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Jian Zhuge

Publications and source records attributed to Jian Zhuge.

18 recordsLinked to original sources
PubMed2006

Increased toxicity by transforming growth factor-beta 1 in liver cells overexpressing CYP2E1.

Ethanol treatment causes an increase in expression of TGF-beta1 and CYP2E1 in the centrilobular area. Alcoholic liver disease is usually initiated in the centrilobular region of the liver. We hypothesized that the combination of TGF-beta1 and CYP2E1 produces increased oxidative stress and liver cell toxicity. To test this possibility, we studied the effects of TGF-beta1 on the viability of HepG2 E47 cells that express human CYP2E1, and C34 HepG2 cells, which do not express CYP2E1. E47 cells underwent greater growth inhibition and enhanced apoptosis after TGF-beta1 treatment, as compared to the C34 cells. There was an enhanced production of reactive oxygen species (ROS) and a decline in reduced glutathione (GSH) levels in the TGF-beta1-treated E47 cells and the enhanced cell death could be prevented by antioxidants. The CYP2E1 inhibitor diallyl sulfide prevented the potentiated cell death in E47 cells validating the role of CYP2E1. Mitochondrial membrane potential declined in the TGF-beta1-treated E47 cells, prior to developing toxicity, and cell death could be prevented by trifluoperazine, an inhibitor of the mitochondrial membrane permeability transition. TGF-beta1 also produced a loss of cell viability in hepatocytes from pyrazole-treated rats with elevated levels of CYP2E1, compared to control hepatocytes. In conclusion, increased toxic interactions by TGF-beta1 plus CYP2E1 can occur by a mechanism involving increased production of intracellular ROS and depletion of GSH, resulting in mitochondrial membrane damage and loss of membrane potential, followed by apoptosis. Potentiation of TGF-beta1-induced cell death by CYP2E1 may contribute to mechanisms of alcohol-induced liver disease.

Animals↗
PubMed2006

Biochemical properties of a thermostable phytase from Neurospora crassa.

A gene (Ncphy) encoding a putative phytase in Neurospora crassa was cloned and expressed in Pichia pastoris, and the biochemical properties of the recombinant protein were examined in relation to the phytic acid hydrolysis in animal feed. The recombinant phytase (rNcPhy) hydrolyzed phytic acid with a specific activity of 125 U mg-1, Km of 228 micromol L-1, Vmax of 0.31 nmol (phosphate) s-1 mg-1, a temperature optimum of 60 degrees C and a pH optimum of 5.5 and a second pH optimum of 3.5. The enzyme displayed pH stability around pH 3.5-9.5 and showed satisfactory thermostability at 80 degrees C. The phytase from N. crassa has potential for improving animal feed processing at higher temperatures.

6-Phytase↗
PubMed2006

[Stimulatory effects of some amino acids on glycerol production by Candida glycerinogenes].

By using some intermediate metabolites in EMP pathway and TCA cycle as the control, the effects of amino acid supplements on glycerol production by Candida glycerinogenes in shake-flask fermentations with urea as nitrogen resource were investigated. The results showed that ten kinds of amino acids, including L-glutamic acid, L-glutamine, L-aspartic acid, L-asparagine, L-glycin, L-lysine, L-tyrosine, L-proline, L-histidine, and L-serine, had strong promotional effects on glycerol production; The optimal concentrations of these amino acids were 0.40, 0.45, 0.36, 0.35, 0.39, 0.36, 0.35, 0.45, 0.26, and 0.45 g/L, respectively. Accordingly the optimum contents of pyruvic acid, a-oxoglutarate, oxaloacetic acid, citrate, and succinate were 0.24, 0.42, 0.40, 0.37, and 0.38 g/L, respectively. The advantageous opportunities of supplement were as follows: L-lysine at the beginning of fermentation; pyruvic acid and oxaloacetic acid at the fourteenth hour; L-glutamic acid, L-glutamine, L-histidine, L-proline, L-aspartic acid, L-tyrosine, L-glycin, alpha-oxoglutarate, and succinate at the thirtieth hour; and L-asparagine, L-serine, and citrate at the forty-eighth hour. The addition of each stimulant at the optimal conditions could significantly promote glycerol production, with the glycerol yield on initial glucose and its increased speed exceeding 60% and 16%, respectively. The possible stimulatory mechanism due to the amino acid supplements is that the increased intermediate metabolites levels from amino acids degradation may have enhanced the flux through TCA cycle, which improve cell energetics. Meanwhile, the shift of carbon metabolism flux at the glyceraldhyde-3-phosphate node can result in the incremental flux through glycerol biosynthesis pathway.

Amino Acids↗
PubMed2006

[Comparison of different transformation methods for Monascus sp].

In order to facilitate the producer of polyketide pathway, four different transformation methods were tested and compared in an attempt to develop the genetic transformation system of Monascus sp. Using vector pBC-Hygro, the fungus was transformed to be hygromycin B-resistant, by conventional transformation as well as electroporation based on protoplast, electroporation based on germinated conidia, and restriction enzyme-mediated integration (REMI). Electroporation based on germinated conidia was found to be inappropriate for transforming Monascus sp. due to a low transformation frequency. The conventional transformation and electroporation technique based on protoplasts were thought not to be fit for transforming Monascus sp., due to a low stability of transformants though they yielded up to 135 transformants and 125 transformants per microgrammol/Lol/Le DNA, respectively. Transformant number was increased by 20-fold by REMI (2,500 transformants per microgrammol/Lol/Le DNA) and 70%-75% of them were stable. REMI technique would be very beneficial to the establishment of the genetic transformation system of Monascus sp.

DNA, Recombinant↗
PubMed2006

[Construction of an engineering strain producing alkaline pectate lyase with pHSh].

The structure gene PL from Bacillus subtilis WSHB04-02 encoding pectate lyase was amplified by PCR. The pET22b(+) vector, with leader sequence PelB, harboring PL gene was constructed. From pET22b(+) PL, the fragment of PL and leader sequence PelB was amplified by PCR together, which was transformed into E. coli JMI109. The expression of PL in E. coli JM109 was not evidently different from E. coli BL21DE3 (pET22b(+) PL) which promoter is T7. SDS-PAGE analysis showed that the molecular weight of expressed recombinant PL was about 43 kDa which was the same as calculated value. The results indicated the expression of pHsh PL in E. coli JM109, Hsh as a promoter, was satisfied and low-cost. It is significant for large-scale fermentation of pectate lyase.

Bacillus subtilis↗
PubMed2005

Serum deprivation-induced HepG2 cell death is potentiated by CYP2E1.

Induction of oxidative stress plays a key role in serum deprivation-induced apoptosis. CYP2E1 plays an important role in toxicity of many chemicals and ethanol and produces oxidant stress. We investigated whether CYP2E1 expression can sensitize HepG2 cells to toxicity as a consequence of serum deprivation. The models used were HepG2 E47 cells that express human CYP2E1, and C34 HepG2 cells which do not express CYP2E1. E47 cells showed greater growth inhibition and enhanced cell death after serum deprivation, as compared to the C34 cells. DNA ladder and flow cytometry assays indicated that apoptosis occurred at earlier times after serum deprivation in E47 than C34 cells. Serum withdrawal-induced E47 cell death could be rescued by antioxidants, the mitochondrial permeability transition inhibitor cyclosporine A, z-DEVD-fmk, and a CYP2E1 inhibitor 4-methylpyrazole. Increased production of reactive oxygen species (ROS) and lipid peroxidation occurred in E47 cells after serum deprivation, and there was a corresponding decline in the E47 cell mitochondrial membrane potential and reduced glutathione (GSH) levels. We propose that the mechanism of this serum withdrawal plus CYP2E1 toxicity involves increased production of intracellular ROS, lipid peroxidation, and decline of GSH levels, which results in mitochondrial membrane damage and loss of membrane potential, followed by apoptosis. Potentiation of serum deprivation-induced cell death by CYP2E1 may contribute to the sensitivity of the liver to alcohol-induced ischemia and growth factor deprivation.

Antidotes↗
PubMed2005

Isolation and sequence analysis of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Candida glycerinogenes WL2002-5, an industrial glycerol producer.

The URA3 gene of Candida glycerinogenes WL2002-5, an industrial glycerol producer encoding orotidine-5'-phosphate decarboxylase enzyme, was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 786 bp, encoding a 262 amino acid protein, which shares 71.65% amino acid sequence similarity to the S. cerevisiae URA3 protein. Furthermore, the cloned ORF fully complemented the ura3 mutation of S. cerevisiae, confirming that it encodes for the C. glycerinogenes Ura3 (CgUra3) protein.

Amino Acid Sequence↗
PubMed2005

[Glyoxylate cycle is required for the overproduction of glutamate but is not essential for Corynebacterium glutamicum growth on glucose].

The glyoxylate cycle was hypothesed to be indispensable for glutamate overproduction in coryneform bacteria, for it was thought to fulfill anaplerotic functions and to supply energy during the growth phase. During glutamate overproduction phase, however, it has been noted that the high level of the cycle is detrimental to the glutamate production. In order to clarify the relationship between the glutamate production and the glyoxylate cycle, a chromosomal aceA-disrupted mutant of wild-type C. glutamicum ATCC 13032 was constructed. The isocitrate lyase (ICL) activity of the parental strain was 0.011 u/mg of protein and reached 1.980 u/mg of protein after acetate induction; the mutant strain WTdeltaA, however, had no detectable ICL activity and was no longer able to grow on minimal medium with acetate as the sole carbon source. Compared with the wild-type C. glutamicum WT, the mutant strain WTdeltaA, exhibited the same growth rate with glucose as the sole carbon source, indicating glyoxylate cycle is not required for its growth on glucose. On the contrary, the glutamate production in WTdeltaA was severely impaired and more residual glucose was found in the fermentation broth at the end of fermentation with the mutant strain than with the wild-type strain. Further investigations into the relationship between the glutamate production and the glyoxylate cycle are under the way, which may help to elucidate the mechanism of glutamate overproduction.

Corynebacterium glutamicum↗
PubMed2005

[Construction of novel recombinant Escherichia coli capable of producing 1,3-propanediol].

The 1,3-propanediol oxidoreductase isoenzyme encoding gene (yqhD) from E. coli was amplified by PCR. yqhD was inserted in pEtac to yield the recombinant expression vector pEtac-yqhD. Over-expression of yqhD in E. coli JM109 was achieved with pEtac-yqhD. SDS-PAGE analysis showed an over-expressed recombinant product at about 43 kD, consistent with the molecular weight predicted from gene sequence. Compared with E. coli JM109 (pEtac), the 1,3-propanediol oxidoreductase isoenzyme activity of the recombinant E. coli (pEtac-yqhD) reached 120 u/mg protein under the induction of 1.0 mmol/L IPTG at 37 degrees C for 4 hours; at similar conditions, enzyme activity of E. coli JM109 (pEtac) was only 0.5 u/mg protein. The recombinant E. coli JM109 (pUCtac-dhaB, pEtac-yqhD) was constructed. After induction with 1.0 mmol/L IPTG, the recombinant strain could transform 50 g/L glycerol to 38 g/L 1,3-propanediol under aerobic conditions. This work demonstrated firstly that the 1,3-propanediol oxidoreductase isoenzyme could show high activity under aerobic conditions.

Aerobiosis↗
PubMed2004

Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue.

AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced. Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of 17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced. RESULTS: One of the CYP2D6 cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4), and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples, only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6 cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6 gene were detected. The third variant was the skipped exon 3 , and 153 bp was lost. CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.

Alternative Splicing↗
PubMed2004

Stable expression of human cytochrome P450 2D6*10 in HepG2 cells.

AIM: Over 90% of drugs are metabolized by the cytochrome P-450 (CYP) family of liver isoenzymes. The most important enzymes are CYP1A2, 3A4, 2C9/19, 2D6 and 2E1. Although CYP2D6 accounts for <2% of the total CYP liver enzyme content, it mediates metabolism in almost 25% of drugs. In order to study its enzymatic activity for drug metabolism, its cDNA was cloned and a HepG2 cell line stably expressing CYP2D6 was established. METHODS: Human CYP2D6 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from human liver tissue and cloned into pGEM-T vector. cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A cell line was established by transfecting the recombinant plasmid of pREP9-CYP2D6 to hepatoma HepG2 cells. Expression of mRNA was validated by RT-PCR. Enzyme activity of catalyzing dextromethorphan O-demethylation in postmitochondrial supernatant (S9) fraction of the cells was determined by high performance liquid chromatography (HPLC). RESULTS: The cloned cDNA had 4 base differences, e.g. 100 C-T, 336 T-C, 408 C-G and 1 457 G-C, which resulted in P34S, and S486T amino acid substitutions, and two samesense mutations were 112 F and 136 V compared with that reported by Kimura et al (GenBank accession number: M33388). P34S and S486T amino acid substitutions were the characteristics of CYP2D6*10 allele. The relative activity of S9 fraction of HepG2-CYP2D6*10 metabolized detromethorphan O-demethylation was found to be 2.31 +/- 0.19 nmol/min(-1)/mg(-1) S9 protein (n=3), but was undetectable in parental HepG2 cells. CONCLUSION: cDNA of human CYP2D6*10 can be successfully cloned. A cell line, HepG2-CYP2D6*10, expressing CYP2D6*10 mRNA and having metabolic activity, has been established.

Cell Line, Tumor↗
PubMed2004

Cloning and over-expression of an alkaline protease from Bacillus licheniformis.

The alkaline protease gene, apr, from Bacillus licheniformis 2709 was cloned into a Bacillus shuttle expression vector, pHL, to yield the recombinant plasmid pHL-apr. The pHL-apr was expressed in Bacillus subtilis WB600, yielding a high expression strain BW-016. The amount of alkaline protease produced in the recombinant increased by 65% relative to the original strain. SDS-PAGE analysis indicated a Mr of 30.5 kDa. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 6816.

Amino Acid Sequence↗
PubMed2004

Purification and characterisation of an alkaline protease used in tannery industry from Bacillus licheniformis.

An extracellular alkaline protease produced by Bacillus licheniformis AP-1 was purified 76-fold, yielding a single 28 kDa band on SDS-PAGE. It was optimally active at pH 11 and at 60 degrees C (assayed over 10 min). The protease was completely inhibited by phenylmethylsulfonyl fluoride and diodopropyl fluorophosphate, with little increase upon Ca2+ and Mg2+ addition.

Bacillus↗
PubMed2003

By-product formation by a novel glycerol-producing yeast, Candida glycerinogenes, with different O2 supplies.

Candida glycerinogenes is an aerobe which does not depend on sulphite for production of glycerol. With a sufficient O2 supply, up to 130 g glycerol l(-1) was produced with 2.6 g acetic acid l(-1) as by-product. However, with an insufficient O2 supply--with higher volumes of medium or at higher corn steep liquid concentrations--the glycerol concentration was lower because the by-products, ethanol, pyruvate and lactic acid, were produced in greater amounts, up to 45 g l(-1), 4.3 g l(-1), 1.6 g l(-1), respectively, whereas, less acetic acid (0.6 g l(-1)) was produced. In addition, ethanol decreased to 0.4 g l(-1) and the glycerol yield improved from 34 to 50% (w/w) by adding 50 g sulphite l(-1), nevertheless, acetic acid increased to 7.8 g l(-1).

Acetic Acid↗
PubMed2003

Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line.

AIM: Human cytochrome P-450 2E1 (CYP2E1) takes part in the biotransformation of ethanol, acetone, many small-molecule substrates and volatile anesthetics. CYP2E1 is involved in chemical activation of many carcinogens, procarcinogens, and toxicants. To assess the metabolic and toxicological characteristics of CYP2E1, we cloned CYP2E1 cDNA and established a HepG2 cell line stably expressing recombinant CYP 2E1. METHODS: Human CYP2E1 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2E1 to HepG2 cells. The expression of CYP2E1 mRNA was validated by RT-PCR. The enzyme activity of CYP2E1 catalyzing oxidation of 4-nitrophenol in postmitochondrial supernate (S9) fraction of the cells was determined by spectrophotometry. The metabolic activation of HepG2-CYP2E1 cells was assayed by N-nitrosodiethylamine (NDEA) cytotoxicity and micronucleus test. RESULTS: The cloned CYP2E1 cDNA segment was identical to that reported by Umeno et al (GenBank access No. J02843). HepG2-CYP2E1 cells expressed CYP2E1 mRNA and had 4-nitrophenol hydroxylase activity (0.162 +/- 0.025 nmol.min(-1).mg(-1) S9 protein), which were undetectable in parent HepG2 cells. HepG2-CYP2E1 cells increased the cytotoxicity and micronucleus rate of NDEA in comparison with those of HepG2 cells. CONCLUSION: The cDNA of human CYP2E1 can be successfully cloned, and a cell line, HepG2-CYP2E1, which can efficiently express mRNA and has CYP2E1 activity, is established. The cell line is useful for testing the cytotoxicity, mutagenicity and metabolism of xenobiotics, which may possibly be activated or metabolized by CYP2E1.

Base Sequence↗
PubMed2002

[Purification and properties of thermostable catalase in engineered E. coli].

A thermostable catalase in engineered bacterium E. coli was purified to electrophoretic homogenenity by heat treatment, ammonium sulfate fractionation precipitation, DEAE-A50 ion exchange chromatography, HiPrep 16/10 Phenyl hydrophobic interaction chromatography and Superdex200 HR 10/30 size exclusion chromatography with 187.2-fold purification and 9.8% recovery. The optimum reaction temperature and pH of this recombinant catalase were 70 degrees C and 7.0 respectively. The catalase is stable below 60 degrees C and at pH range 3-8. The residual activity of the catalase was about 60% after treated at 70 degrees C for 60 minutes and 80 degrees C for 10 minutes. The apprant Km and Vmax value of the catalase were 7.75 mmol/L and 27.8 mmol.min-1.mg-1 respectively. The affects of some metal ions and compounds on this enzyme were shown. Zn2+, Ba2+, Mn2+ of 1 mmol/L could completely inactivate the enzyme, EDTA of 50 mmol/L had no affect on activity.

Barium↗
PubMed2002

[Study on fermentation condition of alkaline protease gene engineering strain and the purification and characterization of recombinant enzyme].

In a 5L fermentor the production conditions of alkaline protease gene engineering strain BA071 were investigated. The maximum activity of alkaline protease reached 24,480 u/mL in 40 hours of fermentation by combination of enhancing aeration and changing the agitation rate. The fast purification method of recombinant protease was conducted with FPLC (Fast Protein Liquid Chromatography). The crude enzyme, treated with ammonium sulfate fractionation and decolored with DEAE-A-50 and polyethylene glycol concentration, was purified with CM-Sephadex-C-50 and Sephadex-G-75. The purified enzyme appears homologous on SDS-PAGE. The purity of enzyme was increased 76.2 times. SDS-PAGE analysis showed that the molecular weights of expressed recombinant products were about 28 kD. The optimal reaction pH and temperature of recombinant enzyme were at pH11 and 60 degrees C, respectively. The recombinant enzyme exhibited high temperature tolerance and was stable at a wide range of pH. Ca2+, MG2+ can enhance the stability of the recombinant enzyme. While the protease activity of the enzyme was strongly inhibited by Hg2+, Ag+, PMFS [symbol: see text] DFP, and was not affected by SDS and Urea.

Bacillus↗