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Receptor, ErbB-2 ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Solubility ; Transfection

The aim of this study was to investigate the effect of diallyl disulfide (DADS) on the apoptosis of K562 cells and to explore the mechanism of K562 apoptosis induced by DADS. The K562 cells were treated with different concentrations of DADS for 24, 48 and 72 hours. The concentrations of DADS were as follows: 0 (control group), 10, 20, 40 and 80 mg/L. The morphologic changes of leukemia K562 cells treated with DADS were observed by Hoechst33 258 staining. The apoptosis of K562 cells treated with different concentrations of DADS for 24, 48 and 72 hours was analyzed by flow cytometry. The mRNA expression changes of Fas and FasL were detected by reverse transcription-polymerase chain reaction (RT-PCR) after K562 cells were treated with different concentrations of DADS for 48 hours. The results indicated that the characteristics of apoptosis in K562 cells induced by DADS were as follows: reduction of nucleus, chromatin condensation and nuclear membrane rupture. The flow cytometry with PI straining showed that after 24 hours of DADS treatment the apoptosis rate of K562 cells increased from 11.60 ± 0.83% at the concentration of 10 mg/L to 37.94 ± 0.87% at the concentration of 40 mg/L. The apoptosis rate of K562 cells increased from 37.94 ± 0.87% (24 hours) to 47.02 ± 0.66% (72 hours) after treatment with DADS of 10 mg/L increasing to 40 mg/L DADS. The Fas mRNA expression levels of the related apoptotic genes increased after K562 cells were treated with different concentrations of DADS for 48 hours, while FasL mRNA expression decreased significantly after DADS treatment for 48 hours, compared with those in the control group (p < 0.05). It is concluded that DADS can induce the apoptosis of human leukemia K562 cells in a time-and concentration-dependent manners. The activation of Fas/FasL pathway may play an important role in the K562 cell apoptosis induced by DADS, which is associated with increasing Fas gene expression and decreasing FasL gene expression.
Allyl Compounds ; pharmacology ; Apoptosis ; drug effects ; Disulfides ; pharmacology ; Fas Ligand Protein ; metabolism ; Gene Expression Regulation, Leukemic ; Humans ; K562 Cells ; Signal Transduction ; fas Receptor ; metabolism

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

Diallyl disulfide (DADS) induced apoptosis through the caspase-3 dependent pathway in leukemia cells was earlier reported from this laboratory. In this study, we investigated the involvement of Ca(2+) in DADS-induced apoptotic cell death of HCT-15, human colon cancer cell line. DADS induced the elevation of cytosolic Ca(2+) by biphasic pattern; rapid Ca(2+) peak at 3 min and following slow and sustained elevation till 3 h after the addition of DADS. Production of H(2)O(2) was also observed with its peak value at 4 h. Apoptotic pathways including the sequence of caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation by DADS were completely blocked by various inhibitors such as specific caspase-3 inhibitor, free radical scavenger, and intracellular Ca(2+) chelator. N-acetylcystein and catalase treatment prevented the accumulation of H2O2 and later caspase-3 dependent apoptotic pathway. However, these radical scavengers did not block the elevation of intracellular Ca(2+). Treatment of cells with 1, 2-bis (2-aminophenoxyethane)-N, N, N-tetraacetic acid tetrakis -acetoxymethyl ester (BAPTA-AM), cellular Ca(2+) chelator, resulted in a complete blockage of the caspase-3 dependent apoptotic pathway of HCT-15 cells. It abolished the elevation of intracellular Ca(2+), and furthermore, completely inhibited the production of H(2)O(2). These results indicate that cytosolic Ca(2+) elevation is an earlier signaling event in apoptosis of HCT-15 cells. Collectively, our data demonstrate that DADS can induce apoptosis in HCT-15 cells through the sequential mechanism of Ca(2+) homeostasis disruption, accumulation of H(2)O(2), and resulting caspase-3 activation.
Allyl Compounds/*pharmacology ; Apoptosis/*drug effects ; Calcium/*metabolism ; Caspases/metabolism ; Colonic Neoplasms/*metabolism/*pathology ; Disulfides/*pharmacology ; Enzyme Activation/drug effects ; Human ; Hydrogen Peroxide/metabolism ; Tumor Cells, Cultured

This study was purposed to investigate the reversal effect of garlicin, erythromycin alone or combination of garlicin with erythromycin on K562/A02 and its possible mechanisms, so as to provide experimental evidence for combination reversal strategies. Cytotoxicity and the reversal effect of garlicin and erythromycin alone and combination of this two drugs were detected by MTT assay. The expression of mdr1 gene of K562/A02 was detected by RT-PCR. The P-gp expression was observed by immunohistochemical technique. Flow cytometry was used to detect intracellular drug concentration. The results showed that the sensitivity of K562/A02 to ADM increased somewhat in the presence of 1, 4, 8 mg/L garlicin, the reversal multiples at 1, 4, 8 mg/L garlicin were 1.80, 2.26 and 2.82 respectively in dose-dependent manner. The reversal multiple of erythromycin 60 mg/L was 2.20. The combination of two drugs could increase the reversal multiple to 4.94, and had no more cytotoxin. Both of garlicin and erythromycin alone could down-regulate the expression of mdr1 and P-gp of K562/A02 and elevate the intracellular concentrations of ADM in K562/A02 cells. Meanwhile, the effects described above were enhanced when garlicin was combined with erythromycin. It is concluded that the garlicin and erythromycin alone under cytotoxic dose both can reverse the MDR of K562/A02 cells effectively. Moreover, the combination of two drugs is more effective than that in use alone. Combination of these two drugs shows synergistic actions in regulating the expression of mdr1/P-gp and increasing the intracellular concentrations of ADM in K562/A02 cell.
Allyl Compounds ; pharmacology ; Antineoplastic Agents ; pharmacology ; Disulfides ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Erythromycin ; pharmacology ; Humans ; K562 Cells