Enhanced anti-tumor effects achieved in a murine tumor model using combination therapy of recombinant human manganese superoxide dismutase and adriamycin.

Manganese superoxide dismutase (MnSOD) is the only primary antioxidant enzyme in mitochondria that scavenges superoxide radicals. Overexpressing MnSOD in cancer cells by cDNA transfection suppresses tumor formation and reverses malignant growth. In this study, we examined the effect of recombinant human manganese superoxide dismutase (rhMnSOD) alone and in combination with adriamycin (ADR) against solid tumors of sarcoma 180 in Institute of Cancer Research (ICR) mice. Administration of rhMnSOD alone and in combination with ADR significantly inhibited tumor growth in a dose-dependent manner. The use of rhMnSOD in combination with ADR enhanced ADR's anti-tumor potency without increasing toxicity. Histopathological examination provided evidence of the anti-tumor effect. In addition, we found lymphocyte infiltration of the tumors, with an increase in both CD4- and CD8-positive cells in the treated tumors. The expression of CD4 and CD8 was up-regulated with increasing dose of rhMnSOD, and the combination treatment with ADR further enhanced this up-regulation. Collectively, these data indicate that rhMnSOD may exhibit an anti-tumor effect by stimulating the immune system and promoting the recruitment of lymphocytes into the tumor to kill tumor cells. Thus MnSOD may constitute a potential new therapeutic agent to be exploited as an adjuvant in cancer therapy.

[Antioxidant activity of natural and cultured Cordyceps sp].

OBJECTIVE: Investigating the antioxidant activities of water and ethanol extracts of natural Cordyceps sinensis and Cordyceps militaris and their fermentation preparations. METHOD: The samples were tested through 6 assays: inhibition ability of linoleic acid oxidation; scavenging activity of DPPH, hydrogen peroxide, hydroxyl radical and superoxide anion; and metal chelating activity. RESULT: Samples showed different antioxidant ability, and there was not an extract that exhibited high activity in all assays; however, water extract of natural C. militaris could be regarded as the most powerful antioxidant among 8 samples. It had high activity in inhibition of linoleic acid oxidation, chelating metal ions, and scavenging DPPH and hydroxyl radical. The research also indicated that the contents of phenolic compounds in water and ethanol extracts of natural and cultured Cordyceps sp. had huge difference. CONCLUSION: Natural Cordyceps sp. and its fermentation preparations could be used as potential natural antioxidants. The fermented process affected the antioxidant ability of cultured Cordyceps sp., and the antioxidant activity of both natural and cultured Cordyceps sp. did not significantly related with the quantity of phenolics.

Two observed regions in B lymphocyte stimulator important for its biological activity.

B lymphocyte stimulator (BLyS), a member of the tumor necrosis factor superfamily of ligands, is a crucial survival factor for B cells. We successfully constructed seven mutants of the functional soluble fragment of human BLyS (named cBLyS, amino acid 134-285), including three deletion mutants and four site-directed mutants. All the mutant proteins were expressed in Escherichia coli and purified by Ni-NTA affinity chromatography. The biological activities of these mutants were assessed by the ligand-receptor binding assay, B cell proliferation assay and immune effect response in vivo. Our results indicated that four residues, H218, F220, T228 and L229, are indispensable for the biological activity of cBLyS, whereas two regions, amino acid 134-148 and amino acid 271-285, are related to the biological activity of BLyS. The protein of deletion of amino acid 134-148 leads to a complete defection in raising the antigen-specific IgM titer. The deletion of amino acid 271-285 reduces the effectiveness compared with the native cBLyS. This indicates that the region of amino acid 134-148 is indispensable for cBLyS to function normally.

Effect of nutrition factors on the synthesis of superoxide dismutase, catalase, and membrane lipid peroxide levels in Cordyceps militaris mycelium.

Effect of carbon, nitrogen, and metal ion sources on superoxide dismutase (SOD), catalase (CAT) activities, and lipid perioxide (LPO) levels in Cordyceps militaris mycelium were investigated at stationary growth phase by step supplementing with these nutrition factors in shake-flask cultures. Mycelium was cultivated in several growth media containing different carbon sources. The observed highest SOD and CAT activities were 44.3 U/mg protein in the presence of 20% potato broth plus 2% glucose medium and 93.7 U/mg protein in presence of 20% potato broth plus 1% glucose medium, respectively. By supplementing with either yeast extract or tryptone in 0.1-0.5% concentration range, the highest SOD and CAT activities were 21.1 U/mg protein in medium supplemented with 0.1% yeast extract and 20.7 U/mg protein in medium supplemented with 0.1% tryptone, respectively. Supplementing with Cu(2+), Zn(2+), and Mn(2+) caused a stimulation of SOD synthesis. The minimum LPO level was observed at media presented Zn(2+). The time course of SOD and CAT biosynthesis showed two maxima, which correspond to the maximum of biomass. High SOD levels and low LPO levels in the medium described above indicated that the appropriate metal ions could provide a suitable protection for cells against oxygen radical damage.

Effects of shark hepatic stimulator substance on the function and antioxidant capacity of liver mitochondria in an animal model of acute liver injury.

This study was carried out to investigate whether shark hepatic stimulator substance (HSS) can prevent acute liver injury and affect mitochondrial function and antioxidant defenses in a rat model of thioacetamide (TAA)-induced liver injury. The acute liver injury was induced by two intraperitoneal injections of TAA (400 mg/kg) in a 24 h interval. In the TAA plus shark HSS group, rats were treated with shark HSS (80 mg/kg) 1 h prior to each TAA injection. In this group, serum liver enzyme activities were significantly lower than those in the TAA group. The mitochondrial respiratory control ratio was improved, and the mitochondrial respiratory enzyme activities were increased in the TAA plus shark HSS group. The mitochondrial antioxidant enzyme activities and glutathione level were higher in the TAA plus shark HSS group than in the TAA group. These results suggest that the protective effect of shark HSS against TAA-induced acute liver injury may be a result of the restoration of the mitochondrial respiratory function and antioxidant defenses and decreased oxygen stress.

On-column refolding of an insoluble His6-tagged recombinant EC-SOD overexpressed in Escherichia coli.

The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile. Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.

Expression of C-peptide multiple gene copies in Escherichia coli and stabilities of C-peptide in aqueous solution.

A gene fragment encoding three copies of proinsulin C-peptide was synthesized and expressed in E. coli and the recombinant proinsulin C-peptide was produced through site-specific cleavage of the resulting gene products. The fusion protein was expressed at high level, about 80 mg/L, as a soluble product in the cytoplasm. Ni-NTA affinity chromatography efficiently separated the expressed fusion protein from the supernatant, to obtain about 37.5 mg/L of the fusion protein with 70% purity. Enzymatic digestion by trypsin and carboxypeptidase B of the fusion protein efficiently released native C-peptide, the overall yield of recombinant C-peptide at a purity over 95% was 1.5 mg/L. The good agreement of amino acids composition, together with shown similarities of the recombinant C-peptide to C-peptide standard in the comparative RP-HPLC analysis and IMMULITE C-Peptide quantitative assay, suggested that the recombinant C-peptide obtained in this report was the native human C-peptide. The investigation of the chemical stability of recombinant human C-peptide in aqueous solutions by RP-HPLC was also reported. The degradation of the recombinant C-peptide showed a marked dependence on pH and temperature. The degradation reaction of C-peptide occurred immediately in pH 3 or pH 9 buffered solution. The degradation reaction of C-peptide followed first-order kinetics in pH 3 buffered solution at 37 degrees C or 70 degrees C, only 40.3% of C-peptide was remained after 10 h at 70 degrees C. The maximum stability was achieved at pH 7.4, more than 90% of C-peptide were detected at pH 7.4 and 37 degrees C after 10 h and at pH 7.4 and 70 degrees C after 5 h. 99% and 96% of C-peptide was remained at pH 7.4 and 37 degrees C after 10 h with and without 10 g/L BSA respectively.

[Coexpression of caiB and caiE with two plasmids in E. coli].

Recombinant bacteria exhibiting high enzymatic activities were obtained by cloning and coexpression of both caiB gene and caiE gene in the host of E. coli Bl21(DE3), which encode carnitine dehydratase and a protein related to the synthesis of cofactor for carnitine dehydratase, respectively. In order to coexpress these two genes, compatible and incompatible two plasmids system were used, the difference between them was also studied. After induction with IPTG, both caiB and caiE genes were coexpressed in both compatible two plasmids system and incompatible two plasmids system. In the former system, the expressed products accounted for 17% and 10% of the total proteins in the host; in the later system, the proportion was 39% and 20%, respectively. The activity of carnitine dehydratase in E. coli Bl21(DE3) with either coexpression system is about 2.3 times than that in E. coli Bl21(DE3) with only pET28-caiB. The plasmids stabilities in these two systems were the same, all needed the help of antibiotic selective pressure.

High-level expression of human extracellular superoxide dismutase in Escherichia coli and insect cells.

Much is known about the physical properties of the Cu,Zn- and Mn-superoxide dismutases (SODs). However, the biochemical characteristics and pharmacological properties of extracellular (EC)-SOD have been severely limited due to difficulties in obtaining and purifying the enzyme. The EC-SOD cDNA was inserted into the Escherichia coli expression plasmid pET-28a(+) which contains the T7 promoter and transformed into the E. coli BL21(DE3). After induction with 1 mmol/L isopropyl beta-D-thiogalactoside, the recombinant human EC-SOD was highly expressed as inclusion bodies. SDS-PAGE analysis revealed that recombinant EC-SOD accumulated up to 26% of the total soluble protein of E. coli cells. The expression product was purified by a Ni(2+)-IDA-Sepharose 6B column. After the denaturing and refolding processes, the recombinant human EC-SOD retains the specific enzymatic activity of 920 U/mg of the purified product. The gene encoding human EC-SOD mature peptide was also inserted into the donor plasmid pFastBacHTb. After transposition, transfection, and amplification were performed, the recombinant baculoviruses infected the Tn-5B1-4 cells and EC-SOD was highly expressed in Tn-5B1-4 cells. SDS-PAGE and Western blot analysis revealed that the subunit molecular weight of the expression product is 28 kDa. Furthermore, recombinant human EC-SOD retains the enzymatic specific activity of 200 U/mg of the Tn-5B1-4 cell lysates.

Cloning and Expression of Targeting CuZn-SOD to Central Nervous System.

Oxygen-derived free radicals are thought to be involved in the pathogenesis of a wide range of neurological disorders. Targeted delivery of CuZn-SOD to neurons in central nervous system may have therapeutic value in such diseases. The gene encoding human CuZn-SOD was fused to tetanus toxin fragment C geneto construct a fusion gene, then it was cloned into prokaryotic expression vector pET-22b( ) and baculovirus vector pFastBacHTb, and was expressed in E.coli and Tn-5B1-4 cells, respectively. The recombinant fusion protein has a subunit molecular mass of 68 kD and is recognized by both anti-CuZn-SOD and anti-tetanustoxin antibody. By pyrogallol autooxidation assay, it is shown that the CuZn-SOD moiety retains substantial enzymatic activity, where the TTC moiety might deliver the fusion protein to neurons in central nervous system. So, CuZn-SOD/TTC may be a useful agent for the targeted delivery of CuZn-SOD to neurons.

Cloning and Expression of Tetanus Toxin Fragment C in E.coli.

The fragment C of tetanus toxin was amplified from Clostridium tetani DNA by PCR. This fragment was cloned into expression vector pET-28a(+),under the control of the T7 promoter. Expression of this plasmid in E.coli resulted in the production of a protein consisting of 6xHis of the vector fused to the N-terminal 451 amino acids of tetanus toxin. After induction with 1 mmol/L IPTG, TTC was expressed in E.coli BL21(DE3). The protein product accounted for 8.2% of the bacteria total protein in soluble form, SDS-PAGE and Western blot analysis of TTC recombinant protein confirmed this result. The expression products were also purified by Ni(2+)-IDA-Sephrose 6B column. Immunization of mice with rTTC resulted in the production of antibodies that were able to protect mice against a challenge with tetanus toxin furthermore, rTTC in vivo appeared to be able to undergo retrograde axonal transport.

Cloning, Sequencing and Expression of Human Copper, Zinc-superoxide Dismutase cDNA.

The cDNA encoding the human copper, zinc-superoxide dismutase(Cu, Zn-SOD) was amplified from the human liver by RT-PCR and sequenced. The cloned human Cu, Zn-SOD cDNA was ligated into expression vector pET-22b(+) under T7 promotor. After 3 h induction with 1 mmol/L IPTG, human Cu, Zn-SOD was highly expressed in E.coli BL21(DE3). The expression product was up to 30% of the total protein of the bacteria in soluble form, which had specific SOD activity.

Cloning and Expression of Human Manganese-superoxide Dismutase cDNA.

The cDNA encoding human manganese-superoxide dismutase was amplified from the human liver cell (L02) total RNA by RT-PCR and sequenced. The human Mn-SOD cDNA was ligated into expression vector pET-24a(+) under T7 promoter. After 5 h induction with 1 mmol/L IPTG 800 mol/L Mn(2+) human Mn-SOD was highly expressed in E.coli BL21(DE3). The protein product was up to 50% of the bacteria total protein in soluble form and had specific SOD activity.

Structure Prediction and Analyzing of the Molecular Chaperone TCP1.

An improved Chou and Fasman method and the Robson method from Prosis software were applied to four TCP1s and five homologous proteins. The predictive results showed that there may exist two structural domains in TCPI with a "hinge region" between them, suggesting that the two structural domains may be alpha/beta barrels. The putative ATP-binding domain, whose motif centres around the absolute conserved GDGTT sequence at position 87-91 in TCP1Hu, was found to be a coil region in the predictive results.