Restriction Enzymes XhoI Research Articles

Cloning and Optimization of Intracellular Expression of Human Interferon β-1a in Pichia pastoris GS115.

Interferon-beta (IFN-β) is a cytokine with a wide range of biological and pharmaceutical applications, including multiple sclerosis (MS), cancer, some autoimmune disorders, and viral infectious diseases. Thus, many studies have been performed to develop novel strategies for the high-yield production of functional IFN-β in a cost-effective approach. Here, we aimed to improve the intracellular expression of IFN-β-1a in Pichia pastoris. The gene of IFN-β-1a was successfully sub-cloned into the pPICZA vector. The recombinant vector was transfected to P. pastoris GS115 cells by electroporation. After screening positive P. pastoris transformants, the expression of IFN-β-1a was evaluated and the cultivation conditions, including temperature, time of incubation, and methanol concentration, were optimized. The protein expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The double digestion with EcoRI and XhoI restriction enzymes and sequence analysis confirmed the correct sub-cloning of the IFN-β-1a gene into pPICZA. SDS-PAGE analysis showed that the highest level of IFN-β-1a (25 mg per 1 L of yeast culture) was produced with 2% methanol at 28°C after 72 h incubation. Optimization of cultivation conditions for intracellular expression of IFN-β-1a was successfully performed. This approach can be generally applied to improve the production yield and quality of other recombinant proteins in P. pastoris.

A reverse vaccinology and immunoinformatics approach for designing a multiepitope vaccine against SARS-CoV-2.

Since 2019, the world was involved with SARS-CoV-2 and consequently, with the announcement by the World Health Organization that COVID-19 was a pandemic, scientific were an effort to obtain the best approach to combat this global dilemma. The best way to prevent the pandemic from spreading further is to use a vaccine against COVID-19. Here, we report the design of a recombinant multi-epitope vaccine against the four proteins spike or crown (S), membrane (M), nucleocapsid (N), and envelope (E) of SARS-CoV-2 using immunoinformatics tools. We evaluated the most antigenic epitopes that bind to HLA class 1 subtypes, along with HLA class 2, as well as B cell epitopes. Beta-defensin 3 and PADRE sequence were used as adjuvants in the structure of the vaccine. KK, GPGPG, and AAY linkers were used to fuse the selected epitopes. The nucleotide sequence was cloned into pET26b(+) vector using restriction enzymes XhoI and NdeI, and HisTag sequence was considered in the C-terminal of the construct. The results showed that the proposed candidate vaccine is a 70.87 kDa protein with high antigenicity and immunogenicity as well as non-allergenic and non-toxic. A total of 95% of the selected epitopes have conservancy with similar sequences. Molecular docking showed a strong binding between the vaccine structure and tool-like receptor (TLR) 7/8. The docking, molecular dynamics, and MM/PBSA analysis showed that the vaccine established a stable interaction with both structures of TLR7 and TLR8. Simulation of immune stimulation by this vaccine showed that it evokes immune responses related to humoral and cellular immunity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00251-021-01228-3.

Evaluation and Comparison of Clostridium Epsilon-Alpha Fusion Gene Expression Using Different Commercial Expression Vector.

Clostridium perfringens and Clostridium septicum are gram-positive, anaerobic, spore-forming rods and pathogens for humans and livestock, which are widespread in nature as well as human and animal digestive systems. C. perfringens produces numerous different exoproteins, which are various systems of action. The major C. perfringens toxins include alpha, beta, epsilon, and iota. C. perfringens are classified into five groups (A-E) on the basis of the production of these lethal toxins. Furthermore, toxins secreted from C. septicum include alpha, beta, delta, and gamma. Epsilon and alpha toxins of C. perfringens and C. septicum are the major causes of enterotoxemia and braxy in sheep and goats, respectively. The production of recombinant immunogenic proteins of these bacteria using suitable expression vectors and expression prokaryotic hosts can be a convenient method for the reduction of the costs and production time of clostridial anaerobic vaccines. In the present study, recombinant Escherichia coli strain TOP10 containing pJETεα was used for the evaluation of C. perfringens type D and C. septicum epsilon-alpha fusion protein using different commercial vectors. After the extraction of pJETεα from the recombinant cell, it was digested by NdeI and XhoI restriction enzymes and subcloned into pET22b (+), pET26b (+), and pGEM-B1 expression vectors in E. coli/Rosetta and E. coli/BL21 (DE3). The expression of recombinant fusion toxin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting in three different temperatures, various isopropyl β-D-1-thiogalactopyranoside (IPTG) gradients, and different times using pGEMεα, pET22εα, and pET26εα vectors in E. coli/Rosetta and E. coli/BL21 (DE3). According to the obtained results, recombinant E. coli/Rosetta/pET22εα showed better expression at a temperature of 37°C after 6 h of induction by IPTG.

Expression, purification, crystallization and preliminary X-ray crystallographic studies of a mitochondrial membrane-associated protein Cbs2 from Saccharomyces cerevisiae

BackgroundMitochondria are unique organelles that are found in most eukaryotic cells. The main role of the mitochondria is to produce ATP. The nuclear genome encoded proteins Cbs1 and Cbs2 are located at the mitochondrial inner membrane and are reported to be essential for the translation of mitochondrial cytochrome b mRNA. Genetic studies show that Cbs2 protein recognizes the 5′ untranslated leader sequence of mitochondrial cytochrome b mRNA. However, due to a lack of biochemical and structural information, this biological process remains unclear. To investigate the structural characteristics of how Saccharomyces cerevisiae (S. cerevisiae) Cbs2 tethers cytochrome b mRNA to the mitochondrial inner membrane, a preliminary X-ray crystallographic study was carried out and is reported here.MethodsThe target gene from S. cerevisiae was amplified by polymerase chain reaction. The PCR fragment was digested by the NdeI and XhoI restriction endonucleases and then inserted into expression vector p28. After sequencing, the plasmid was transformed into Escherichia coli C43 competent cells. The selenomethionine derivative Cbs2 protein was overexpressed using M9 medium based on a methionine-biosynthesis inhibition method. The protein was first purified to Ni2+-nitrilotriacetate affinity chromatography and then further purified by Ion exchange chromatography and Gel-filtration chromatography. The purified Se-Cbs2 protein was concentrated to 10 mg/mL. The crystallization trials were performed using the sitting-drop vapor diffusion method at 16 °C. The complete diffraction data was processed and scaled with the HKL2000 package and programs in the CCP4 package, respectively.ResultsCbs2 from S. cerevisiae was cloned, prokaryotic expressed and purified. The analysis of the size exclusion chromatography showed that the Cbs2 protein peaked at a molecular weight of approximately 90 KDa. The crystal belonged to the space group C2, with unit-cell parameters of a = 255.11, b = 58.10, c = 76.37, and β = 95.35°. X-ray diffraction data was collected at a resolution of 2.7 Å. The Matthews coefficient and the solvent content were estimated to be 3.22 Å 3 Da-1 and 61.82%, respectively.ConclusionsIn the present study Cbs2 from S. cerevisiae was cloned, expressed, purified, and crystallized for structural studies. The molecular weight determination results indicated that the biological assembly of Cbs2 may be a dimer.The preliminary X-ray crystallographic studies indicated the presence of two Cbs2 molecules in the asymmetric unit. This study will provide an experimental basis for exploring how Cbs2 protein mediates cytochrome b synthesis.

Expression, Purification, and Bioactivity of a Soluble Recombinant Ovine Interferon-tau in Escherichia Coli.

IntroductionOvine interferon-tau (oIFN-τ) is a newly discovered type I interferon. This study used biochemical techniques to transform the oIFN-τ gene into Escherichia coli to obtain the mass and soluble expression of the recombinant protein.Material and MethodsFirst, total RNA was extracted from fresh sheep embryonic tissues with TRIzol reagent and then used as a template to reverse transcribe and amplify the mature oIFN-τ gene with RT-PCR. The amplified product was next digested with the HindIII and XhoI restriction enzymes and inserted into the pET-32a(+) vector to construct the prokaryotic expression plasmid. The corrected in-frame recombinant plasmid, pET-32a(+)-oIFN-τ, was transformed into E. coli Rosetta (DE3) competent cells. After induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant protein was detected in bacteria. Finally, the bacteria were lysed by sonication, and the recombinant protein was purified by nickel affinity chromatography and DEAE anion exchange chromatography.ResultsThe protein was confirmed to be oIFN-τ, which mainly existed in the soluble lysate fraction, as proven by SDS-PAGE and Western blot assays.ConclusionPurified IFN-τ exists mostly in a soluble form, and its anti-vesicular stomatitis virus (VSV) activity reached 7.08×10(6)IU/mL.

Discrimination of Kratom Products by an Improved PCR-RFLP Method

In Japan, mitragynine, 7-hydroxymitragynine and Mitragyna speciosa Korth. (M. speciosa, "Kratom") were controlled as Designated Substances under the Pharmaceutical and Medical Device Act from March 2016. In this study, the origins of 16 Kratom products obtained from the illegal drug market in Japan were investigated by DNA analyses and LC-MS analyses. When the PCR-restriction fragment length polymorphism (RFLP) was performed using the restriction enzyme XmaI (as reported by Sukrong et al. to be able to distinguish M. speciosa), the same DNA fragment patterns were obtained from all 16 products. On the other hand, as a result of the identification of the plant species of each product by nucleotide sequence analyses, the sequences of M. speciosa were detected in only 14 products. Despite the facts that mitragynine and 7-hydroxymitragynine were detected also in the other two products by the LC-MS analyses, M. speciosa DNAs were not amplified from these products by the PCR. Moreover, the DNA amplicons of the other psychotropic plant (Mesembryanthemum sp., e.g. "Kanna") were detected. This plant PCR amplicon has the restriction site for the XmaI at the same position of the M. speciosa PCR amplicon and it is difficult to distinguish "Kratom" and "Kanna" by the conventional PCR-RFLP. When the restriction enzyme XhoI was used simultaneously with the Xmal, the specific DNA fragment was only observed from the M. speciosa amplicon and it was possible to distinguish both species using this improved PCR-RFLP method. This method is useful to identify the origin of Kratom products distributed in the illegal drug market.

Signal Peptide Optimization, Cloning, Expression and Characterization of Ce16B Cellobiohydrolase in Lactococcus lactis

Cellobiose is one of the most abundant carbohydrates available in nature which can be used as biofuel in various applications such as food industry, energy production, paper production, etc. with no harm to the environment. One way for a safe decomposition the cellobiose is enzymatic hydrolysis. Cellobiohydrolase enzyme, which is naturally present in some yeasts and bacteria, is capable of decomposing carbohydrates. Because of its low production in its original organism and also high volumes of requirements to this enzyme for industrial usage, there is strong desired to mass-produce of this enzyme using biotechnological approaches. In this study, several signal peptide sequences with Lactococcus originality, with the aim of binding to the cellobiohydrolase gene, were evaluated. In this regard, signalP server, a valid and accurate online tool, was used to determine the most important details including N-terminal (n), hydrophobic (h) and carboxy-terminal (c) regions of signal peptides and their probability. Thereafter, Portparam and Solpro online tools were used to investigate the physico-chemical properties of each signal peptide. Finally, the best peptide signal with the best specificity and the highest D-score was selected for binding to the cellobiohydrolase gene sequence aimed at expression in the Gram-positive bacterium Lactococcus lactis. Final frame of target gen was synthetized and ligated in pBU003 expression vector using double digestion by SalI and XhoI restriction enzymes. Recombinant expression vector was transferred to MC1061 for replication and then transferred into the MG1363 as expressing strain of Lacticoccus lactis. Recombinant proteins were purified from M17 medium using the Ni-NTA Agarose column. Their quantification and qualification examined through Bradford assay and 12% SDS-PAGE respectively. Phosphoric acid-treated cotton as a substrate was used to determine the Ce16B cellobiohydrolase activity at 450 nm absorbance. In silico investigating the signal peptides have led to the choice of the USP45 signal peptide as a powerful secretory signal. Recombinant proteins were successfully expressed in Lactococcus lactis. The concentration of each recombinant protein was determined 0.7 g/l (R2 = 0.989). In vitro assessment of enzymatic activity or recombinant proteins on PC showed 1.89 U (R2: 9941).

Potentiation Effect Of 5FU by Fragaceatoxin C Pore-Forming Toxin in MCF-7 Cell Line

Introduction: Chemotherapy has been restricted due to the high-dose side effects. In the present study, acceleration of the chemotherapeutic drug (5FU) entrance into MCF-7 cells has been explored by using a recombinant form of Fragaceatoxin C (FraC) pore-forming toxin. Methods: In this experimental study, the gene for FraC toxin was order from a commercial source and was sub-cloned into pET28a vector using NcoI and XhoI restriction enzymes. Expression of the protein was induced by IPTG and purification was performed using Ni-NTA affinity chromatography. Hemolysis was tested using a turbidimetric assay. Cytotoxic and potentiation effects of the recombinant toxin on 5FU chemotherapeutic drug were analyzed using trypan blue and MTT assays. T-student test was used to analyze the data using the SPSS v16 software. Significance was defined as P≤0.05 Results: Recombinant FraC toxin was produced in E. coli with 86% purity. Cytotoxic effects of the FraC toxin in MCF-7 cells were dose and time-dependent. the IC50 of FraC toxin on MCF-7 cells was 3.3 mg/ml. Non-toxic concentrations of recombinant FraC toxin enhanced cytotoxic effects of 5FU by a factor of 4 to 6 in different exposure times (24-72 h). Conclusion: Potentiation of the 5FU chemotherapeutic effect by acceleration of its entrance into cancer target cells using FraC pore-forming toxin can reduce its side effects of the chemotherapy.

Molecular characterization of Babesia microti seroreactive antigen 5-1-1 and development of rapid detection methods for anti-B. microti antibodies in serum

Babesiosis has become a new global threat impacting human health, and most human babesiosis cases are caused by Babesia microti. Until now few antigens of B. microti have been described which can be used for the diagnosis of human babesiosis. In the present study, we report on the bioinformatic analysis, cloning and expression of the sequence encoding the B. microti seroreactive antigen 5-1-1 to investigate its potential incorporation in serologic diagnostic tools for babesiosis. Bioinformatic analysis and recombinant gene expression were performed to molecularly characterize seroreactive antigen 5-1-1. Enhanced chemiluminescence (ECL)-Western blot methods were used to detect specific antibodies in infected mice. Immunofluorescence antibody assays (IFA) were performed to detect the localization of BmSA5-1-1 in B. microti parasites. ELISA and immunochromatographic (ICT) tests were developed using recombinant BmSA5-1-1 to evaluate its potential use in rapid detection methods for B. microti antibodies and for the diagnosis of babesiosis. A recombinant expression plasmid was constructed by inserting the target gene fragment in the pET28a vector after double digestion with BamHI and XhoI restriction enzymes. The recombinant BmSA5-1-1 protein was expressed in Escherichia coli (rBmSA5-1-1) and purified by means of Ni-nitrilotriacetic acid (NTA) agarose columns. Polyclonal antibodies were generated against rBmSA5-1-1. Based on indirect immunofluorescence assay results, BmSA5-1-1 appeared to localize on the surface of B. microti. ELISA tests using the rBmSA5-1-1 antigen detected specific antibodies from infected mice as early as 4 days post-infection. Our results indicate that the two methods we developed can detect specific antibodies in mice at different stages of infection with sensitivities of 100% (rBmSA5-1-1 ELISA) and 90% (ICT). The specificity of the two methods was 100%. Sera of patients suffering from other closely related parasitic diseases, such as malaria and toxoplasmosis, produced negative results. In conclusion, seroreactive antigen 5-1-1, a member of the BMN1 protein family, is expressed on the outer surface of B. microti and is a promising candidate antigen for the early diagnosis of babesiosis. rBmSA5-1-1 ELISA and ICT methods show good potential for detecting specific antibodies in mice at different stages of infection.

SUB-CLONING OF GENES ENCODINGCYTHOCHROME P450 MONOOXYGENASE (CYP71AVI) INTOEXPRESSION VECTOR IN ESCHERICHIA COLI

Objective: Cytochrome P450 monooxygenase (CYP71AVI) is a key enzyme involved in the artemisinin biosynthesis pathway.In this research, sub-cloning gene encoding CYP71AVI into pETDUET1 vector in Escherichia coli has been done and then the expression products characterized with SDS-PAGE.Methods: Gene construction started with sub-cloning of cyp71avi gene from pJexpress401_cyp into pETDUET1 through restriction site NdeI and XhoI to get pETDUET1_cyp. Overproduction of CYP71AVI at temperature 37 °C has conducted by IPTG induction.Results: Confirmation of the recombinant vector pETDUET1_cyp was done by migration, restriction site and sequencing analysis. The result of pETDUET1_cyp restriction analysis with XhoI restriction enzyme showed one DNA band with experimental size 6585 bp.The CYP71AVI protein has been produced and characterized with SDS-PAGE method. Based on experimental calculation from SDS-PAGE analysis obtained molecular weight of CYP71AVI band was 57.55 kDa.Conclusion: Construction of gene encoding CYP71AVI into pETDUET1 as the co-expression vector in Escherichia colihas been succesfully and confirmed by migration, restriction site and sequencing analysis. The result of overproduction showed protein bands on SDS-PAGE analysis indicated as CYP71AVI.

Abstract 4794: Oligoamide-based mimics of double-stranded B-DNA as a new class of DNA topoisomerase I catalytic inhibitors

Abstract Since the discovery of Topoisomerase I as a specific target for the treatment of cancers more than 30 years ago, only two inhibitors derived from the natural compound camptothecin (CPT) have been approved in the clinic for the treatment of colon, lung and ovarian cancers: topotecan and irinotecan. The cytotoxicity of these Top1 poisons relies on their capability to stabilize covalent Top1-DNA complexes, leading to replication-mediated DNA double-strand breaks. However tumor cells develop multiple resistance mechanisms that are limiting their efficacy. Though new CPT derivatives and other Top1 poisons with various chemical structures have been developed, they share the same resistance mechanisms. Aside from conventional approaches focus on compounds with higher potency to stabilize Top1-DNA complexes, we investigated the possibility to inhibit Top1 binding to DNA and/or DNA cleavage by using DNA mimics that would act as decoys, a strategy that was never explored before. To this aim, single chain oligoamides composed by iteration of mQQ units (Q: 8-amino-2-quinoline carboxylic acid; mQ: 8-aminomethyl-2-quinoline carboxylic acid) were synthesized. These molecules fold into helices that are stabilized by electrostatic repulsions and hydrogen bonds between the amide functions and endocyclic nitrogen atoms at adjacent residues. When Q and mQ precursors are functionalized by negatively charged residues, the positions of these residues in the folded structure match the position of phosphate residues in duplex B-DNA. Because distances between residues in mQQ and QmQ units are slightly different, it is possible to define a major groove and a minor groove, as in B-DNA, with the possibility to impact on groove width by changing the positions of the substituents (Q4 or Q5 for substitution in position 4 or 5 of the quinoline monomer, respectively), clearly establishing (mQQ5)n and (mQQ4)n as potential DNA-mimics of an unprecedented kind. We found that both (mQQ5)n and (mQQ4)n oligomers inhibited the activity of Top1-mediated relaxation of supercoiled DNA in vitro. Inhibition increased with the length of the oligoamide to reach an IC50 value (concentration inhibiting 50% of Top1-mediated DNA relaxation) around the nM range for the longest oligomer that was tested i.e. (mQQ4)16, corresponding to a double-strand DNA of 16 base pairs. By comparison, CPT had an IC50 of ∼10 μM in the same conditions. Top1 inhibition was rather selective as a (mQQ4)8 oligomer moderately inhibited Top2-mediated activity and did not affect the activity of various DNA interacting enzymes such as restriction enzymes XhoI and NdeI or nucleases such as DNAse I, S1 nuclease, benzonase or Flap-endonuclease I. Because these oligomers are resistant to proteases and nucleases and can be synthesized rapidly by solid phase synthesis with the possibility to modify its substituents without altering their helicity, they represent good candidates for drug development. Citation Format: Philippe Pourquier, Krzysztof Ziach, Céline Chollet, Vincent Parissi, Mathieu Marchivie, Panchami Prabhakaran, Partha P. Bose, Katta Laxmi-Reddy, Frédéric Godde, Stéphane Chaignepain, Jean Marie Schmitter, Ivan Huc. Oligoamide-based mimics of double-stranded B-DNA as a new class of DNA topoisomerase I catalytic inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4794.

Cloning and investigating GFP (green fluorescent protein) allowing higher expression in Bacillus subtilis

Gfp gene coding for protein GFP (green fluorescent protein) from the jellyfish Aequorea victoria is the most popular indicator protein, due to (i) easy to recognize the expression through the fluorescent ability, (ii) without substrate or cofactor, (iii) less impact on the fusion protein target form. A lot of modifications of GFP have been studied and improved, in which GFP superfolder containing beneficial properties such as rapid folding, structural stability and strong fluorescence showed great applicability. However, GFP superfolder has just been developed in Escherichia coli. It is difficult to apply this version for all other strains since codon usage and the folding ability of each host species. In this study, with the aim of generating a GFP gene which is suitable for Bacillus subtilis, we mutated in the gene gfp+ to delete general BamHI, XhoI restriction enzyme site for facilitating in cloning step; as well asmodified Y39N, N105T, I171V codon that is compatible with B. subtilis. The results showed that the mutations have made the new version of GFP stronger in fluorescence and sequences of restriction enzymes have also been removed.

Generation of divalent DNA vaccine based on p39 and shiga-like toxin 2 (Stx2) genes

The virulence factors such as shiga-like toxin (Stx) and immunogenic P39 protein in Escherichia coli and Brucella melitensis are related to disease of digestive system in human worldwide. In the present study the stx2 and p39 genes were cloned into expression plasmid pEEF1D-FLAG (pcDNA 3.1+) as a divalent DNA vaccine candidate. The Enterohemorrhagic E. coli ATCC 3081 and smooth virulent B. melitensis strain M5 were obtained and cultured on specific media. Bacterial DNA was extracted from colonies and was used for p39 and stx2 genes amplification by PCR. The amplified products on 2% agarose gel electrophoresis were revealed 285 and 1220 bp fragments for stx2 and p39 genes, respectively. Each amplified genes were T/A cloned into pGEM-T easy vector and pGEM-T-stx2 and pGEM-T-p39 were produced. The stx2 and p39 genes were sub-cloned in linearized expression vector (pcDNA 3.1+) using HindIII, XhoI and XbaI restriction enzymes and pCDNA3-stx2-p39 was generated. This final construct was confirmed by PCR and enzymes digestion. The results were showed stx2 and p39 genes were sub-cloned, successfully into pcDNA 3.1+ to generate pcDNA 3.1+-stx2-p39 recombinant vector. According to these findings novel recombinant pcDNA 3.1+-stx2- p39 construct that was produced in this study could be useful as DNA vaccine candidate in animal models against shiga-like toxin producing E. coli and virulence B. melitensis strains in future studies.

Molecular fingerprinting of peppermint (Mentha piperita) and some Mentha hybrids by sequencing and RFLP analysis of the 5S rRNA Non-Transcribed Spacer (NTS) region

Hybridization of species belonging to the genus Mentha is quite common. However, the indicators of hybridity are many and make Mentha hybrids' identification difficult. By using the same molecular strategy that allowed us to unequivocally identify some Mentha species, we amplified the Not-Transcribed-Spacer (NTS) of the 5S-rRNA gene to characterize the industrial crop peppermint, M. × piperita and some important Mentha interspecific hybrids: M. × dalmatica, M. × dumetorum, M. × rotundifolia, M. × maximilianea, M. × smithiana, M. × verticillata, M. × villosa. DNA amplification, sequence and cluster analysis revealed differences in the 5S-rRNA NTS region of Mentha hybrids. Peppermint and all other hybrids were unequivocally discriminated by RFLP analysis by using TaqI restriction enzyme, while a further discrimination between M. × dumetorum and M. × verticillata was obtained by XhoI restriction enzyme. Essential oil composition showed clustering patterns similar to DNA fingerprint, with a clear discrimination between plants producing menthofuran (e.g. M. aquatica and its related hybrids, including peppermint) and those containing piperitenone oxide (M. longifolia and its related hybrids).

Development and Practical Use of RT-PCR for Seed-transmitted Prune dwarf virus in Quarantine.

Among imported plants, seeds are the items that have many latent pathogens and are difficult to inspect. Also, they are the import and export items whose market is expected to expand. The biggest problem with seeds is viruses. Prune dwarf virus (PDV) is the virus that is commonly inspected in Prunus cerasifera, P. persica, P. armeniaca, P. mandshurica, P. cerasus, P. avium or P. serotina seeds. In this study, two RT-PCR primer sets, which can promptly and specifically diagnose plant quarantine seed-transmitted PDV, were developed; and nested PCR primers, where products amplify 739 and 673 nucleotides (nt), and an nested PCR-product, 305 nt, can be obtained as these products are amplified again, were developed. Also, a modified-positive control plasmid was developed, where the restriction enzyme XhoI, which can identify the contamination of samples from the control, was inserted. The method developed in this study has detected PDV in 18 cases since 2007, and is expected to continuously contribute to the plant quarantine in Korea.

Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity

The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments.

Role of nuclear factor of activated T-cells in regulating ADAMTS -4 promoter activity in nucleus pulposus cells

Objective To observe the regulatory effect of nuclear factor of activated T-cells( NFAT)-1,4,5 on ADAMTS-4 promoter activity in nucleus pulposus cells,so as to discuss the underlying molecular mechanism of intervertebral disc degeneration. Methods The ADAMTS-4-pβ-gal-Basic was inserted into PGL3-Basic vector after double digestion by the restriction enzyme XhoⅠ and HindⅢ. The purified PGL3-ADAMTS-4 plasmid was obtained through screening. Rat nucleus pulposus cells were cultured in vitro and,via Lipofectamine2000 transfection reagent,transfected with NFAT-1,NFAT-4,NFAT-5 or DN-TonEBP expressing plasmids with or without appropriate backbone vector and 175 ng ADAMTS-4 promoter. The normal cells were taken as controls and transfected cells were taken as treatment group. Dual-LuciferaseTMreporter assay system was used to detect the effect of NFAT-1,4,5( TonEBP) on ADAMTS-4 promoter activity. Results We successfully constructed PGL3-ADAMTS-4 plasmid and identified two NFAT-Bind elements in the promoter. Compared with the control group,ADAMTS-4 promoter activity was significantly inhibited in NFAT-1 group( P 0. 05). While the activities of ADAMTS-4 promoter in NFAT-4,NAFT-5 and DN-TonEBP groups showed no significant changes( P 0. 05). Conclusion NFAT-1 can regulate ADAMTS-4 expression,which may play a role in modulating aggrecan content in the intervertebral disc physiology and / or intervertebral disc degeneration,providing a new strategy for biological treatment of intervertebral disc degeneration.

Thermal and Spectroscopic Characterization of a Proton Pumping Rhodopsin from an Extreme Thermophile

So far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. In this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus, which lives in a hot spring at around 75 °C. The gene for the retinylidene protein, named thermophilic rhodopsin (TR), was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of Escherichia coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans-retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 °C, and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (200 ×), Haloquadratum walsbyi bacteriorhodopsin (by 10-times), and Gloeobacter rhodopsin (100 ×). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have high stability. TR should allow the investigation of the molecular mechanisms of ion transport and protein folding.

Diabody Mixture Providing Full Protection against Experimental Scorpion Envenoming with Crude Androctonus australis Venom

Androctonus australis is primarily involved in envenomations in North Africa, notably in Tunisia and Algeria, and constitutes a significant public health problem in this region. The toxicity of the venom is mainly due to various neurotoxins that belong to two distinct structural and immunological groups, group I (the AahI and AahIII toxins) and group II (AahII). Here, we report the use of a diabody mixture in which the molar ratio matches the characteristics of toxins and polymorphism of the venom. The mixture consists of the Db9C2 diabody (anti-group I) and the Db4C1op diabody (anti-AahII), the latter being modified to facilitate in vitro production and purification. The effectiveness of the antivenom was tested in vivo under conditions simulating scorpion envenomation. The intraperitoneal injection of 30 μg of the diabody mixture protected almost all the mice exposed to 3 LD(50) s.c. of venom. We also show that the presence of both diabodies is necessary for the animals to survive. Our results are the first demonstration of the strong protective power of small quantities of antivenom used in the context of severe envenomation with crude venom.

Loss of Hypermethylated in Cancer 1 (HIC1) in Breast Cancer Cells Contributes to Stress-induced Migration and Invasion through β-2 Adrenergic Receptor (ADRB2) Misregulation

The transcriptional repressor HIC1 (Hypermethylated in Cancer 1) is a tumor suppressor gene inactivated in many human cancers including breast carcinomas. In this study, we show that HIC1 is a direct transcriptional repressor of β-2 adrenergic receptor (ADRB2). Through promoter luciferase activity, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments, we demonstrate that ADRB2 is a direct target gene of HIC1, endogenously in WI-38 cells and following HIC1 re-expression in breast cancer cells. Agonist-mediated stimulation of ADRB2 increases the migration and invasion of highly malignant MDA-MB-231 breast cancer cells but these effects are abolished following HIC1 re-expression or specific down-regulation of ADRB2 by siRNA treatment. Our results suggest that early inactivation of HIC1 in breast carcinomas could predispose to stress-induced metastasis through up-regulation of the β-2 adrenergic receptor.