• BMB Reports
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• v.35 no.3
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• pp.262-266
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• 2002

A non-isotopic neutravidin-based reverse transcriptase (RT) assay adapted for high throughput screening of HIV activity is described. Using a 96-well microtitre plate, HIV particles are lysed and the RT enzyme released into a reaction mixture containing poly(A) RNA, biotinylated oligo d(T) and fluorescein-labelled dUTP (FI-dUTP). With poly(A) as a template and oligo d(T) as primer, the viron RT incorporates FI-dUTP into an elongating DNA strand. The resulting product is captured on a neutravidin-coated 96-well plate and the unincorporated nucleotides removed by a series of washing steps. A simple ELISA is subsequently performed using a monoclonal antifluorescein antibody conjugated to alkaline phosphatase. Quantification of RT activity is facilitated by a colorimetric readout. The assay was validated in the context of a diagnostic HIV-1 phenotyping assay. Using supernatants from HIV-1 infected lymphocyte cultures the assay was shown to be as sensitive as a radioactive assay and the RT activity correlated well with levels of cell-asociated HIV-p24. Importantly, even minor reductions of RT activity by virus variants with reduced fitness could be distinguished.

• Archives of Pharmacal Research
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• v.19 no.6
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• pp.497-501
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• 1996

A gene coding for triosephosphate isomerase (TPI) from a rat skeletal muscle cDNA library was cloned and its nucleotide sequence was determined. The 1, 348-bp cDNA clone contains 24 bp $5^I$ noncoding region, the entire 750 bp coding region corresponding to a protein of 249 amino acids, $547bp 3^I$ noncoding region and part of a poly(A) tail. It also contains a polyadenylation signal, AATAAA, starting from 17 bp upstream of the poly(A) tail. The calculated molecular weight of rat TPI is 27.8 kDa and the net charge is +4. The deduced amino acid sequence from rat TPI CDNA sequence has 93% and 94% homology with that of mouse and human clones, respectively. The amino acids at the residue of Asn12, Lys14, His96, Glu 166, His96, His101, Ala177, Tyr165, Glu13O, Tyr2O9, and Ser212 in catalytic site are completely identical, confirming that the functional residues in TPI proteins are highly conserved throughout evolution. The most profound characteristic of rat TPI enzyme, compared with other TPIs, is that there are five cysteine substitutions at the residue of 21, 27, 159, 195 and 204. A Glu123 instead of Gly was found in rabbit, rhesus, mouse and human sequences. Through the method of RT-PCR, the mRNA transcription level of TPI gene was found to be different among various tissues and was highest in muscle.

• BMB Reports
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• v.56 no.6
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• pp.347-352
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• 2023

The protein family of poly (ADP-ribose) polymerases (PARPs) is comprised of multifunctional nuclear enzymes. Several PARP inhibitors have been developed as new anticancer drugs to combat resistance to chemotherapy. Herein, we characterized PARP4 mRNA expression profiles in cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines. PARP4 mRNA expression was significantly upregulated in cisplatin-resistant ovarian cancer cell lines, and this upregulation was associated with the hypomethylation of specific cytosine-phosphate-guanine (CpG) sites (cg18582260 and cg17117459) on its promoter. Reduced PARP4 expression was restored by treating cisplatin-sensitive cell lines with a demethylation agent, implicating the epigenetic regulation of PARP4 expression by promoter methylation. Depletion of PARP4 expression in cisplatin-resistant cell lines reduced cisplatin chemoresistance and promoted cisplatin-induced DNA fragmentation. The differential mRNA expression and DNA methylation status at specific PARP4 promoter CpG sites (cg18582260 and cg17117459) according to cisplatin responses, was further validated in primary ovarian tumor tissues. The results showed significantly increased PARP4 mRNA expressions and decreased DNA methylation levels at specific PARP4 promoter CpG sites (cg18582260 and cg17117459) in cisplatin-resistant patients. Additionally, the DNA methylation status at cg18582260 CpG sites in ovarian tumor tissues showed fairly clear discrimination between cisplatin-resistant patients and cisplatin-sensitive patients, with high accuracy (area under the curve = 0.86, P = 0.003845). Our findings suggest that the DNA methylation status of PARP4 at the specific promoter site (cg18582260) may be a useful diagnostic biomarker for predicting the response to cisplatin in ovarian cancer patients.

• Journal of fish pathology
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• v.25 no.1
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• pp.11-20
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• 2012

C-type lectins are crucial for pathogen recognition, innate immunity, and cell-cell interactions. In this study, a C-type lectin gene was cloned from the rock bream. The full-length RbCTL cDNA was 729 bp with a 429 bp ORF encoding a 164-residue protein. The deduced amino acid sequence of RbCTL had all of the conserved features crucial for its fundamental structure, including the four cysteine residues involved in sulfide bridge formation and potential $Ca^2+$/carbohydrate-binding sites. RbCTL contains a signal peptide one single carbohydrate recognition domain. It showed 29.4% similarity to the C-type lectin of rainbow trout. RbCTL mRNA was predominately expressed in gill and head-kidney tissue and expressed less in peripheral blood leukocytes, trunk-kidney, spleen, liver, intestine and muscle. Expression of RbCTL was differentially upregulated in rock bream stimulated with LPS, Con A/PMA and poly I:C.

• Korean Journal Plant Pathology
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• v.11 no.3
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• pp.271-277
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• 1995

The RNA nucleotide sequences of the 3/-noncoding regions (3'-NCRs) of two Korean strains of turnip mosaic virus (TuMV), Ca and cqs, have been determined from their cDNA clones that encompassed the 3'-terminal regions of the viral genomic RNAs. The 3'-NCRs of both strains were 209 nucleotides long, terminated with GAC residues and poly (A) tails. The potential polyadenylational signal motif, UAUGU, was located 140 nucleotides upstream from the poly (A) tail in each of the virus. A highly conserved hexanucleotide sequence [A G U G A/U G/C], which was common in the 3'-NCRs of the potyvirus RNAs, was also found at the regions of 119 bases upstream from the 3'-end. Comparison of the 3'-NCRs of the two Korean isolates with those of four strains from Canada, China and Japan showed significantly identical genotypes (94.3∼99.5%). The secondary structure of three loops with long stems was found within the 3'-NCRs by sequence analysis. The substituted bases in the region among the six TuMV strains did not alter their secondary structures. Length of the 3'-NCRs of the know 11 potyviral RNAs and TuMV RNAs was different from one another and their nucleotide sequences showed 55.7% to 24.0% of homology. The 3'-NCR, therefore, is considered to be useful for phylogenetic studies in potyviruses.

• Animal cells and systems
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• v.15 no.1
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• pp.1-8
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• 2011

Perturbation of metabolism with increased expression of lipogenic enzymes is a common characteristic of human cancers, including prostate cancer. In the present work the overexpression of stearoyl-CoA desaturase (SCD) in LNCaP cells led to increased mRNA levels of fatty acid synthase (FAS) and acetyl-CoA-carboxylase-a, whereas micro RNA-mediated silencing of SCD inhibited the expression of these lipogenic genes in LNCaP cells. Treatment with the FAS-specific inhibitor cerulenin inhibited SCD induction of LNCaP cell proliferation. In addition, a transient transfection assay revealed the capability of cerulenin to suppress SCD and dihydrotestosterone induction of androgen receptor transcriptional activity. Furthermore, overexpression of SCD in LNCaP cells produced marked resistance to ceramide-induced cell death with reduced poly(ADP-ribose) polymerase (PARP) cleavage. In contrast, silencing of SCD expression increased Bax protein in LNCaP cells. Furthermore, addition of ceramide to SCD knockdown LNCaP cells increased cell death and caspase-3 activity with drastic increase of PARP cleavage. Together, the data indicate that SCD may provide resistance of prostate cancer cells to ceramide-induced cell death.

• Korean Journal of Medicinal Crop Science
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• v.12 no.2
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• pp.135-140
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• 2004

Total RNAs were isolated from cultured roots of Scopolia parviflora, $poly(A)^+$ RNA was obtained through the mRNA purification, cDNA library of Hnh6h was constructed. Recombinant baculoviruses in Spodoptera frugiperda (Sf) cells were constructed by use of the transfer vector pBacPAK, which has the AcNPV sequence under the polyhedrin promoter. The expression vector carrying Hnh6h gene was transferred to S. parviflora and obtained transgenic hairy root lines. Our results confirmed the over expression of the H6H protein was used by anti-pBacPAK about cDNAs of S. parviflora. This study will served for production of tropane alkaloids by metabolic engineering.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.175-179
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• 2006

A bacterium that produced a large amount of poly-$\gamma$-glutamate (PGA) was isolated from the compost and designated as Bacillus subtilis CH-10. The optimum temperature and pH for PGA production were at $37^{\circ}C$ and 7.5, respectively. The maximum amount of PGA production (18.84 mg/ml) was obtained when it was grown in a medium containing 3% L-glutamate and 5% sucrose at $37^{\circ}C$ with shaking. The result that the L-glutamate significantly induced PGA production indicates that it produces a PGA by the glutamate dependent manner. Some properties of the PGA obtained at different times of cultivation were investigated by SDS-PAGE and ninhydrin analysis. The PGA production was elongated along with cultivation time and maximum amount was achieved at 96 h. Average molecular weight of PGA was estimated to be 1100 kDa by FDNB method.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.8
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• pp.1366-1372
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• 2018

Objective: A disintegrin and metallopeptidase with thrombospondin motifs type 8 (ADAMTS8) is crucial for diverse physiological processes, such as inflammation, tissue morphogenesis, and tumorigenesis. The chicken ADAMTS8 (chADAMTS8) gene was differentially expressed in the kidney following exposure to different calcium concentrations, suggesting a pathological role of this protein in metabolic diseases. We aimed to examine the molecular characteristics of chADAMTS8 and analyze the gene-expression differences in response to toll-like receptor 3 (TLR3) stimulation. Methods: The ADAMTS8 mRNA and amino acid sequences of various species (chicken, duck, cow, mouse, rat, human, chimpanzee, pig, and horse) were retrieved from the Ensembl database and subjected to bioinformatics analyses. Reverse-transcription polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) experiments were performed with various chicken tissues and the chicken fibroblast DF-1 cell line, which was stimulated with polyinosinic-polycytidylic acid (poly[I:C]; a TLR3 ligand). Results: The chADAMTS8 gene was predicted to contain three thrombospondin type 1 (TSP1) domains, whose amino acid sequences shared homology among the different species, whereas sequences outside the TSP1 domains (especially the amino-terminal region) were very dif­ferent. Phylogenetic analysis revealed that chADAMTS8 is evolutionarily clustered in the same clade with that of the duck. chADAMTS8 mRNA was broadly expressed in chicken tissues, and the expression was significantly up-regulated in the DF-1 cells in response to poly(I:C) stimulation (p<0.05). These results showed that chADAMTS8 may be a target gene for TLR3 signaling. Conclusion: In this report, the genetic information of chADAMTS8 gene, its expression in chicken tissues, and chicken DF-1 cells under the stimulation of TLR3 were shown. The result suggests that chADAMTS8 expression may be induced by viral infection and correlated with TLR3-mediated signaling pathway. Further study of the function of chADAMTS8 during TLR3-dependent inflammation (which represents RNA viral infection) is needed and it will also be important to examine the molecular mechanisms during different regulation, depending on innate immune receptor activation.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.6
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• pp.892-897
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• 2005

Macrophage colony-stimulating factor (M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocyte lineage. Total and 16 poly (A) mRNA of bovine M-CSF were isolated from healthy bovine peripheral mononuclear cells stimulated by phobol 12-myristste 13-acetate (TPA). The more compatible cultured mononuclear cells were 5${\times}$10/ml for RNA isolation. TPA-activated mononuclear cells increased the level of M-CSF-mRNA more than concanavalin A (Con A) and lipopolysaccharide (LPS). The optimal analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) for14 Macrophage colonystimulating factor (M-CSF) as a growth factor required for bovine M-CSF was denaturation at 94$^{\circ}C$ for 1 minute, annealing at 57$^{\circ}C$ for 1 minute, extension at 72$^{\circ}C$ for 1 minute for 30 cycles. The size of cDNA of bovine M-CSF by RT-PCR was 774 base pairs. A 774 base pairs cDNA encoding bovine M-CSF was synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). Ligated cDNA was transformed to competent cells and then plasmid isolation and digestion was performed. Molecular cloning and sequencing were performed for cDNA of bovine M-CSF. The size of cloned cDNA of bovine M-CSF was 774base pairs. The homology of base sequence and amino acid sequence was 88% and 86% compared with known human M-CSF, respectively. From a high degree of sequence similarity, the obtained cDNA of bovine M-CSF is thought be a specific gene of bovine M-CSF.