OBJECTIVETo clone the pokeweed anti-viral protein (PAP) gene, to express it in Pichia pastoris, and to study the inhibitory effect of PAP on U251 in vitro.

METHODSThe cDNA sequence encoding PAP was cloned by Real-time PCR from Phytolacca americana. The recombinant PAP was subcloned into the expression vector pPICZaA and expressed in Pichia pastoris GSI15 after methanol induction. SDS-PAGE analysis showed that the expressed PAP existed in the yeast culture supernatant. The drug cytotoxicity to U251 cells was assessed using MTT assay and the obvious apoptotic nuclei of the tumor cells detected using the method of single cell gel electrophoresis.

RESULTSThe full-length PAP gene was cloned. The recombinant expression plasmid pPICZaA-PAP was constructed successfully. SDS-PAGE analysis showed that the relative molecular mass (M) of the recombinant protein was about 35 kDa. The degradation of the genome of the apoptotic cells induced by PAP was detected using the method of single cell gel electrophoresis. PAP possessed very high ability to inhibit the growth of U251. The anti-tumor activities (IC50) to U251 cells of PAP was 81.0 microg/mL.

CONCLUSIONPAP could be a potent anti-tumor candidate for inhibiting the growth of U251 and inducing its apoptosis.

Antineoplastic Agents, Phytogenic ; biosynthesis ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Humans ; Pichia ; metabolism ; Ribosome Inactivating Proteins, Type 1 ; biosynthesis ; pharmacology

OBJECTIVETo clone luffin-a cDNA from the seeds of Luffa cylindrical, and to obtain bioactive recombinant luffin-a protein using the expression vector pET-44a (+) in E. coli.

METHODSThe cDNA sequence encoding luffin-a was cloned from the fresh seeds of Luffa cylindrical by RT-PCR. The target DNA fragments were sequenced after T-A cloning. The luffin-a expression plasmid was constructed by inserting the luffin-a cDNA fragment into vector pET-44a (+). Luffin-a was expressed in E. coli by addition of IPTG into final concentration 1.0 mmol/L. The recombinant luffin-a was identified by SDS-PAGE. The biological activity of luffin-a protein was evaluated by using the MTT assay in HepG2 cells following fluid-phase endocytosis.

RESULTSIn comparison with the reported luffin-a, the homology of nucleotide sequence of the cloned luffin-a gene was 99.73%, while their amino acid sequences were identical. The solubility of recombinant protein was analyzed by SDS-PAGE, and the luffin-a was mainly produced in inclusion bodies. The recombinant luffin-a, renatured by dialysis of the denatured products, showed a similar cytotoxicity to ricin A chain.

CONCLUSIONThe cDNA of luffin-a has been successfully cloned. The recombinant luffin-a protein expressed by E. coli is bioactive.

Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Luffa ; chemistry ; Molecular Sequence Data ; Plant Proteins ; biosynthesis ; genetics ; Polymerase Chain Reaction ; Ribosome Inactivating Proteins, Type 1 ; Seeds ; chemistry

OBJECTIVETo construct mature protein curcin gene prokaryotic expression vectors in Escherichia coli and choose the optimal inducing condition of the recombinant strains.

METHODThe gene encoding of curcin was amplified from the genome of Jatropha curcas seeds by PCR and cloned into the expression vectors pQE-30 and pET-32 obtaining recombinant vectors pQE-R and pET-R respectively. The two vectors were transferred into E. coli BL21 (DE3) and the recombinant strains PRM and PRB were attained respectively. PRM and PRB were induced by different revulsants and under different temperature and time.

RESULTThe gene encoding of mature protein curcin was amplified by PCR and the recombinant strains PRM and PRB were obtained.

CONCLUSIONThe results showed that PRB could not produce recombinant protein under such conditions. However, PRM could highly produce recombinant protein induced by 0.5 mmol x L(-1) IPTG at 28 degrees C for 6 h.

Cloning, Molecular ; Escherichia coli ; genetics ; Gene Expression ; drug effects ; Genetic Vectors ; genetics ; metabolism ; Genome, Plant ; genetics ; Jatropha ; genetics ; Ribosome Inactivating Proteins, Type 1 ; biosynthesis ; genetics ; Temperature

The cDNA sequence encoding pokeweed antiviral protein-II was cloned from the fresh summer leaves of phytolacca amercana by RT-PCR. The recombinant PAP-II was subcloned into the expression vector pET-28a(+) and expressed in E. coli BL21 after IPTG induction. SDS-PAGE analysis showed that the expressed PAP-II existed in the form of inclusion bodies. The purified fusion protein was obtained after a series of steps including cell break, inclusion body solubilization, protein refolding and purification through BBST NTA resin column. The non-radioactive ELISA-based HIV-1 integrase assay showed that the recombinant pokeweed antiviral protein-II and RTA were able to inhibit HIV-1 integrase to some extent (IC50 = 303 microg/mL, 220 microg/mL respectively). MTT assay showed that cytotoxicity of pokeweed antiviral protein II for HEP-G2 cells and Hela cells was in a dose-dependent manner with IC50 s of 93 microg/mL and 102 microg/mL, respectively. The results suggested that pokeweed antiviral protein-II is a potent anti-tumor candidate. The finding of integrase inhibitory activity and the discovery of cytotoxicity provide more insights into the anti-HIV and the anti-tumor activities of PAP-II.
Cloning, Molecular ; HIV Integrase ; drug effects ; HeLa Cells ; Humans ; Phytolacca americana ; genetics ; Plant Leaves ; genetics ; Plasmids ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology ; Ribosome Inactivating Proteins, Type 1 ; genetics ; isolation & purification ; pharmacology

OBJECTIVETo develop an ELISA-based method for analyzing biologic activities of HIV-1 integrase and for high throughput screening of integrase inhibitors.

METHODSAfter expression, renaturation and purification of integrase, the bioactivity of integrase and the inhibition of luffin-a were evaluated with an in vitro assay based on biotin-avidin EILSA and chemiluminescent substrates.

RESULT(1) The specific activity of the purified integrase was 54.92 units/mg of protein. (2)IC(50) (concentration causing 50% inhibition of integrase) of luffin-a was (0.63 +/- 0.026) micromol/L.

CONCLUSIONThe non-radioactive assay can be used for analysis of bioactivities and high throughput screening of inhibitors of HIV-1 integrase.

Catalysis ; drug effects ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; methods ; HIV Integrase ; chemistry ; metabolism ; Humans ; Kinetics ; Luminescent Measurements ; Ribosome Inactivating Proteins, Type 1 ; pharmacology ; Substrate Specificity

Eight saporin peaks were obtained from the purification of seed extracts of Saponaria officinalis L. Saporin peak No. 6 (SAP-6) showed the highest activity in the inhibition of protein synthesis (98%) in an in vitro translation study. An immunotoxin (IT) was prepared from SAP-6 conjugated to a monoclonal anti-CEA antibody 26/5/1 (mab B) using N-succinimidyl pyridyl dithiopropionate (SPDP) and 2-iminothiolane as a cross linker. Under thermal stability study by a DSC (differential scanning calorimetry), the IT showed a denature temperature of 75 degrees C. In in vitro translation studies, the purified IT showed the same activity as SAP-6 at 10(-7) M and 10(-9) M protein concentration at 0, 30 and 60-min incubation effects with mab B and SAP-6 not conjugated at 24-hr incubation periods on human promyelocytic cell line HL 60 and on human colon adenocarcinoma cell lines which were SW 403, LoVo and LS 174 T. SAP-6, mab B and IT had no cytotoxic effect on HL-60. The IT showed a higher cytotoxic effect than SAP-6 in CEA-positive cell lines. The IT demonstrated the highest cytotoxic effect of 51% inhibition of control at 10(-7) M on the LS 174 T.
Antibodies, Monoclonal/*therapeutic use ; Antineoplastic Agents, Phytogenic/administration & dosage/*pharmacology ; Carcinoembryonic Antigen/biosynthesis/*immunology ; Colonic Neoplasms/*pathology/therapy ; Hot Temperature ; Humans ; Immunotoxins/*therapeutic use ; *N-Glycosyl Hydrolases ; Plant Proteins/administration & dosage/*pharmacology ; Ribosome Inactivating Proteins, Type 1 ; Tumor Cells, Cultured/drug effects

We evaluated the effect of the conjugate of basic fibroblast growth factor (FGF2) and saporin (FGF2-SAP) on proliferation of cultured keratocytes. Cultured rabbit and human keratocytes were incubated in medium containing 0.01 to 100 nM of chemical conjugate of EGF2 conjugated by disulfide bond to saporin (CCFS1), FGF2 genetically fused to saporin (rFGF2-SAP), FGF2, or saporin for three hours or four days and cell proliferation was quantified four days after the drug treatment. Proliferation of rabbit and human keratocytes was effectively inhibited by three hour and by four day exposure to CCFS1 and rFGF2-SAP in a dose-dependent manner, whereas it was affected minimally by four day exposure to saporin. Their inhibitory effects were detected at concentrations above 0.1 or 1 nM, and were most prominent in serum-stimulated rabbit keratocytes. These results suggest a potential role for FGF2-SAP in limiting proliferation of keratocytes during corneal wound healing.
Animals ; Antineoplastic Agents, Phytogenic/*pharmacology ; Cell Division/drug effects ; Cell Line ; Cells, Cultured ; Corneal Stroma/*cytology/drug effects ; Dose-Response Relationship, Drug ; Fibroblast Growth Factor 2/*pharmacology ; Humans ; Immunotoxins/pharmacology ; *N-Glycosyl Hydrolases ; Plant Proteins/*pharmacology ; Rabbits ; Ribosome Inactivating Proteins, Type 1

PURPOSE: Reduced brain glucose metabolism and basal forebrain cholinergic neuron degeneration are common features of Alzheimer's disease and have been correlated with memory function. Although regions representing glucose hypometabolism in patients with Alzheimer's disease are targets of cholinergic basal forebrain neurons, the interaction between cholinergic denervation and glucose hypometabolism is still unclear. The aim of the present study was to evaluate glucose metabolism changes caused by cholinergic deficits. MATERIALS AND METHODS: We lesioned basal forebrain cholinergic neurons in rats using 192 immunoglobulin G-saporin. After 3 weeks, lesioned animals underwent water maze testing or were analyzed by 18F-2-fluoro-2-deoxyglucose positron emission tomography. RESULTS: During water maze probe testing, performance of the lesioned group decreased with respect to time spent in the target quadrant and platform zone. Cingulate cortex glucose metabolism in the lesioned group decreased, compared with the normal group. Additionally, acetylcholinesterase activity and glutamate decarboxylase 65/67 expression declined in the cingulate cortex. CONCLUSION: Our results reveal that spatial memory impairment in animals with selective basal forebrain cholinergic neuron damage is associated with a functional decline in the GABAergic and cholinergic system associated with cingulate cortex glucose hypometabolism.
Acetylcholine/metabolism ; Alzheimer Disease ; Animals ; Antibodies, Monoclonal/*pharmacology ; Basal Forebrain/*drug effects/metabolism ; Cholinergic Agents/administration & dosage/*pharmacology ; Cholinergic Neurons/*drug effects/metabolism ; Fluorodeoxyglucose F18 ; GABAergic Neurons/*drug effects/metabolism ; Glucose/*metabolism ; Gyrus Cinguli/*drug effects/metabolism ; Humans ; Injections ; Maze Learning ; Motor Activity/physiology ; Positron-Emission Tomography ; Rats ; Ribosome Inactivating Proteins, Type 1/*pharmacology

OBJECTIVETo investigate the effects of different PAP domains on hepatitis B virus replication.

METHODSThe full length and two truncated PAP mutants were cloned into a eukaryotic expression plasmid, and were transfected into HepG2.2.15 cells using lipofectamine 2000. 3 days after transfection, the medium and cells were collected. HBsAg and HBeAg were measured using ELISA. The titers of HBV DNA were quantified using fluorogenic quantitative PCR (FQ-PCR). HepG2 cells were used to determine the cytotoxicity of the plasmids transfection by MTT assays.

RESULTSThe inhibitory effect on HBV replication of the C-terminal 25 amino acids deleted PAP mutant (pXF3H-PAP14) was not significantly different from that of the full length PAP (pXF3H-PAP12) (Chi-square test = 0.5, 2.0, 0.02, probability value more than 0.05), however, the cytotoxicity of pXF3H-PAP14 was lower than that of pXF3H-PAP12 (Chi-square test = 7.7, probability value less than 0.01). Both N-terminal 69 amino acids deleted mutant and C-terminal 25 amino acids deleted mutant had no cytotoxicity and no antiviral activity.

CONCLUSIONC-terminal 25 amino acid of PAP is related to cytotoxicity but not related to antiviral activity of PAP. N-terminal 69 amino acid of PAP is related to the anti-HBV effect of PAP.

Amino Acid Sequence ; Antiviral Agents ; pharmacology ; Blotting, Western ; DNA, Viral ; drug effects ; metabolism ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; drug effects ; genetics ; physiology ; Humans ; Liposomes ; Plasmids ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Ribosome Inactivating Proteins, Type 1 ; genetics ; metabolism ; pharmacology ; Sequence Deletion ; Transfection ; Virus Replication ; drug effects

OBJECTIVE: Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells. METHODS: A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined. RESULTS: The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells. CONCLUSION HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.
Animals ; Dependovirus/genetics* ; Genetic Therapy ; Genetic Vectors/genetics* ; HEK293 Cells ; Humans ; Mice ; MicroRNAs/genetics* ; Trichosanthin