Lentivirus-mediated short hairpin RNA targeting the APRIL gene suppresses the growth of pancreatic cancer cells in vitro and in vivo.

RNA interference (RNAi) is an evolutionarily conserved process of gene silencing in multiple organisms, which has become a powerful tool for investigating gene function by reverse genetics. Herein, we constructed a short hairpin RNA (shRNA) lentiviral expression vector targeting a proliferation-inducing ligand (APRIL) gene in the CFPAC-1 cell (a type of cell strain of human pancreatic cancer) in order to observe the inhibitory effect of APRIL gene's shRNA on the growth of the CFPAC-1 cell in vitro and in vivo. The results showed that lentivirus-mediated RNAi effectively inhibited the expression of APRIL mRNA and protein in CFPAC-1 cells. Moreover, it can inhibit the growth of pancreatic cancer cells in vitro and in vivo. Our study indicates that lentivirus-mediated gene therapy is an attractive strategy in the treatment of pancreatic cancer and justifies the use of lentivirus in cancer gene therapy studies.

[Effect of EPHA2-siRNA plasmid on biological behavior of human osteosarcoma cells in vitro].

OBJECTIVE: To investigate the role of EPHA2 in regulating apoptosis, proliferation and vasculogenic mimicry of osteosarcoma cells, by gene silencing through RNA interference. METHODS: EPHA2-siRNA plasmids were achieved by gene cloning. The plasmids were transfected into human osteosarcoma cells (MG63). The expression level of EPHA2 protein was measured by Western blotting. The proliferation, apoptosis and vasculogenic mimicry features of osteosarcoma MG63cells were assessed by light microscopy, MTIP assay, flow cytometry, annexin V-FITC/PI and HE staining, respectively. RESULTS: The EPHA2-siRNA plasmid was confirmed by DNA sequencing. After treatment with Sequence-specific siRNA targeted EPHA2, the protein level of the transfected group decreased significantly. As compared to non-siRNA transfected cells, the transfected group showed lower proliferation, higher and earlier apoptosis and less osteosarcoma-generated vasculogenic mimicry. CONCLUSION: EPHA2 gene may be involoved in apoptosis and proliferation of osteosarcoma cells, and may be necessary for vasculogenic mimicry. Down-regulation of EPHA2 expression by sequence-specific siRNA may be considered as a new option in the treatment of EPHA2 over-expressing cancer including osteosarcoma in future.

[Preparation and identification of immune serum against recombinant fusion protein of rhoptry 2 and major surface protein 1 from Toxoplasma gondii].

OBJECTIVE: To prepare and identify immune serum against the recombinant fusion protein of rhoptry 2 (ROP2) and major surface protein 1(P30) from Toxoplasma gondii. METHODS: The constructed recombinant plasmid of pET28b/ROP2-P30 was transformed to a bacterium BL21-Codon Plus (DE3)-RIL strain and was expressed under IPTG induction. Cells were lysed by multiple rounds of sonication to obtain supernatant and inclusion body respectively. The inclusion body was washed with 2 mol/L and 4 mol/L urea to remove the nonspecific protein. The washed products dissolved in 8 mol/L urea were received by SDS-PAGE. Two rabbits were immunized with the fusion protein rROP2-P30 and sera from the rabbits were collected. Immune diffusion test, indirect ELISA and Western-blot were used to detect antibody titer and specificity of the immune serum against rROP2-P30. RESULTS: Immune diffusion test demonstrated that specific immune serum were obtained. Indirect ELISA confirmed that the antibody titer in the serum reached 1:12 800 and the rROP2-P30 was recognized by specific IgG in this serum by Western-blot analysis. CONCLUSION: Specific immune serum against the recombinant fusion protein rROP2-P30 has been prepared.

Cost-effective method of siRNA preparation and its application to inhibit hepatitis B virus replication in HepG2 cells.

AIM: To find a cost-effective method of preparation of short interfering RNAs based on cloning, fermentation, digestion and purification (CFDP) and test its feasibility to inhibit hepatitis B virus replication in cell culture. METHODS: We constructed an expression vector containing T7 and tac promoter in a head-to-head orientation. cDNA fragment of interest was cloned into this vector between the opposing promoters. dsRNAs were expressed with this vector in Escherichia coli, and purified by affinity chromatography using CF 11 column. They were digested by RNase III in a buffer containing manganese ions, then separated on 15% non-denaturing PAGE, and the siRNAs about 25 bp in length were recovered. siRNAs prepared with CFDP were co-transfected with target gene expression plasmid into human cell lines with lipofectamine 2,000 to test their inhibition efficiency. RESULTS: siRNAs corresponding to part of the hepatitis B virus polymerase gene (siHBVP) prepared by CFDP specifically and dramatically suppressed the virus protein expression. The HBsAg expression level was reduced to 10% that of the control by co-transfection of 60 nmol/L siHBVP in SMMC7721 cells. Dose-dependent effect on suppression of HBsAg and HBeAg expression was observed in HepG2 cells. The highest inhibition rate was kept at 70% during the six days after transfection of 7.5 nmol/L siHBVP. CONCLUSION: We show CFDP is a very promising method to prepare therapeutic agents in anti-virus applications.

Isolation and characterization of a novel strain of Natrinema containing a bop gene.

A novel member of extremely halophilic archaea, strain AJ2, was isolated from Ayakekum Lake located in Altun Mountain National Nature Reserve of Xinjiang Uygur Autonomous Region in China. The strain AJ2 requires at least 10% (w/v) NaCl and grows 10% to 30% (optimum at 20%). Phylogenetic analysis based on 16S rDNA sequence comparison revealed that strain AJ2 clustered to three Natrinema species with less than 97.7% sequence similarities, suggesting AJ2 is a novel member of Natrinema. A bacteriorhodopsin-encoding (bop) gene was subsequently detected in the AJ2 genome using the polymerase chain reaction technique. The cloning and sequencing of a 401 base pairs fragment indicated the deduced amino acid sequence of bop from AJ2 is different from that reported for bacteriorhodopsins. This is the first reported detection of a bop gene in Natrinema.

[Cloning and expression of the fused gene of rhoptry protein ROP2 and major surface protein P30 from Toxoplasma gondii].

OBJECTIVE: To clone and express the fused gene fragment coding rhoptry protein ROP2 and major surface protein P30 from Toxoplasma gondii as a preparation for the construction of the complex ROP2-P30 antigen by gene engineering. METHODS: The gene fragment encoding P30 was amplified by PCR from T. gondii RH strain and subcloned into the recombinant plasmid pUC119/ROP2 already constructed. The recombinant plasmid pUC119/ROP2-P30 was digested by Sac I/HindIII and inserted into the same site of expression vector pET28b. The recombinant plasmid of pET28b/ROP2-P30 was transformed to E. coli and expressed under the induction of IPTG. RESULTS: The gene fragment 700 bp encoding P30 was obtained from the total DNA of T. gondii by PCR. The recombinant plasmid pET28b/ROP2-P30 was successfully constructed, which was highly expressed in E. coli, a fusion protein with molecular weight of 69000. CONCLUSION: The fusion gene encoding the rhoptry protein ROP2 and the major surface protein P30 of T. gondii has been successfully cloned and expressed to be an expected recombinant fusion protein ROP2-P30 with molecular weight 69000.

Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum.

AIM: To study the effect of co-expression of ppsA, pckA, aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strain for L-phenylalanine. METHODS: ppsA and pckA genes were amplified from genomic DNA of E. coli by polymerase chain reaction, and then introduced into shuttle vectors between E coli and Brevibacterium flavum to generate constructs pJN2 and pJN5. pJN2 was generated by inserting ppsA and pckA genes into vector pCZ; whereas pJN5 was obtained by introducing ppsA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes. The recombinant plasmids were then introduced into B. flavum by electroporation and the transformants were used for L-phenylalanine fermentation. RESULTS: Compared with the original B. flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably. pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold. Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly. CONCLUSION: Co-expression of ppsA and pckA genes in B. flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pckA, aroG, pheA and tyrB of E. coli in B. flavum was a feasible approach to construct a strain for phenylalanine production.

Purification and Characterization of an Alkaline Lipase from Penicillium cyclopium PG37.

An extracellular, novel alkaline lipase produced by Penicillium cyclopium PG37 was purified by centrifugation, ammonium sulfate precipitation, and phenyl-Sepharose CL-4B, DEAE Sepharose fast flow and Sephadex G-75 column chromatographies. A 16.5-fold purification of the enzyme was achieved which had a specific activity of 5 200 u/mg protein, and the recovery of the activity was 33.2%. The purified enzyme exhibited a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and polyacrylamide gel electrophoresis (PAGE). The molecular weight of the native lipase was estimated to be about 29 kD by gel filtration using Sephadex G-150, and that of the denatured lipase was determined to be about 27.5 kD by its mobility on SDS-PAGE, indicating that the lipase was a monomer. The N-terminal amino acid sequence was determined with automatic protein sequencer to be ATADAAAFPD, which has no homology with other sequences of known lipases. The optimum temperature of the action of this enzyme was 25 degrees and the lipase was stable below 30 degrees, but only 30% of its activity remained after 20 min incubation at 40 degrees. The enzyme was stable at pH from 6.5 to 10.5, and its optimal pH for activity is 10.0. Low concentration of alkaline proteinase has little effect on the lipase PG37, therefore these two enzymes can be used as ingredients that are added to commercial detergents simultaneously.

Purification and characterization of a novel chitinase from Bacillus brevis.

An extracellular chitinase secreted by Bacillus brevis was purified to homogeneity by a combination of ammonium sulfate precipitation, Phenyl-Sepharose hydrophobic-interaction chromatography and DEAE anion-exchange chromatography. On SDS-polyacrylamide gel electrophoresis analysis, the purified enzyme showed a mass of 85 kD even in the presence of beta mercaptoethanol, but shifted to 48 kD when heated in boiling water or treated with 8 mol/L urea at 50 degrees for 10 min. The depolymerization of subunits was accompanied with the loss of chitinase activity, and removing denaturing factors by dialysis could restore the dimer structure and enzymatic activity. The enzyme had an isoelectric point of 5.5 and an optimal temperature of 60 degrees, and was most active at pH 8.0. The enzymatic activity was stable at pH 6-10, and inhibited by Ag(+). Ten N-terminal amino acids were determined to be AVSNSKIIGY, demonstrating that the purified enzyme was a novel one. The hydrolysis pattern of the purified enzyme indicated that the chitinase was an endochitinase. The extraordinary thermo-stability and high resistance to proteolysis provide the enzyme with a good prospect to be used as a new tool for biocontrol.

Expression of Human Soluble CD40 Ligand in Pichia pastoris and Its Effects on Dendritic Cells and Malignant B Cells.

CD40 ligand (CD40L) is a member of the tumor necrosis factor (TNF) superfamily and is expressed primarily on the activated CD4( )T lymphocytes. The CD40 molecule, the cognate receptor of CD40L presents on many immunocytes such as B lymphocytes, dendritic cells (DCs) as well as on some neoplastic cells. Triggering of CD40 through CD40L plays a central role in the initiation and regulation of the human immune response. In order to further investigate the possible biological roles of CD40 signaling triggered by CD40L, we subcloned the DNA fragment encoding the extracellular region of human CD40L into the pSK plasmid. After being sequenced, the target fragment was introduced into the pPICZalphaA plasmid to construct the pPICZalphaA-sCD40L expressing vector which was then transduced into Pichia pastoris GS115 cells by electroporation. The tansformant expressed sCD40L in culture supernatants with a maximum yield of about 35 mg/L. Furthermore, we found that the recombinant human soluble CD40 ligand (rhsCD40L) could effectively induced human peripheral blood monocytes(PBMCs) in vitro in the absence of TNFalpha into dendritic cells (DCs) with the typical morphology and special surface markers of dendritic cells including CD1a, CD80, CD83, and HLA-DR etc. To our surprise, the rhsCD40L also could inhibit directly in vitro proliferation of the CD40-positive multiple myeloma cell line XG-2 and the B lymphoma cell line Daudi significantly at an optimal concentration from 2.5 to 15.0 mg/L, while CD40 negative ovarian carcinoma cell lines, SKB and SKR, were not effected by either high or low concentration of rhsCD40L. Moreover, rhsCD40L had the same effects as CD40L-transfected cell in inducing XG2 cell apoptosis. Our results demonstrated that functional human soluble CD40L could be successfully expressed in the Pichia pastoris system and that the recombinant human soluble CD40L might be a potential immune adjuvant and a new powerful molecule for tumor bio-therapy.

Effect of F209S Mutation of Escherichia coli AroG on Resistance to Phenylalanine Feedback Inhibition.

In Escherichia coli, 80% of the 3-deoxy-D-arabino-heptulosonate 7-phosphate(DAHP) synthase was encoded by aroG gene. The aroG gene was amplified by polymerase chain reaction(PCR) from strain K-12 and a mutant strain resistant to phenylalanine analogues. The PCR products were cloned and subject to DNA sequence analysis. A single base mutation of Tright curved arrow C was detected at nucleotide 625, which causes a substitution of Phe(209) by Ser in the gene product. The gene was expressed on pTrc99A in E.coli strain JM105. Under the induction of IPTG, distinct band with the expected molecule weight was detected on SDS-polyacrylamide gel electrophoresis and the specific activity of DAHP of the crude extract of the transformed cells increased by 1.8-fold. Enzyme activity inhibition analysis revealed the high resistance of mutant AroG to feedback inhibition by phenylalanine. JM105 cells harboring with mutant aroG gene showed were able to grow on medium containing higher concentration of analogues than that carrying normal aroG gene. Discussion was focused on the varieties of mutations contributing to desensitization of feedback inhibition.

[Determination of the D- and L-configurations of amino deoxy sugars by (S)-TBMB carboxylic acid, a fluorescent chiral derivatization reagent].

D- and L- amino sugars were coupled with (S)-TBMB (S)-TBMB = (S)-2-tert-butyl-2-methyl-1, 3-benzodioxole carbonyl chloride, a fluorescent chiral reagent, followed by per-O-acetylation. The reactions yielded diastereomeric per-O-acetylated N-(S)-TBMB carbonyl amino sugars. Their (1)HNMR signals, especially the strong singlet peaks of tert-Bu and Me groups were diagnostic for the determination of the D-, L- configurations of amino sugar. Furthermore, a simple and highly sensitive method for the determination of the D-, L- configuration of amino deoxy sugars was developed based on the same fluorescent labeling method and reverse phase HPLC. The total time in analysis is less than two hours and the detection limit of the method is 0.2 picomolar.

The Isolation and Purification of Archaerhodopsin from Halobacterium sp. XZ515.

The claret membrane, instead of the purple membrane, was isolated when the conventional method for isolating purple membrane from Halobacterium halobium was applied to Halobacterium sp. XZ515. The SDS-PAGE results showed that only one protein, archaerhodopsin, existed in claret membrane with M. W. similar to BR. The method for isolating and purifying the archaerhodopsin, an intrinsic membrane protein, by detergent dissolution and hydrophobic chromatography on the column of octylsepharose, was also introduced. The pure archaerhodopsin showed the absorption spectrum similar to BR and was able to produce the M412 photoproduct in the photocycle under illumilation. It was concluded that this archaerhodopsin was a BR-like retinal protein.

Cloning and Expression of aroG Gene of E. coli and Its Co-expression with pheA and tyrB Genes.

3-Deoxy-D-arabino-heptulonate-7-phosphate synthetase (DAHP) is one of the key enzymes in phenylalanine biosynthesis pathway. In E. coli, DAHP is encoded by aroG Gene. In this work, aroG was cloned from an E. coli mutant strain resistant to m-fluro-L-phenylalanine (mPF) and p-fluro-L-phenylalanine (pPF) by PCR. The gene was expressed under the control of lambda phage promoter p(R) in P2392 strain of E. coli. Distinct band was detected as the product of aroG on SDS-PAGE. The specific activity in crude extract of DAHP was raised to 1.7-fold. Based on the cloning and expression of pheA (encoding both chorsmate mutase CM and prephenate dehydratase PD) and tyrB (encoding phenylalanine aminotransferase PAT) genes, aroG, pheA and tyrB genes were constructed and expressed in P2392. The results showed that the specific activities of DAPH, CM/PD and PAT in crude extracts were increased by 1.7, 13.9/7.8 and 2.3-fold, respectively.

Increasing bioactivity of Flt3 ligand by fusing two identical soluble domains.

Flt3 ligand (FL) is a hematopoietic growth factor, initiating its in tracellular signaling cascade by binding to counterpart receptor and driving receptor dimerization. The native form of soluble FL in vivo is mainly monomeric. In this study, we constructed a rFL-FL fusion protein cDNA by linking two copies of cDNA encoding the soluble domain of FL in tandem and expressed it in Pichia pastoris. On SDS-polyacrylamide gel electrophoresis, the rFL-FL fusion protein showed a molecular weight of 43 kD, agreeing well with the predicted value. The 43 kD protein was further confirmed by Western blot using polyclonal rabbit anti-human FL antibody. The rFL-FL fusion protein exhibited about 10-fold increment in its activity on colony formation of bone marrow progenitor cells. RFL-FL fusion protein also exerted more potent effect than monomeric FL on extending the survival of starving Raji cells.

Expression of Human Flt3 Ligand in Pichia pastoris and Its Biological Characteristics.

Flt3 ligand(FL) is a cytokine that stimulates the proliferation and differentiation of hematopoietic stem cells/progenitors. In order to obtain high level of recombinant human soluble FL(rhFL) production, an artificial gene for rhFL was synthesized by using favored genetic codons of the yeast Pichia pastoris. Then the gene was cloned into the vector pPICzalphaA and the resulting construct was introduced into Pichia pastoris for expression. It was found that it was possible to obtain biologically active rhFL with a yield of over 30 mg/L of yeast culture. The rhFL stimulated colony formation from cord blood. rhFL, SCF, GM-CSF and IL-3 are the most promising combination for the in vitro expansion of stem/progenitor cells. It was also showed that rhFL increased the induction of dendritic cells from cord blood in combination with GM-CSF, TNFalpha and IL-4. Interestingly, rhFL stimulated the growth of an endothelial cell line this effect has not been reported before.

Expression of recombinant human ICOS and in vitro characterization of its bioactivity on B lymphocytes.

Inducible costimulator (ICOS) is a novel costimulatory molecule expressed in activated T cell and has critical regulation effect on special immune response. In this study, the cDNA encoding human ICOS was cloned from activated tonsil cells via RT-PCR, and was expressed in E. coli on pET28 expression vector. The recombinant ICOS protein expressed from E. coli showed a molecular weight of 14 kD on SDS-polyacrylamide gel electrophoresis and was further confirmed by Western blot. In presence of IL-10, the purified rhICOS significantly increased in vitro B cell growth stimulated by pokeweed mitogen (PWM), and enhanced the secretion of IgG from B cells.

[Analysis of partial sequence of the gene for a novel Br protein].

A strain of halophilic archaeum AB1 was isolated and purified from Aibi Lake located in the north of Xinjiang Uygur Autonomous Region. Partial DNA fragment encoding a bacteriorhodopsin (Br) protein as well as 16S rRNA of AB1 was amplified by PCR, and their nucleotide sequences were determined subsequently. On the basis of homology and phylognetic analysis about 16S rRNA gene (16S rDNA), it could be speculated that the strain AB1 is a novel member of the genus Natronococcus. The hydropathy analysis of Br fragment revealed that the AB1 Br had a transmembrane heptahelical structure similar to that of other Brs. On the other hand, homology alignment using the deduced partial amino acid sequence of Br protein of AB1 with other Br proteins showed that AB1 Br protein is obviously different to others. These facts indicated that the Br in halophilic archaeum AB1 is a new Br protein.

DCA increases the antitumor effects of capecitabine in a mouse B16 melanoma allograft and a human non-small cell lung cancer A549 xenograft.

PURPOSE: Capecitabine is one of the few chemotherapy drugs with high oral availability. Recently, sodium dichloroacetate (DCA) has shown great potential as an anticancer agent. In the present study, we assessed the anticancer effect of DCA in combination with capecitabine for cancers that modestly expressed TP. METHODS: A mouse B16 melanoma allograft and a human non-small cell lung cancer A549 xenograft were used to assess the effect of DCA and capecitabine combined treatment. Histology and immunohistochemistry were used to detect the apoptosis and proliferation of cancer cells. Real-time PCR and Western blot were carried out to detect the expression of TP and caspases, respectively. RESULTS: For the first time, we report that DCA increased the antitumor effects of capecitabine in a mouse B16 allograft and a human A549 xenograft by promoting apoptosis of tumor cells. DCA has little effect on the expression of TP. CONCLUSIONS: Our finding suggests that DCA in combination with capecitabine might be potential as a new therapeutic regimen against some cancers.

Oren-gedoku-to and its constituents with therapeutic potential in Alzheimer's disease inhibit indoleamine 2, 3-dioxygenase activity in vitro.

A well-known traditional Chinese medicinal prescription, Oren-gedoku-to (OGT), has been used in clinical therapies for many types of dementia in China and Japan. Additionally, it ameliorates the age-related deterioration of learning and memory in an Alzheimer's disease (AD) rat model. Indoleamine 2, 3-dioxygenase (IDO-1) is the first and rate-limiting enzyme in the kynurenine pathway of tryptophan catabolism, which ultimately leads to the production of the excitotoxin quinolinic acid (QUIN). IDO-1 has recently been established as one of the key players involved in the pathogenesis of AD. OGT is indicated to prevent cholinergic dysfunction and reduce oxidative stress; however, the exact mechanism underlying its ability to improve cognitive ability remains elusive. Here we present a novel mechanism of OGT's therapeutic potential in AD. We demonstrated that OGT significantly inhibited recombinant human IDO-1 (rhIDO-1) activity in vitro, and its four main constituents (i.e., berberine, palmatine, jatrorrhizine, and baicalein) were potent IDO-1 inhibitors. IC50 values, obtained from a cell-based assay, of HEK 293 cells and an enzymatic assay were much lower than the most commonly used IDO-1 inhibitor, 1-methyl tryptophan (1-MT). Berberine was the best inhibitor and had IC50 values of 7 µM (cell-based assay) and 9.3 µM (enzymatic assay). Jatrorrhizine and palmatine exhibited irreversible inhibition of rhIDO-1, whereas berberine and baicalein behaved as uncompetitive, reversible inhibitors with Ki values of 8 µM and 215 µM, respectively. In conclusion, constituents of OGT show strong IDO-1 inhibitory activity and may have significant therapeutic potential for AD.