• BMB Reports
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• v.53 no.12
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• pp.611-621
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• 2020

Bacterial endoribonuclease toxins belong to a protein family that inhibits bacterial growth by degrading mRNA or rRNA sequences. The toxin genes are organized in pairs with its cognate antitoxins in the chromosome and thus the activities of the toxins are antagonized by antitoxin proteins or RNAs during active translation. In response to a variety of cellular stresses, the endoribonuclease toxins appear to be released from antitoxin molecules via proteolytic cleavage of antitoxin proteins or preferential degradation of antitoxin RNAs and cleave a diverse range of mRNA or rRNA sequences in a sequence-specific or codon-specific manner, resulting in various biological phenomena such as antibiotic tolerance and persister cell formation. Given that substrate specificity of each endoribonuclease toxin is determined by its structure and the composition of active site residues, we summarize the biology, structure, and substrate specificity of the updated bacterial endoribonuclease toxins.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.5
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• pp.695-701
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• 1999

The hypothalamus is the classic site of synthesis of arginine vasotocin as neurohypophyseal hormone in the chicken. However, high concentrations of arginine vasotocin were also measured in ovarian tissues by radioimmunoassay. At first, we observed specific positive signal of mRNA encoding AVT in the hypothalamus by Northern hybridization. However, we could not find any specific bands in ovarian and uterine tissues. For evidence of transcription of the arginine vasotocin gene ingonadal tissues of the chicken, this study has applied the polymerase chain reaction as a highly sensitive assay. The hypothalamus, the four largest preovulatory ovarian follicles and the shell gland (uterus) were collected at 4 h and 20 h before oviposition. The ovarian follicular tissues were separated into granulose theca interns and theca externa layers. The uterine tissues were separated into myometrium and endometrium The extracted mRNA was converted to cDNA by reverse-transcriptase using oligo-$d(T)_{15}$ primer. Then, the cDNA was amplified by Vent polymerase and arginine vasotocin specific primers. The amplification reaction was incubated by 30 cycles successively, $95^{\circ}C$, $55^{\circ}C$ and $72^{\circ}C$ earth for 1 min. Te comparisons of the mRNA levels encoding arginine vasotocin between the tissues were determined by semi-quantification methods. After amplification of the cDNA, the PCR products were detected in hypothalamus, ovarian tissues and uterine tissues. The results of semi-quantification showed that the levels of arginine vasotocin mRNA in ovarian iud uterine tissues were about from 1/50 to 1/1000 when compared to that in the hypothalamus. The very low levels of mRNA encoding arginine vasotocin in ovarian and uterine tissues probably led us to conclude that arginine vasotocin may play a role of local mediate acting autocrine and/or paracrine.

• Parasites, Hosts and Diseases
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• v.37 no.4
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• pp.257-263
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• 1999

Identification of the genes responsible for the recovery of virulence in brain-passaged Acanthamoeba culbertsoni was attempted via mRNA differential display polymerase chain reaction (mRNA DD-PCR) analysis. In order to identify the regulatory changes in transcription of the virulence related genes by the brain passages, mRNA DD-PCR was performed which enabled the display of differentially transcribed mRNAs after the brain passages. Through mRNA DD-PCR analysis. 96 brain-passaged amoeba specific amplicons were observed and were screened to identify the amplicons that failed to amplify in the non-brain-passaged amoeba mRNAs. Out of the 96 brain-passaged amoeba specific amplicons, 12 turned out to be amplified only from the brain-passaged amoeba mRNAs by DNA slot blot hybridization. The clone, A289C, amplified with an arbitrary primer of UBC ＃289 and the oligo dT$_{11}$-C primer, revealed the highest homology (49.8％) to the amino acid sequences of UPD-galactose lipid transferase of Erwinia amylovora, which is known to act as an important virulence factor. The deduced amino acid sequences of an insert DNA in clone A289C were also revealed to be similar to cpsD, which is the essential gene for the expression of type III capsule in group B streptococcus. Upregulated expression of clone A289C was verified by RNA slot blot hybridization. Similar hydrophobicity values were also observed between A289C (at residues 47-66) and the AmsG gene of E. amylovora (at residues 286-305: transmembrane domains). This result suggested that the insert of clone A289C might play the same function as galactosyl transferase controlled by the AmsG gene in E. amylovora.a.

• Journal of Plant Biology
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• v.39 no.4
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• pp.279-286
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• 1996

To know the changes of rbcL mRNA level by illumination, Northern hybridization analysis was performed with maize (Zea mays L.cv. Golden X Bantam). The average level of rbcL. mRNA in the light-grown shoots was 3.1 times higher than that of the dark-grown shoots after 6 to 10 growth days. The maximum difference of rbcL mRNA level between the dark-grown and the light-grown shoots was 5.1 folds. These results indicate that accumulation of rbcL mRNAin maize shoots is induced by light. Since the transcriptional DNA binding proteins and their cognate promoter elements, we carried out gel-retardation assays to elucidate the specific binding proteins on the rbcL promoter. It was found that plastid proteins of light-grown shoots bound to the R2 DNA fragment (-33 to -229) and R3 DNA fragment (-230 to -418 from ATG) of the rbcL promoter. From the results of competitive binding assays and heat or protease treatments, it was demonstrated that the bindings were sequence-specific DNA-protein interactions. Therefore, it could be concluded that the rbcL promoter region has at least two specific recognition sites for plastid proteins.

• Development and Reproduction
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• v.14 no.3
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• pp.207-214
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• 2010

The tremendous changes of uterine endometrium are observed during early pregnancy and protease and their inhibitors are involved in regulation of cell proliferation and remodeling of the tissues through remodeling the extracellular matrix (ECM). Some of the proteases and protease inhibitors have been suspected to a factor in endometrial changes but many parts of their expression profiles and the physiological roles are not uncovered. To evaluate the functional roles of them, in this study the expression profiles of proteases and protease inhibitors were analyzed using real-time quantitative PCR analysis. Mmp9 (matrix metalloproteinase 9) mRNA levels peaked on day 4 at the time of implantation. On the other hand, Ela2 (neutrophil elastase, NE) mRNA levels were peaked on day 2 of pregnancy. Its expression were decreased until day 4 of pregnancy but increased rapidly until day 7 of pregnancy and decreased again. NE inhibitor Slpi (secretory leukocyte protease inhibitor, SLPI) mRNA levels were related with the implantation stage and with the levels of Ela2. At the time of implantation the expression levels of Slpi mRNA were about 5 times higher than the Ela2 mRNA in the uterus. In the implantation stage embryos, Mmp9 specific mRNA was only detected at the blastocyst. On the other hand, the expression level of SLPI was higher than that of the Ela2 mRNA at blastocyst and 4.5 day p.c. embryos. Based on these results it is suggested that MMP9, SLPI, and NE have important physiological role in embryo implantation both in uterus and embryos.

• Animal cells and systems
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• v.1 no.1
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• pp.115-121
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• 1997

To precisely analyze the role of ovarian steroids in the regulation of laminin chain gene expression in mouse uterine tissues, the ovariectomized mouse model was used. Ovariectomized mice received a single injection of steroid hormones and total RNA was isolated from whole uterine tissues. Messenger RNA levels of each laminin chain (A, 81, and 82) were determined by competitive RT-peR procedures. Estradiol decreased mRNA levels of laminin 81 chain about two-fold, and 82 chain rather moderately. Estradiol-induced inhibition of laminin 81 and 82 chain mRNA levels were completely blocked by pretreatment with estrogen receptor antagonist tamoxifen. Estriol, a short acting estrogen which cannot induce hyperplastic responses of rodent uterine tissues, also showed an inhibitory effect on 81 and 82 chain mRNA levels, while estrone, an inactive estrogen, failed to influence either 8 chain mRNA levels. Effects of steroids on A chain mRNA level were quite different from those on 8 chains. Laminin A chain mRNA level was slightly increased by estradiol treatment, but negatively affected by progesterone. Progesterone treatment greatly increased both 8 chain mRNA levels, but slightly decreased A chain mRNA level compared to the control. The effect of progesterone on laminin chain-specific mRNA levels was further increased by co-injection of estradiol in a time-dependent manner. Progesterone-induced 81 and 82 chain mRNA transcription was inhibited by RU486, a synthetic anti-progesterone /anti-glucocorticoid. The present study demonstrates for the first time that steroids are able to regulate laminin gene expression in mouse uterine tissues, indicating that steroid-regulated laminin gene expression is involved in uterine growth and probably differentiation.

• Genomics & Informatics
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• v.7 no.4
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• pp.181-186
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• 2009

Myotonic dystrophy type 1 (DM1), which is a dominantly inherited neurodegenerative disorder, results from a CTG trinucleotide repeat expansion in the 3'-untranslated region (3'-UTR) of the myotonic dystrophy protein kinase (DMPK) gene. Retention of mutant DMPK (mDMPK) transcripts in the nuclei of affected cells has been known to be the main cause of pathogenesis of the disease. Thus, reducing the RNA toxicity through elimination of the mutant RNA has been suggested as one therapeutic strategy against DM1. In this study, we suggested RNA replacement with a trans -splicing ribozyme as an alternate genetic therapeutic approach for amelioration of DM1. To this end, we identified the regions of mDMPK 3'-UTR RNA that were accessible to ribozymes by using an RNA mapping strategy based on a trans-splicing ribozyme library. We found that particularly accessible sites were present not only upstream but also downstream of the expanded repeat sequence. Repair or replacement of the mDMPK transcript with the specific ribozyme will be useful for DM1 treatment through reduction of toxic mutant transcripts and simultaneously restore wild-type DMPK or release nucleus-entrapped mDMPK transcripts to the cytoplasm.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.707-713
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• 2000

Myasthenia gravis (MG) is caused mainly by autoantibodies directed against acetylcholine receptors located in the postsynaptic muscle cell membrane. Using in vitro selection techniques, we isolated an RNA containing 2'-fluoro pyrimidines that can specifically and avidly ($K_d$ ~25 nM) bind rat monoclonal antibody called mAb198, which recognizes the main immunogenic region on the acetylcholine receptors. This RNA can act as a very effective decoy and block mAb198 binding to the receptors in vitro. Furthermore, this RNA decoy can prevent the antigenic modulation of the acetylcholine receptor caused by mAb198 in human muscle cell cultures with and $IC_{50} $of approximately $2.4{\mu}M$. These results indicate that the RNA selected in this study is a more potent decly inhibitor of myashthenic antibodies than the previously identified RNA with 2'-amino pyrimidines [11]. Moreover, this RNA cross-reacts with autoantibodies from patients with MG and can protect human cells from the effects of these antibodies. These observations have important implications for developing an antigen-specific treatment of autoimmune diseases including MG, which is based on decoy RNAs selected in vitro.

• 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 Genetic Medicine
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• v.2 no.1
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• pp.31-34
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• 1998

The N-methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, removes N-methylpurine and other damaged purines induced in DNA. Tissue-specific mRNA levels of the N-methylpurine-DNA glycosylase (MPG) were investigated in Balb/c mice of four different growing stages; newborn, 1, 4 and 8-weeks postpartum. MPG expressions in the newborn and the 8-week-old mice were the highest in thymus and testis, respectively. The tested tissues of the newborn mice had consistently higher MPG mRNA level than 8-week-old adults except in testis and thymus. The MPG mRNA level in testis was the lowest in the newborn mice, but it attained the highest in the 8-week-old mice. The levels of MPG mRNA among the different tissues in the newborn and the 8-week-old mice were more than 9.0 and 19.0-fold respectively. These results suggest that the of MPG expression was dependent on the growing stage and had tissue-specificity.