[Effect of cultivation in zinc media on the growth of and the degree of zinc in organic form in transgenic metallothionein mushroom].

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich and metal-binding functional proteins. Transgenic MT mushroom can be used as functional food additives, but its zinc-enriching ability has not been studied systemically until now. The zinc contents in mycelia of transgenic MT mushroom (Pleurotus ostreatus) and wild type mushroom mycelia cultivated in different zinc concentration media were analyzed by ICP-OES. The growth status, zinc-enriching ability and degree of zinc in organic form (DZOF) were also analyzed. Results showed that MT mushroom mycelia grew rapidly, but the growth was inhibited when the zinc content in solid media was higher than 1.6 mmol x L(-1). MT mushroom mycelia could enrich more zinc than that of wild type, and the zinc content in MT mushroom mycelia could be 2.56-27.49 mg x kg(-1) when it was cultivated in a liquid media with 0.6-1.2 mmol x L(-1) zinc. DZOF of MT mushroom mycelia in a liquid media with 0.6 mmol x L(-1) zinc at 7 d was significantly higher (88.7%) than that in the wild type (82.1%, alpha = 0.05), but there was no significant difference in DZOF when the MT mushroom mycelia was cultivated in a liquid media with different zinc content at 7 d.

A potential thrombus diagnosis reagent based on P-selectin monoclonal antibody SZ-51 light chain.

INTRODUCTION: P-selectin is a well characterized platelet adhesion molecule that can shift from the secretory granules to the surface of activated platelets, which makes it a potential target in thrombus diagnosis and therapy. SZ-51 is a monoclonal antibody against P-selectin. MATERIALS AND METHODS: To build a potential thrombus diagnosis reagent, we expressed the light chain of SZ-51 (SZ-LC) in P. pastoris and the protein was highly purified by the procedure combining nickel affinity purification, Q-column and Superdex 75 column chromatography. The purified recombinant SZ-LC was labeled with (99m)Tc and used for blood clearance, in vitro platelet binding and dog thrombus binding assay. RESULTS AND CONCLUSION: The yield of SZ-LC by the expression and purification method reached above 70 mg/L culture. We found that the nucleotide from (99m)Tc-SZ-LC was removed quickly through animal kidney, and (99m)Tc-SZ-LC could bind specifically to the activated human platelet in vitro. More importantly, with this recombinant protein, we successfully detected the fresh thrombus that was induced in dog vein. These results suggested that the recombinant SZ-LC expressed by P. pastoris was functional active and a potential reagent for thrombus diagnosis.

[Decontamination of heavy metals in wastewater by transgenic Synechococcus sp. PCC 7002 with mouse metallothionein-I gene].

The growth characteristics and the decontamination of heavy metals in analogous heavy metals wastewater by transgenic Synechococcus sp. PCC 7002 with mouse metallothionein-I gene were studied. The results show that transgenic Synechococcus sp. PCC 7002 not only has a higher tolerance to heavy metals, but also has a higher growth rate than wild strain. The concentration of Cd2+, Pb2+ and Hg2+ decreases with the progress of cultivation, and its maximum decreasing extent occurs at 1 - 3 day. After three days of cultivation, the absorption of Cd2+, Pb2+ and Hg2+ by transgenic Synechococcus sp. PCC 7002 is 10.75, 58.89 and 112.61 mg g(-1) of dried cells respectively, which is 3.16, 2.18 and 100.45 times higher than wild cells. The mono-factor and overall model developed fit the experiment data well.

[Distribution of Pb and Zn in transgenic metallothionein tobacco].

Transgenic metallothionein (MT) plant can clear the heavy metals from soil and environment, but the distribution of metals in plants has not been studied systematically. The Pb and Zn contents in different parts of transgenic MT tobacco plant of sixth generation and traditional plant (same culture variety as control) were analyzed. The Pb and Zn contents in total transgenic plant were 21.8% and 27.2% higher than control, respectively. The distribution of Pb and Zn in different organs varied in these two types of plants. The Pb and Zn contents in old leaves, stem and root in transgenic plants were significantly higher than those in wild type tobacco, while there was no significant difference in young leaves. The Pb contents in old leaves and root were 30.2% and 47.8% higher than those in the control, and the Zn contents in old leaves, stem and root were 4.7%, 29.2% and 21.6% higher than those in the control. These data showed that Pb was accumulated in old leaves and root easily, while Zn was accumulated in old leaves and stem easily.

Improving zinc content and antioxidant activity in transgenic tomato plants with expression of mouse metallothionein-I by mt-I gene.

Metallothioneins (MTs), as a family of low-molecular-weight, cysteine-rich, and metal-binding proteins, show potential for utilization in functional food. Tomato plants were transformed with gene constructs that contained mt-I encoding the mouse MT-I, similar in sense orientation with the constitutively active double 35S promoter from cauliflower mosaic virus. Three independent transformants, which had copies of the gene in their genomes, were obtained. In these transgenic lines, high-level expression of MT-I, high zinc content, and some antioxidant enzyme activities were detected in leaves. The average zinc content in transgenic tomato leaves was 32.7 mg/100 g FW, which about 1.6 times higher than that in wild-type. The superoxide dismutase activity was also higher (68.6, 66.9, and 66.1 U/g FW in the three transformants) than that in wild-type (57.4 U/g FW). In particular, the levels of superoxide free radical scanvenging in the three transformants were 14.2%, 14.6%, and 13.7%, respectively, which about 1.5 times higher than that in control (5.6%). Transgenic MT tomato may potentially be used as an antioxidant and for zinc supplementation.

Survival of human metallothionein-2 transplastomic Chlamydomonas reinhardtii to ultraviolet B exposure.

Solar ultraviolet (UV) radiation has a great influence on green organisms, especially plankton like Chlamydomonas. A human metallothionein-2 gene, which is generally considered to have an anti-radiation function by its coding product, was transferred into the chloroplast genome of Chlamydomonas reinhardtii. To dynamically measure the UV effects on Chlamydomonas cells grown in liquid tris-acetate-phosphate medium, a new method was developed based on the relationship between the chlorophyll content of an algal culture and its absorbance at 570 nm after the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In this experiment, both the wild-type and the transplastomic C. reinhardtii cells were cultivated in 96-well microplates containing liquid tris-acetate-phosphate medium in the absence or presence of zinc, copper, cadmium and cysteine. The transgenic C. reinhardtii showed a higher resistance than wild-type to UV-B exposure under all the examined conditions. Metals in the medium had positive impacts on both types of cells, but had significant influence only on the transplastomic cells. However, the high cell viability of the transgenic alga at the end of the 8 h UV-B treatment disappeared after a 20-h recovery culture. Cysteine did not protect cells from UV-B damage, but clearly enhanced the growth of both wild-type and transgenic C. reinhardtii.

Studies on the epitope of neuronal growth inhibitory factor (GIF) with using of the specific antibody.

Human neuronal growth inhibitory factor (GIF), a metalloprotein classified as metallothionein-3, is specifically expressed in mammal central nervous system (CNS). In these Studies the specific antibody to human GIF was prepared and used to search the epitope of human GIF by enzyme-linked immunosorbent assay (ELISA) and sequence comparison. The result of ELISA showed the epitope of human GIF may locate on a octapeptide (EAAEAEAE) in the alpha-domain of human GIF, and the result of nerve cell culture indicated that the biological activity of GIF may be affected by the specific antibody.

Proteomic analysis of corticobasal degeneration: a case study of corticobasal degeneration at the proteome level.

Corticobasal degeneration (CBD) is an adult-onset progressive neurodegenerative disorder. However, its pathogenic mechanisms underlying this disorder remain poorly understood. The authors examined changes of proteome profiles between three nondemented comparison brains and a CBD brain based on two-dimensional gel electrophoresis. Two up-regulated proteins in the CBD brain were identified as protein-L-isoaspartate (D-aspartate) O-methyltransferase and cofilin 1 (non-muscle), and six down-regulated proteins were identified as carbonyl reductase [NADPH] 1, two of peptidyl-prolyl cis-trans isomerase A, ubiquitin carboxyl-terminal hydrolase isozyme L1, phosphoglycerate mutase 1 (brain) and chain A of human peroxiredoxin 5. Subsequently, the possible relevance of these changes was analyzed.

Effect of metallothionein on cell viability and its interactions with cadmium and zinc in HEK293 cells.

Metallothioneins (MTs) are thought to participate in a wide variety of physiological roles, but the mechanisms involved are still unclear. The study was designed to examine the possible factors related to these mechanisms. Methods, including transfection, MTT assay and flow cytometry, were used to investigate the effect of MTs on cell viability and their interactions with cadmium and zinc in HEK293 cells. The results showed that transient overexpression of human MT1A, MT2 and MT3 genes dynamically affected cell viability, and the effect was influenced by zinc and cadmium ions. Overexpressed MTs with added zinc showed a greater inhibitory effect on cell viability. Overexpressed MTs protected cells against low concentrations of cadmium ions (10 microM), but increased cell death in response to high concentrations (20-50 microM). Out of the three MTs, MT1A was more efficient than MT2 and MT3 in its resistance to cadmium (10 microM), and MT3 together with zinc showed more cell growth inhibition than MT1 and MT2. These results indicate that both of the divalent metal ions that could bind MTs, as well as the individual MT isoforms, affect the role of MTs on cell viability, which may explain in part why the comprehensive effect of MTs on the cells was elusive.

High-level expression of human TFF3 in Escherichia coli.

A strategy for expression and purification of recombinant N-terminal human trefoil factor family-domain peptide 3 (hTFF3) in Escherichia coli was established. The gene of hTFF3 was synthesized to substitute the low-usage condons with corresponding high-usage synonymous condons. At the same time, the signal peptide of DsbC was added to the N-terminus of the hTFF3 gene. The mature recombinant hTFF3 was located in the periplasm of E. coli, which can be released by sonication. The protein was further purified by a two-step cation exchange chromatography mentod. The yield is about 14-15 mg/l of culture. The biological activity of purified hTFF3 was analyzed by cell-based apoptosis assay, which shows that the recombinant hTFF3 is biologically active.

Expression of an antibody-targeted plasminogen activator in Escherichia coli using chemically induced stress responses.

A recombinant gene coding for an antibody-targeted urokinase-type plasminogen activator was constructed for the purpose of enhancing the thrombolytic specificity of urokinase. The recombinant gene was cloned into prokaryotic expression vector pTrcHisA, and transformed into Escherichia coli strain Rosetta (DE3). Less than 4 mg of the desired protein l(-1) could be obtained in the form of inclusion bodies. Of various inducers and enhancers of stress responses, the heat-shock enhances, streptomycin, the osmotic stress inducers, D-arabinose and sucrose, and the cold-shock enhancer, tetracycline, simulated the expression of the antibody-targeted plasminogen activator by 2-5-fold.

[Molecular cloning and identification and biological activity of GIF-interacting protein(s)].

In order to examine the elusive functional mechanism of GIF (Neuronal growth inhibitory factor, GIF) and elucidate the possible relationship between GIF and Alzheimer's disease, we constructed bait1 plasmid (pHyblex-GIF) by cloning GIF cDNA directly in frame with plasmid pHyblex, and used the yeast two-hybrid system to screen Alzheimer's disease human brain cDNA library and found the GIF-interacting proteins. The final results from coimmunoprecipitation and western blotting experiments confirmed that interacting proteins specifically binds to GIF. After sequencing the nucleotide of the putative positive plasmids and searching for homologues, we found that one of these is the part of human nuclear dUTPase protein sequence. Then the dUTPase genes are cloned into pGEX-4T-1, the fusion expression vector of GST,and highly expressed in E. coli BL21. The proteins dUTPase and GIF were purified and obtained by affinity chromatography, thrombin digestion and gel filtration on Sephacryl S100. It demonstrated that the proteins dUTPase and GIF had the growth inhibitory activity on co-cultured neuron in vitro. The inhibitory curve was very similar to the GIF. It's possible that dUTPase is one of the proteins interacting with GIF in Alzheimer's disease human brain extracts.

Functional properties of a recombinant chimeric plasminogen activator with platelet-targeted fibrinolytic and anticoagulant potential.

The construction, purification, and characterization of dscuPA33khC, a bifunctional protein designed for thrombosis treatment is described. The chimera was designed to consist of a decorsin (platelet aggregation inhibitor), a low molecular mass (33kDa) single-chain urokinase (scuPA-33k), and a thrombin inhibitory domain. We have successfully produced this recombinant protein in the Escherichia coli expression system, in which the target protein exists in the form of inclusion bodies. After refolding by dilution in vitro, the chimeric protein was purified to homogeneity by immobilized metal affinity chromatography, ion-exchange chromatography, and gel filtration chromatography. The dscuPA33khC could directly activate plasminogen following Michaelis-Menten kinetics with K(m) = 1.52 microM and K(2) = 0.0024 s(-1). The specific activity of the chimera detected by fibrin plate determination was 11,000 IU/mg, which suggested a high thrombolysis effect. However, the chimeric dscuPA33khC bound the activated platelet and significantly increased affinity to platelet clots as compared to fibrin clots. It was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner as well as it exhibits antithrombin activity. These results suggest that the chimeric protein not only has platelet-targeted thrombolytic activity but also obtains anti-thrombus function.

Enhanced expression of an antibody-targeted plasminogen activator in Escherichia coli by fusion to decorsin.

To improve the thrombolytic specificity of plasminogen activators, an antibody-targeted plasminogen activator was constructed consisting of a single-chain variable fragment of a monoclonal antibody SZ-51 raised specifically against human P-selectins on activated platelets and a low molecular weight single-chain urokinase. After fusion to the 3' end of the gene coding for decorsin, originally isolated from the leech Macrobdella decora, expression of the antibody-targeted plasminogen activator gene in E. coli strain Rosetta (DE3) pLysS was greatly enhanced.

Bacterial expression and purification of biologically active human TFF3.

A glutathione S-transferase (GST) fusion protein expression system for the production and purification of recombinant human trefoil factor family-domain peptide 3 (hTFF3) was established. The hTFF3 gene, prepared by PCR, was cloned into a pBluescript KS(+) plasmid, and inserted into a pGEX-4T-1 GST fusion vector. The GST-hTFF3 fusion protein was expressed in Escherichia coli, and hTFF3 was purified with Glutathione Sepharose 4B affinity chromatography, yielding about 3-4 mg of pure hTFF3 in one liter of culture broth. The biological activity of purified hTFF3 was tested in two previously reported rat gastric ulcer models. Oral administration of recombinant hTFF3 has a dose dependent protective effect against ethanol-induced or pylorus ligation-induced gastric mucosa injury in rat, which indicates that our recombinant hTFF3 is biologically active.

Expression and characterization of ARSP1 from Eisenia fetida.

To study the characterization of a protease ARSP1 (apoptosis-related serine protease) of Eisenia fetida, a recombinant ARSP1 was constructed. ARSP1 was produced in E. coli BL21-CodonPlus (DE3)-RIL after IPTG induction and exited in inclusion body. After refolding in vitro, the protein was purified by DEAE-Sepharose F.F. and Sephacryl S-100 chromatography in sequence. ARSP1 showed high sequence identity to other chymotrypsin-like serine proteases and the catalytic triad was His41-Asp90-Ser188. ARSP1 could degrade casein following Michaelis-Menten kinetics with a Vmax of 43.9 U/mg protein and a Km for casein of 0.83 g/l. Studies with inhibitors indicated that ARSP1 was a chymotrypsin-like serine protease. Experiments in vitro demonstrated that ARSP1 could not only hydrolyze fibrinogen and fibrin directly, but also activate plasminogen to plasmin. ARSP1 inhibited thrombin activity and ADP-induced platelet aggregation in a dose-response correlation. These results showed that ARSP1 has thrombolytic activity and also an anti-thrombus function.

Attenuation of zinc-induced neuronal death by the interaction of growth inhibitory factor with Rab3A in rat hippocampal neurons.

We examined the protective effect of growth inhibitory factor (GIF) against zinc-induced neuronal death in rat hippocampal neurons. In an in vitro cell culture system, 300 microM Zn(2+) readily induced death of hippocampal neuronal cells, which was characterized by massive necrosis and a minor degree of apoptosis. Neither the addition of recombinant GIF nor Rab3A alone could rescue these cells from death. However, the combination of GIF with Rab3A could significantly enhance the survival of the hippocampal neurons. This result was supported by both Annexin -V FITC/propidium dual staining and chromosomal DNA analysis. These findings suggest that GIF may inhibit Zn(2+)-induced neuronal death via its interaction with Rab3A.

Differential display proteome analysis of PC-12 cells transiently transfected with metallothionein-3 gene.

Metallothionein-3 (MT-3), also known as growth inhibitory factor, possesses several unique properties other than the common features of metallothionein family. To investigate the mechanisms underlying its multifaceted roles in the central nervous system, we employed differential display proteomics techniques to study holistic protein changes of PC-12 cells induced by transient transfection of MT-3. Ten significantly and reproducibly changed proteins were identified and their functional implications are discussed in some detail.

[Affection of metallothionein-3 to dUTPase's accommodating cellular toxicity of dUTP].

Metallothionein-3 (MT-3), renamed as growth inhibitory factor (GIF), is a brain specific member of the metallothionein family. Human dUTPase is a recently found protein in brain that can interact with hMT-3. They have the growth inhibitory activity on neuron cell by interaction. To study the affection of hMT-3 to dUTPase's eliminating the cellular toxicity caused by dUTP, the pSVHA-dUTPase and pFLag-hMT-3 genes have been transfected into HEK293 cells. In addition, the dUTPase and hMT-3 proteins were expressed in BL21 to study the role of hMT-3 on the hydrolyzation of dUTP by dUTPase. The results demonstrate that the cells co-transfected with dUTPase and hMT-3 genes have more strong resistibility to dUTP than the cells transfected only with dUTPase gene. And that the hMT-3 protein can accelerate the hydrolyzation of dUTP by dUTPase. All these indicate that hMT-3 can cooperate with dUTPase to protect better the 293 cells from dUTP. This research offered the theoretic elements for the application of hMT-3 and dUTPase in chemic cure.