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

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

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

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

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

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

OBJECTIVETo explore the effect of diallyl disulfide (DADS) on hippocampal synapses and learning and memory abilities in a mouse model of A1zheimer's disease (AD).

METHODSMouse models of AD established by agglutinated Aβ1-42 injection in the lateral cerebral ventricle were randomized into 4 groups and treated with DADS at the daily doses of 0, 10, 50 and 100 mg/kg by gavage for 30 consecutive days. The learning and memory abilities of the mice were assessed with Morris water maze test; the structures of the dendritic spines and synapses in CA1 region of the hippocampus were observed under transmission electron microscope with silver staining; PSD95 and SYP protein and mRNA expressions in the hippocampus were detected with Western blotting and RT-PCR.

RESULTSCompared with the AD model mice, the mice treated with 50 and 100 mg/kg DADS showed enhanced learning and memory abilities in Morris water maze test. The dendritic spines and synapses in CA1 region of the hippocampus increased obviously and hippocampal expressions of PSD95 and SYP were enhanced in mice treated with 50 and 100 mg/kg DADS.

CONCLUSIONDADS at the daily doses of 50 and 100 mg/kg can improve the learning and memory abilities and increase the number of dendritic spines and synapses in the hippocampus in mouse models of AD.

Allyl Compounds ; pharmacology ; Alzheimer Disease ; drug therapy ; Animals ; CA1 Region, Hippocampal ; drug effects ; Disease Models, Animal ; Disulfides ; pharmacology ; Learning ; Male ; Memory ; Mice ; Synapses ; drug effects