Enzymes BamHI Research Articles

Predicting Diffusion Coefficients of DNA-Protein Complexes by Convex Hull Modelling

The recent development of single-molecule diffusometry establishes the diffusion coefficient as a new observable in single-molecule experiments to visualize biomolecular interactions. To decipher the rich information available from these experiments, accurate modeling of the hydrodynamic properties of biomolecular complexes is needed. Currently available tools for predicting diffusion coefficients of biomolecules, such as Hydropro and HullRad, rely on the existence of atomic coordinates for the molecule of interest. Composite molecular complexes that lack detailed Protein Data Bank entries, but can be modeled structurally from their constituent parts (i.e. a complex with multiple proteins bound to a single DNA) are frequently encountered in experiments but cannot yet be properly modeled for hydrodynamic properties. We present a simple program to predict the diffusion coefficients of composite DNA-protein complexes. Our algorithm extends the computational framework introduced by Fleming et al. in the HullRad software to construct a convex hull of the molecular complex and then uses an ellipsoidal approximation to compute hydrodynamic parameters. In order to construct the convex hull of the whole complex, the algorithm incorporates a priori knowledge of the complex such as the crystal structures of the constituent biomolecules, binding site positions, geometry and mechanics of DNA duplexes. The program is computationally efficient, integrates naturally with PyMOL, and outputs the 3D convex hull for visualization. We validate the program by accurately predicting hydrodynamic parameters of known crystal structures of DNA-protein complexes. We then predict diffusion coefficients of the restriction enzyme BamHI bound to double strand DNA of different lengths and compare these predictions with single-molecule diffusometry measurements. We envision that this program will aid researchers in using diffusion coefficient to identify and characterize relevant biological structures at the nanometer scale.

Studies on the synthesis, characterization, cytotoxic activities and plasmid DNA binding of platinum(II) complexes having 2-subsituted benzimidazole ligands

The aim of this study was to synthesize and evaluate the cytotoxic activities and plasmid DNA interaction of some new platinum(II) complexes, which may have potent cytotoxic activity and low side effects, with benzimidazole derivatives containing some amino acid residues as substituents in their 2 position. Seven 2-subtituted benzimidazole derivatives (1–7) (2-(H, methyl isopropyl, isobutyl, sec-butyl 1H-imidazol-4-ylmethyl or 4-hydroxybenzyl)benzimidazole, respectively), seven platinum(II) complexes with two chlorido leaving groups (1a–7a) and six platinum(II) complexes with an oxalato leaving group (2b–7b) bearing the benzimidazole carrier ligand were synthesized. The chemical structures of the compounds were characterized by their elemental analysis, IR, 1H NMR, and HRMS spectra. The compounds were evaluated for their cytotoxic activities against human HeLa cervical cancer cell lines. The interaction of all the ligands and their complexes with plasmid DNA and their restriction endonuclease reactions with BamHI and HindIII enzymes were investigated by agarose gel electrophoresis. Compounds 1a, 3a, 3b, 6, 7a and 7b, having 2-(H, isopropyl, 1H-imidazol-4-ylmethyl or 4-hydroxybenzyl)benzimidazole carrier ligands, have moderate in vitro cytotoxic activity, close to that of carboplatin.

Specific cytotoxicity of epidermal groth factor variantIII oriented chimeric antigen receptor-engineered T cells on SMMC-7721

Objective To observe the specific killing effect of anti-epidermal groth factor variantⅢ (EGFRvⅢ) chimeric antigen receptor modified T cells (EGFRvⅢ/2CAR-T) on liver cancer cell SMMC-7721. Methods Expression cassette of EGFRvⅢ/2CAR (1 374 bp) was inserted into XbaⅠ and BamHⅠ sites of piggybac transposon vector PB513B-1 (PB-EGFRvⅢ/2CAR), the latter together with transposase plasmid was then transducted into human peripheral blood T cells by electroporation (1×20 ms, 840 V). The expression of EGFRvⅢ/2CAR on T cells was detected by flow cytometry. The expression of EGFRvⅢ in SMMC-7721 was detected by Western blotting. After EGFRvⅢ/2CAR-T cells were co-cultured with SMMC-7721, the killing effect was detected by lactate dehydrogenase (LDH) release test and cytokine interferon (IFN)-γ secretion. Results The recombinant vector PB-EGFRvⅢ/2CAR was confirmed by XbaⅠ and BamHⅠ enzyme digestion, electrophoresis and DNA sequencing analysis. The expression of EGFRvⅢ/2CAR on T cells was detected by Flow cytometry (55.5%). Western blotting analysis showed that a protein band of approximate 145 000 molecular weight was observed in SMMC-7721 cells, which corresponds to the calculated molecular weight of EGFRvⅢ. EGFRvⅢ/2CAR-T cells were co-cultured with SMMC-7721 for 24 h, LDH release test showed that tumor specific killing was positively correlated with E∶T ratio (E∶T=1∶1, 5∶1, 10∶1, 15∶1, 20∶1); enzyme linked immunosorbent assay (ELISA) showed that cytokine IFN-γ secretion was (1 060.1±89.0) pg/ml, which was significantly higher than control group (P=0.000). Conclusion Anti-EGFRvⅢ CAR-T cells can kill specifically EGFRvⅢ+ liver cancer cell SMMC-7721, providing research basis for immunotherapy of hepatic cancer. Key words: Epidermal groth factor variantⅢ; Chimeric antigen receptor; Immunotherapy; PiggyBac transposon

Prevalence of CYP2C19 Gene Polymorphism and Its Influence In Omeprazole Metabolism As Predictors Of Drug Inoxification In Malay Ethnic In South Sumatra

Cytochrome P450 2C19 (CYP2C19) is an enzyme complex that plays a role in the metabolism of several drugs and is part of the super family of cytochrome P450. Genetic polymorphisms in these enzymes are associated with the emergence of poor metabolic phenotypes (poor metabolizers / PMs and intermediate metabolizers / IMs) that have a poor ability to metabolize the drugs that become substrates. Genotypes and phenotypes were analyzed using PCR-RFLP and bio-analysis of omeprazole levels in 30 subjects from ethnic Malay living in South Sumatra. Markers used to assess the presence of polymorphisms in the CYP2C19 gene are two polymorphic sites of exon 5 (CYP2C19 * 2) and exon 4 (CYP2C19 * 3). 321 bp DNA bands for exon 5 and 271 bp for exon 4 will be produced after DNA amplification by PCR method under denaturation for 5 min at 95oC; followed by 60 seconds at 95oC, 60sec at 53oC and 60sec at 72oC for 30 cycles; as well as the final polymerization for 5 minutes at 72 ° C. Furthermore, DNA cutting was done using the restriction enzyme endonuclease SmaI (CYP2C19 * 2) at 30oC and BamHI (CYP2C19 * 3) with incubation at 37oC for 3 hours. Bioanalysis of omeprazole levels in the blood with LC-MS. The results of this study indicate the presence of polymorphisms on both sites will eliminate the enzyme sites SmaI and BamHI. The results showed that 46.7% of South Sumatran Malay populations were classified as PM consisting of 13.3% homozygous mutandan mutant 33.4% heterozygotes. The high phenotype of PM enzyme CYP2C19 in ethnic Malays in South Sumatra predicted to influence metabolism of drugs become substrates. However, based on spearman correlation analysis, the correlation value was 0.035 with p = 0.875. This means that between the CYP2C19 gene polymorphism and the omeprazole levels in the blood there is a weak and meaningless correlation. The results of this study provide an overview of the high genetic polymorphisms of dyspepsia syndrome patients from the Malay population, ie almost half of the study subjects (46.7%). There is a weak and insignificant correlation between polymorphism and omeprazole levels.

Construction of Overexpression Vector of CONSTANS Gene Plant in <i>Brassica napus</i> and Production of Transgenic Plants

The CONSTANS target gene from Brassica napus was cloned, and the full length amplified primer of it was designed and amplified on the sequence released on NCBI. The target gene fragments were sequenced and the restriction loci were analyzed. The enzyme primers (BamHⅠ, SacⅠ) were designed, and the target gene fragments were connected to the eukaryotic expression vector pBI121 through BamHⅠ and SacⅠ double enzyme digestion. PCR identification result indicated that PBI121+CONSTANS overexpression vector was successfully constructed. The overexpression vector was transformed into the competent EHA105 cell, and the seeds of T0 were infected by Arabidopsis thaliana . The positive plants were screened by MS solid medium containing Cara (50 mg/L). PCR identification showed that PBI121+CONSTANS overexpression vector was successfully introduced into Arabidopsis thaliana and 12 transgenic positive seedlings were obtained, which did the preparation for the gene function analysis.

Evidence Suggesting Absence of Mitochondrial DNA Methylation.

Methylation of nuclear genes encoding mitochondrial proteins participates in the regulation of mitochondria function. The existence of cytosine methylation in the mitochondrial genome is debated. To investigate whether mitochondrial DNA (mtDNA) is methylated, we used both targeted- and whole mitochondrial genome bisulfite sequencing in cell lines and muscle tissue from mouse and human origin. While unconverted cytosines were detected in some portion of the mitochondrial genome, their abundance was inversely associated to the sequencing depth, indicating that sequencing analysis can bias the estimation of mtDNA methylation levels. In intact mtDNA, few cytosines remained 100% unconverted. However, removal of supercoiled structures of mtDNA with the restriction enzyme BamHI prior to bisulfite sequencing decreased cytosine unconversion rate to <1.5% at all the investigated regions: D-loop, tRNA-F+12S, 16S, ND5 and CYTB, suggesting that mtDNA supercoiled structure blocks the access to bisulfite conversion. Here, we identified an artifact of mtDNA bisulfite sequencing that can lead to an overestimation of mtDNA methylation levels. Our study supports that cytosine methylation is virtually absent in mtDNA.

Synthesis, characterization and DNA binding studies of platinum(II) complexes with benzimidazole derivative ligands

The aim of this study was to synthesize and evaluate plasmid DNA interaction of new platinum(II) complexes with some 2-substituted benzimidazole derivatives as carrier ligands which may have potent anticancer activity and low toxicity. Twelve benzimidazole derivatives carrying indole, 2-/or 3-/or 4-methoxyphenyl, 4-methylphenyl, 3,4-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 4-methoxybenzyl, 3,4,5-trimethoxybenzyl, 3,4,5-trimethoxystyryl, 3,4,5-trimethoxybenzylthio or dimethylamino ethyl groups in their position 2 and twelve platinum(II) complexes with these carrier ligands were synthesized. The chemical structure of the platinum complexes have been characterized by their elemental analysis and FIR, 1H NMR and mass spectra and their 1H NMR and FIR spectra were interpreted by comparison with those of the ligands. The interaction of all the ligands and their complexes with plasmid DNA and their restriction endonuclease reactions by BamHI and HindIII enzymes were studied by agarose gel electrophoresis. It was determined that complex 1 [dichloro-di(2-(1H-indole-3-yl)benzimidazole)platinum(II)·2H2O] has stronger interaction than carboplatin and complex 10 [dichloro-di(2-(3,4,5-trimethoxystyryl)benzimidazole)platinum(II)·2H2O] has stronger interaction than both carboplatin and cisplatin with plasmid DNA.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

Karakterisasi Molekuler Ikan Gurami Soang (Osphronemus gouramy Lac.) yang Mati pada Rentang Waktu Berbeda Menggunakan PCR-RFLP Gen Major Histocompatibility Complex Kelas II B

Gurami (Osphronemus gouramy Lac.) is a popular fish species among Indonesian people. Several Gurami strains have been cultivated by fish farmer, one of which is Gurami Soang. This strain is belived to have a faster growth rate compared to other strains. However, like other strains, the fingerling of Soang strain have also a low survival and suceptible to disease, especially that caused by Aeromonas hydrophila infection. It has been proved that seeds from a single spawning event show varibale disease resistance. The difference in resistance among individuals is suggested related to the difference in their genetic component. One of the genes responsible for resistance is Major Histocompatibility Complex (MHC) class II B gene. Variability in resistance can be analyzed by using PCR - RFLP technique. PCR-RFLP is a technique that can produce a specific DNA fragments by PCR, followed by cutting the PCR product using restriction enzymes to describe the presence or absence of restriction sites in DNA fragments. This research aims to determine genetic marker to differiantiate between resitant and irresistant individual of Gurami Soang infected by A. hydrophila which die at a different time priod based on PCR-RFLP MHC class IIB gene. The study used survey method with purposive random sampling. The Data of PCR-RFLP band patterns were analyzed descriptively. The result indicated that cutting of the MHC class II B gene using HinfI produce two RFLP bands with 300 bp and 100 bp length in all samples. Meanwhile, the MHC IIB gene was not cuted by PstI, HindIII, BamHI and EcoRI enzymes forall samples. These mean that MHC II gene in all individuals were monomorphic. Therefore,it can be concluded that there is no specific genetic marker to differentiate gurami soang individulas which was dying in different time periods.

Occurrence of BK Virus and Human Papilloma Virus in colorectal cancer.

Colorectal cancer is one of the most common cancers worldwide. In Poland, it is the second most common cancer, regardless of gender. The aim of study was to analyze the incidence of HPV and BKV in the tissue of colorectal cancer and to determine the relationship between the presence of these viruses and the development of this cancer. The experiments were conducted using 50 colorectal cancer tissues collected from histological sections. The clinical material was embedded in paraffin blocks. Next, DNA extraction was performed. Isolates of colorectal cancer tissue were tested for the presence of HPV DNA. BKV DNA was detected by PCR using specific primers and then differentiated from JCV by digestion with BamHI enzyme. In clinical specimens taken from patients with colorectal cancer, HPV DNA was detected in 20% of cases. In 10% of cases the presence of HPV type 18 was confirmed, in the other 90% of the samples HPV type 16 was detected, while the presence of BKV was confirmed in 30% of cases. Coinfection with HPV and BKV was shown in 12% of patients. In one case, BK virus coexisted with HPV type 18, in the remaining 5 cases with HPV type 16. Developing colorectal cancer can show no symptoms, even for many years. This is why it is so important to become familiar with as many etiological factors as possible. The development of many human neoplasms is often initiated by exposure to infectious agents - such as bacterial or viral infections. Similar to the human papillomavirus, the BK virus was detected in clinical specimens. It seems that HPV and BKV infections can contribute to the neoplastic process, which requires detailed studies on a larger group of patients.

A rapid method for detecting protein-nucleic acid interactions by protein induced fluorescence enhancement.

Many fundamental biological processes depend on intricate networks of interactions between proteins and nucleic acids and a quantitative description of these interactions is important for understanding cellular mechanisms governing DNA replication, transcription, or translation. Here we present a versatile method for rapid and quantitative assessment of protein/nucleic acid (NA) interactions. This method is based on protein induced fluorescence enhancement (PIFE), a phenomenon whereby protein binding increases the fluorescence of Cy3-like dyes. PIFE has mainly been used in single molecule studies to detect protein association with DNA or RNA. Here we applied PIFE for steady state quantification of protein/NA interactions by using microwell plate fluorescence readers (mwPIFE). We demonstrate the general applicability of mwPIFE for examining various aspects of protein/DNA interactions with examples from the restriction enzyme BamHI, and the DNA repair complexes Ku and XPF/ERCC1. These include determination of sequence and structure binding specificities, dissociation constants, detection of weak interactions, and the ability of a protein to translocate along DNA. mwPIFE represents an easy and high throughput method that does not require protein labeling and can be applied to a wide range of applications involving protein/NA interactions.

Loop-mediated isothermal amplification based on the mitochondrial COI region to detect Pratylenchus zeae

Pratylenchus zeae parasitizes various crops and damages the host roots, resulting in decreased yield and quality of the host plants. Alignments of mitochondrial DNA (mtDNA) Cytochrome Oxidase I (COΙ) sequences revealed the genetic variation among Pratylenchus species. The results indicated 0.2–2.4% intraspecific variations for mtDNA COI sequences among eight P. zeae populations, and 25.4–35.1% interspecific variations between P. zeae and other Pratylenchus species. Based on the mtDNA COΙ region, a loop-mediated isothermal amplification (LAMP) assay was developed for the rapid and specific detection of P. zeae. The optimal conditions for the LAMP assay were 64 °C for 40 min. The LAMP products were confirmed using conventional polymerase chain reaction (PCR), analysis with the restriction enzyme Bam HI and visual inspection by adding SYBR Green I to the products. The LAMP assay could detect P. zeae populations from different hosts and different geographical origins specifically. The LAMP assay was also sensitive, detecting 0.1 individual P. zeae, which was 10 times more sensitive than conventional PCR. This is the first report of the detection of Pratylenchus spp. using LAMP. In addition, the results also suggested that use of the COI gene might allow for good resolution at the Pratylenchus species level.

Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M.

In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene) from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA gene was amplified (642 bp), and the product was cloned into a pGEM-T vector. The recombinant plasmid pGEMT-benA was digested by double restriction enzymes BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3). The recombinant E. coli was induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at 22˚C to produce benzoate dioxygenase. The enzyme was then purified by ion exchange chromatography after 8 purification folds. The resulting product was 25 kDa, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Benzoate dioxygenase activity was found to be 6.54 U/mL and the optimal pH and temperature were 8.5 and 25°C, respectively. Maximum velocity (Vmax) and Michaelis constant (Km) were 7.36 U/mL and 5.58 µM, respectively. The end metabolite from the benzoate dioxygenase reaction was cyclohexane dione, which was determined by gas chromatography mass spectrometry (GC-MS).

Construction and Transformation of HVA1 Gene Expression Vector into Indonesian Elite Rice Varieties

&lt;p&gt;of drought in rice. HVA1 is one of the Late Embryogenesis&lt;br /&gt;Abundant (LEA) protein group that plays a role on cell&lt;br /&gt;protection during stresses. A study was done with an&lt;br /&gt;objective to construct a plasmid vector expressing HVA1 and&lt;br /&gt;to transform it into Indonesian elite rice varieties. Materials&lt;br /&gt;used in the study were plasmid pBY520 (source of HVA1;&lt;br /&gt;intermediate plasmid pRP9; plasmid pAY560326&lt;br /&gt;(backbone); restriction enzymes BamHI, HindIII, XhoI, and&lt;br /&gt;SpeI; T4 DNA ligase, and gel DNA extraction kit. Methods&lt;br /&gt;used were standard procedure for plasmid vector&lt;br /&gt;construction and molecular biology. Step I: the pBY520 and&lt;br /&gt;pRP9 were cut with BamHI and HindIII, and electrophorated&lt;br /&gt;with 1% agarose gel. DNA fragments of HVA1 and pRP9 were&lt;br /&gt;purified, ligated with T4 DNA ligase, and transformed into&lt;br /&gt;Escherichia coli DH5-α by heat shock. E. coli were grown&lt;br /&gt;onto solid medium (+ kanamycin 100 mg/l). A new plasmid&lt;br /&gt;DNA was isolated from single colony culture of the bacteria,&lt;br /&gt;confirmed, and named pRP9_HVA1. Step II: DNA of&lt;br /&gt;pRP9_HVA1 and pAY560326 were cut with XhoI dan SpeI&lt;br /&gt;enzymes, purified, and ligated. The next procedure was&lt;br /&gt;similar to step I, and the resulted plasmid was confirmed by&lt;br /&gt;PCR and digestion with XhoI dan SpeI enzymes, and named&lt;br /&gt;pAY_HVA1. Step III: pAY_HVA1 was first transformed into&lt;br /&gt;Agrobacterium EHA-105 and then into rive varieties Ciherang&lt;br /&gt;and Inpari 6 using the early infection of scutellum&lt;br /&gt;transformation method. Nine transgenic rice lines that&lt;br /&gt;positively contain HVA1 were obtained.&lt;/p&gt;

Penyisipan Gen Inhibitor α-amilase pada Plasmid Biner pCambia 1301

&lt;p class="p1"&gt;The experiment was conducted at the Molecular Biology Laboratory of the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Bogor. The objective was to construct &lt;em&gt;-ai &lt;/em&gt;gene on a binary plasmid &lt;em&gt;p&lt;/em&gt;Cambia 1301. This experiment was carried out using construction method by ligation process between fragments of &lt;em&gt;α-ai &lt;/em&gt;gene from &lt;em&gt;p&lt;/em&gt;TA&lt;span class="s1"&gt;3 &lt;/span&gt;plasmid and &lt;em&gt;p&lt;/em&gt;Cambia 1301 on &lt;em&gt;Hind&lt;/em&gt;III site. The result of ligant transformation into &lt;em&gt;E. coli &lt;/em&gt;DH5&lt;em&gt;α &lt;/em&gt;was 182 surviving colonies on YEP medium containing kanamycin. DNA samples were obtained from 60 randomly selected colonies. The restriction pattern was tested by digesting each DNA sample using &lt;em&gt;Hind&lt;/em&gt;III showed colonies containing two fragments expected of sizes wich are 11.837 and 4.887 kb. Two colonies are predicted containing of &lt;em&gt;α-ai &lt;/em&gt;gene on its the binary plasmid. Advanced tests using restriction enzymes &lt;em&gt;Bam&lt;/em&gt;HI and &lt;em&gt;Xba&lt;/em&gt;I showed two directions (right and left) of &lt;em&gt;α-ai &lt;/em&gt;gene. The right direction was shown by &lt;em&gt;p&lt;/em&gt;Cambia-&lt;em&gt;α-ai&lt;/em&gt;1 from colony number 43. This plasmid showed expected fragments of sizes 13.485 and 3.219 kb when digested with &lt;em&gt;Bam&lt;/em&gt;HI and two fragments of sizes 15.421 and 1.303 kb when digested with &lt;em&gt;Xba&lt;/em&gt;I. The left direction was shown &lt;em&gt;p&lt;/em&gt;Cambia-&lt;em&gt;α-ai&lt;/em&gt;2 from colony number 58. This plasmid also demon-strated expected fragments of sizes 15.026 and 1.698 kb when digested with &lt;em&gt;Bam&lt;/em&gt;HI and two fragments of sizes 13.082 and 3.642 kb when digested with &lt;em&gt;Xba&lt;/em&gt;I. Both &lt;em&gt;p&lt;/em&gt;Cambia-&lt;em&gt;α-ai&lt;/em&gt;1 and &lt;em&gt;p&lt;/em&gt;Cambia-&lt;em&gt;α-ai&lt;/em&gt;2 were transformed into &lt;em&gt;A. tumefaciens &lt;/em&gt;LBA4404.&lt;/p&gt;

PCR-RFLP and Sequencing of trnS/trnfM Fragment of Enhalus acoriodes from Sanur Coastal Waters, Bali, Indonesia: A Preliminary Study

This study aimed to evaluate the resolution of PCR-RFLP and sequencing of trnS/trnfM in detecting genetic diversity of seagrass Enhalus acoroides at Sanur Beach, Sindhu Beach and Semawang Beach. This research used six samples E. acoroides from each location. DNA extraction method followed Doyle and Doyle. PCR amplifications were done using primers P672/P673, P674/P675 and ITS4/ITS5 followed by digestion using restriction enzymes EcoRI, PstI, HindIII, BamHI, Rsa, Mva and HinfI. Sequencing analysis used PCR products of primers trnS/trnfM. Testing of DNA sequences of E. acoroides were conducted using BLAST (Basic Local Alignment Search Tool). DNA sequences were further analyzed using MEGA 5.2 software (Molecular Evolutionary Genetic Analysis) to evaluate variations of DNA. The sequence alignments were done using ClustalW software to determine the homology between the DNA sequences. The results showed that 18 samples of E. acoroides from Sanur, Sindhu and Semawang Beach have no polymorphism based on restriction enzyme analyses. Furthermore, sequencing of trnS/trnfM region of 18 samples E. acoroides showed that the sequences were identical.

Loop-Mediated Isothermal Amplification for the Detection of Tylenchulus semipenetrans in Soil.

Tylenchulus semipenetrans is an economically important plant-parasitic nematode occurring in all citrus-producing regions of the world and causing a disease called "slow decline". For the rapid detection of this nematode, a loop-mediated isothermal amplification (LAMP) assay was developed, based on the ribosomal DNA internal transcribed spacer sequence. The optimal condition for the LAMP assay was 65°C for 50 min. The LAMP products were confirmed using conventional polymerase chain reaction (PCR) and restriction analysis with the BamHI enzyme, and by adding SYBR Green I to the LAMP products for visual inspection. The LAMP assay was highly specific for the detection of T. semipenetrans populations from different geographical origins. It was also sensitive, detecting a tenth of the DNA from an individual specimen of T. semipenetrans, which was 10 times more sensitive than conventional PCR. The LAMP protocol was applied to natural citrus rhizosphere soil samples from several orchards in China and the results were fast, sensitive, robust, and accurate. This study is the first to provide a diagnostic tool for T. semipenetrans using DNA extracted directly from citrus rhizosphere soils. This LAMP assay could be used as a practical molecular tool to identify T. semipenetrans and diagnose slow decline disease, even in remote locations.

Construction of plvx-cyclooxygenase-2-DsRed vector and its effects on proliferation in cyclooxygenase-2 overexpressed breast cancer cell line

Objective To construct plvx-cyclooxygenase-2(COX-2)-DsRed and establish breast cancer cell line MCF7 which overexpressed COX-2, to explore the effect of COX-2 on breast cancer cell. Methods The full-length COX-2 PCR product was obtained by total COX-2 PCR primers and COX-2 cDNA vector. After the PCR product and lentiviral vector plvx-DsRed-Monomer-N1 were cut simultaneously by restriction enzyme BamH1 and Xhol1, they were connected and sequenced, to get lentiviral vector plvx-COX-2-DsRed. After selected by puromycin, overexpressed COX-2 breast cancer cell line MCF7-plvx-COX-2-DsRed was obtained. The stable cell line was verified by real time PCR and Western blot. The differences of proliferation ability between stable cell line and normal one were compared by colony formation test and Western blot. Results The lentiviral vector plvx-COX-2-DsRed and stable cell line MCF7-plvx-COX-2-DsRed after selecting were obtained. COX-2 expression level of the stable cell line was 75.29 times as high as that of MCF7, and 64.91 times as high as that of cell line MCF7-plvx-DsRed-Monomer-N1 by PCR assay (P< 0.05), which was consistent with the results of Western blot and microscope photo. MTT results showed that cell line MCF7-plvx-COX-2-DsRed had grown faster than cell line MCF7 and MCF7-plvx-DsRed-Monomer-N1 from the 2nd day (P< 0.05), which was accordant with colony formation assay. MCF7-plvx-COX-2-DsRed cell line had higher c-myc expression and lower β-catenin expression than MCF7 cell and cell line MCF7-plvx-DsRed-Monomer-N1 detected by Western blot relative quantification (P< 0.05). Conclusion The plvx-COX-2-DsRed lentiviral vector and cell line MCF7-plvx-COX-2-DsRed are successfully constructed. COX-2 can increase proliferation of MCF7 cells through up-regulating the expression of c-myc. Key words: Cyclooxygenase; Breast neoplasms; Cell line; Lentivirus; Cell proliferation

Phosphorus–nitrogen compounds part 33: in vitro cytotoxic and antimicrobial activities, DNA interactions, syntheses, and structural investigations of new mono(4-nitrobenzyl)spirocyclotriphosphazenes

The condensation reactions of hexachlorocyclotriphosphazene, N3P3Cl6, with N-alkyl-N′-mono(4-nitrobenzyl)diamines (1–3), NO2PhCH2NH(CH2) n NHR1 (R1 = CH3 or C2H5), led to the formation of the mono(4-nitrobenzyl)spirocyclotriphosphazenes (4–6). The tetra-pyrrolidino (4a–6a), piperidino (4b–6b), and 1,4-dioxa-8-azaspiro[4,5]decaphosphazenes (4c–6c) were prepared from(for) the reactions of partly substituted compounds (4, 5, and 6) with excess pyrrolidine, piperidine, and 1,4-dioxa-8-azaspiro[4,5]decane (DASD), respectively. The partly substituted geminal (4d and 5d) and cis-morpholino (6d) phosphazenes were isolated from the reactions of excess morpholine in boiling THF and o-xylene, but the expected fully substituted compounds were not obtained. The structures of all the phosphazene derivatives were determined by elemental analyses, MS, FTIR, 1H, 13C{1H}, 31P{1H} NMR, HSQC, and HMBC techniques. The crystal structures of 4, 6, 4a, and 5a were verified by X-ray diffraction analysis. In addition, in vitro cytotoxic activities of fully substituted phosphazenes (4a–6c) against HeLa cervical cancer cell lines (ATCC CCL-2) and the compounds 4a and 4c against breast cancer cell lines (MDA-MB-231) and L929 fibroblast cells were evaluated, respectively. Apoptosis effect was determined by MDA-MB-231 cancer cell lines and fibroblast cells. The MIC values of the compounds were in the ranges of 9.8–19.5 µM. The compounds 6, 5a, 6a, 5b, and 6d have greater MIC activity against bacterial and yeast strain. The investigation of DNA binding with the phosphazenes was studied using plasmid DNA. The phosphazene derivatives inhibit the restriction endonuclease cleavage of plasmid DNA by BamHI and HindIII enzymes. BamHI and HindIII digestion results demonstrate that the compounds bind with G/G and A/A nucleotides.

First report of natural occurence of Papaya leaf crumple virus on soyabean in India

Soybean (Glycine max, family Fabaceae) is a most important nutrient crop. It is native to East Asia and is grown widely as a pulse rich in protein, and also has numerous other uses. Symptoms of leaf crumple disease at Lalitpur (24.6° N, 78.4° E), India and yellow mosaic disease at Lucknow (26.8° N, 80.9° E), India, were noticed on a number of soybean plants in 2013. The naturally infected soybean plants exhibited yellow mosaic on leaves (Fig. 1a) and leaf crumpling and distortion symptoms (Fig. 1b). Severely infected plants were stunted and bore less number of flowers and pods as compared to the healthy ones. Based on leaf crumple and yellow mosaic symptoms, begomovirus infection was suspected. Twenty-two leaf samples of infected soybean plants were collected from each location and total DNA was isolated using a plant genomic DNA isolation kit (Sigma, USA). The PCRs were performed with begomovirus degenerate primers (Rojas et al., 1) which resulted in expected size amplicon of ~1.2 kb from 16/22 samples of yellow mosaic diseased plants collected from Lucknow and 12/22 samples of leaf crumple diseased plants collected from Lalitpur, but not from the healthy samples (Fig. 2). This indicated the presence of begomovirus in the soybean plants that had been sampled. To identify the complete genome sequence of these begomoviruses, total DNAs isolated from a representative leaf sample exhibiting yellow mosaic and leaf crumple symptoms were subjected separately to rolling circle amplification (RCA, TempliPhi kit, GE Healthcare, USA) followed by digestion with EcoRI, BamHI, HindIII, Sal1, XhoI, PstI and XbaI enzymes. Electrophoresis of products digested only with BamHI resulted in a DNA fragment of ~2.7 kb in both the samples. The products obtained ~2.7 kb were cloned separately into the pCAMBIA1300 vector, sequenced and deposited in GenBank with Accession Nos. KR052025 (MJS-1 isolate from yellow mosaic sample of Lucknow) and KR071789 (MJS-2 isolate from crumpled leaf sample of Lalitpur). The sequence analyses of MJS-1 and MJS-2 isolates showed the typical genome of begomovirus containing six open reading frames (V2 and V1 in sense strand and C1-C4 in antisense strand). BLASTn analysis of nucleotide sequence of begomovirus isolate MJS-1 under study showed 98-99% nucleotide sequence identity with Mungbean yellow mosaic India virus (MYMIV) isolates of Vigna unguiculata (DQ389153) and of G. max (EU523045, KC852204) reported from India. However, begomovirus isolate MJS-2 shared 94-97% identity with isolates of Papaya leaf crumple virus (PaLCrV) from Carica papaya (HM140367, HM140368, HM140369) and Solanum nigrum (KJ028210) both from India, and C. papaya (HE580236) from Pakistan. During phylogenetic analysis by MEGA v6.0 program (Tamura et al., 6) using nucleotide sequences of MYMIV and PaLCrV and some other begomoviruses available in GenBank database, MJS-1 and MJS-2 isolates showed close relationships with MYMIV and PaLCrV isolates, respectively (Fig. 3), hence the begomovirus isolated from soybean were identified as MYMIV and PaLCrV. A literature survey revealed the natural occurrence of MYMIV (Girish and Usha 2), Tomato leaf curl Karnatka virus (Raj et al., 3) and Cotton leaf curl Kokhran virus (Raj et al., 4) on soybean in India. The existence of PaLCrV is reported on C. papaya (Singh-Pant et al., 5), S. nigrum and Andrographis paniculata from India. However, this is the first report of natural occurrence of PaLCrV in soybean from India.