• BMB Reports
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• v.46 no.3
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• pp.163-168
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• 2013

The AML1 gene is an essential transcription factor regulating the differentiation of hematopoietic stem cells into mature blood cells. Though at least 12 different alternatively spliced AML1 mRNAs are generated, three splice variants (AML1a, AML1b and AML1c) have been characterized. Here, using the reverse transcription-polymerase chain reaction with outward-facing primers, we identified a novel non-polyadenylated transcript from the AML1 gene, with exons 5 and 6 scrambled. The novel transcript resisted RNase R digestion, indicating it is a circular RNA structure that may originate from products of mRNA alternative splicing. The expression of the novel transcript in different cells or cell lines of human and a number of other species matched those of the canonical transcripts. The discovery provides additional evidence that circular RNA could stably exist in vivo in human, and may also help to understand the mechanism of the regulation of the AML1 gene transcription.

• YAKHAK HOEJI
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• v.57 no.4
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• pp.250-257
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• 2013

Biosimilars are important drugs in medicine and contain many glycosylated proteins. Thorough analysis of the glycosylated protein is a prerequisite for evaluation of biosimilar glycan drugs. A method to assess the diversity of N-glycosylation sites and N-glycans from biosimilar glycan drugs has been developed using two separate methods, LC-MS/MS and MALDI-TOF MS, respectively. Development of sensitive, accurate, and efficient methods for evaluation of glycoproteins is still needed. In this study, analysis of both N-glycosylation sites and N-glycans of glycoprotein was performed using the same LC-MS/MS with two different nano-LC columns, nano-C18 and nano-porous graphitized carbon (nano-PGC) columns. N-glycosylated proteins, including RNAse B (one N-glycosylation site), Fetuin (three sites), and ${\alpha}$-1 acid glycoprotein (four sites), were used, and small amounts of each protein were used for identification of N-glycosylation sites. In addition, high mannose N-glycans (one type of typical glycan structure), Mannose 5 and 9, eluted from RNAse B, were successfully identified using nano-PGC-LC MS/MS analysis, and the abundance of each glycan from the glycoprotein was calculated. This study demonstrated an accurate and efficient method for determination of N-glycosylation sites and N-glycans of glycoproteins based on high sensitive LC-MS/MS using two different nano-columns; this method could be applied for evaluation of the quality of various biosimilar drugs containing N-glycosylation groups.

• Journal of Life Science
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• v.13 no.6
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• pp.822-828
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• 2003

This study was carried out to investigate the changes of enzymes and micro inorganic nutrients that is associated with flower senescence during flower development in tulip cultivars, ‘Apeldoorn’ and ‘Golden Apeldoorn’. Ribonuclease, peroxidase and protease activities were gradually increased from the stage of early flowering to later Polyphenol oxidase showed the highest activity at stage 5, which the flower was in full bloom indicating that it acts at an initial stage of flower senescence. The protease activity was different in the petal extracts during flower development between the cultivars ‘Apeldoorn’ (red petal) and ‘Golden Apeldoorn’ (yellow petal). This result suggested that protease might relate to pigment biosynthesis in petal of tulip. In contrast to the decrease of inorganic nutrients K, Mn, Zn and P contents during floral development, Ca, Mg and Fe showed the gradual increasement that is similar with ribonuclease, peroxidase and protease. It suggests that they have some interaction during flower senescence.

• The Plant Pathology Journal
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• v.35 no.1
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• pp.41-50
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• 2019

Sugarcane bacilliform viruses (SCBV), which belong to the genus Badnavirus, family Caulimoviridae, are an important DNA virus complex that infects sugarcane. To explore the genetic diversity of the sugarcane-infecting badnavirus complex in China, we tested 392 sugarcane leaf samples collected from Fujian, Yunnan, and Hainan provinces for the occurrence of SCBV by polymerase chain reaction (PCR) assays using published primers SCBV-F and SCBV-R that target the reverse transcriptase/ribonuclease H (RT/RNase H) regions of the viral genome. A total of 111 PCR-amplified fragments (726 bp) from 63 SCBV-positive samples were cloned and sequenced. A neighbor-joining phylogenetic tree was constructed based on the SCBV sequences from this study and 34 published sequences representing 18 different phylogroups or genotypes (SCBV-A to -R). All SCBV-tested isolates could be classified into 20 SCBV phylogenetic groups from SCBV-A to -T. Of nine SCBV phylogroups reported in this study, two novel phylogroups, SCBV-S and SCBV-T, that share 90.0-93.2% sequence identity and show 0.07-0.11 genetic distance with each other in the RT/RNase H region, are proposed. SCBV-S had 57.6-92.2% sequence identity and 0.09-0.66 genetic distance, while SCBV-T had 58.4-90.0% sequence identity and 0.11-0.63 genetic distance compared with the published SCBV phylogroups. Additionally, two other Badnavirus species, Sugarcane bacilliform MO virus (SCBMOV) and Sugarcane bacilliform IM virus (SCBIMV), which originally clustered in phylogenetic groups SCBV-E and SCBV-F, respectively, are first reported in China. Our findings will help to understand the level of genetic heterogeneity present in the complex of Badnavirus species that infect sugarcane.

• Restorative Dentistry and Endodontics
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• v.36 no.1
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• pp.26-36
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• 2011

Objectives: In the present study, three kinds of tissues cells (pulp, gingiva, and periodontal ligament) were investigated if those cells express MMP and TIMP when they were stimulated with neuropeptides (substance P, CGRP) or proinflammatory cytokine, TNF-$\alpha$. Materials and Methods: The cells cultured from human dental pulp (PF), gingiva (GF) and periodontal ligament were (PDLF) stimulated with Mock, SP, TNF-$\alpha$, and CGRP for 24 hrs and 48 hrs. for an RNase protection assay and Enzyme Linked Immunosorbent Assay. Cells (PF, GF and PDLF) seeded in 100 mm culture dish were stimulated with SP ($10^{-5}$, $10^{-8}\;M$) or only with medium (Mock stimulation) for 4hrs and for 24 hrs for RNase Protection Assay, and they were stimulated with CGRP ($10^{-5}\;M$) and TNF-$\alpha$(2 ng/mL) for 24 hrs and with various concentraion of TNF-$\alpha$(2, 10, and 100 ng/mL) for Rnase Protection Assay with a human MMP-1 probe set including MMP 1, 2, 8, 7, 8, 9, 12, and TIMP 2, 3. In addition, cells (PF, GF and PDLF) were stimulated with Mock and various concentraion of TNF-$\alpha$(2, 10, and 100 ng/mL) for 24 hrs and with TNF-$\alpha$(10 ng/mL) for 48 hrs, and the supernatents from the cells were collected for Enzyme Linked Immunosorbent Assay (ELISA) for MMP-1 and MMP-13. Results: The expression of MMPs in PF, GF, PDLF after stimulation with SP and CGRP were not changed compared with Mock stimulation for 4 hrs and 24 hrs. The expression of MMP-1, -12, -13 24 hrs after stimulation with TNF-$\alpha$ were upregulated, however the expression of TIMP-3 in PF, GF, PDLF after stimulation with TNF-$\alpha$ were downregulated. TNF-$\alpha$(2 ng/mL, 10 ng/mL, 100 ng/mL) increased MMP-1 and MMP-12 expression in PF dose dependently for 24 hrs. Conclusions: TNF-$\alpha$ in the area of inflammation may play an important role in regulating the remodeling of dentin, cementum, and alveolar bone.

• Applied Biological Chemistry
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• v.21 no.2
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• pp.84-102
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• 1978

Barley embryoless half seeds were incubated in medium containing $10{\mu}M$ GA. Time course activity changes of ${\alpha}-amylase$ were studied in extract and medium seperately by the addition of $0.1{\mu}M,\;5{\mu}M,\;and\;10{\mu}M$ ABA in midcourse incubation of 10 hours after GA treatment. MAK profiles of nucleic acids in embryoless half seeds were compared either with $10{\mu}M$ GA treatment or concomitant treatment with $10{\mu}M$ GA and $10{\mu}M$ ABA after 10 hours incubation, Time course changes of weight increase, chlorophyll, protein and RNA consent in addition to RNase activity were studied in the presence of $10{\mu}M$ GA or $10{\mu}M$ ABA in barley coleoptile sections. After 20 hours incubation in the presence of plant hormones, MAK profiles of nucleic acids and reactive distribution of polysome and monosome were investigated. The above results were summarized as follows. 1) The production of ${\alpha}-amylase$ by treatment with GA alone increased at a linear rate in the incubation period and the active secretion of ${\alpha}-amylase$ began from 18 hours incubation in embryoless half seeds. 2) On the contrary to the partial inhibition by addition of $0.1{\mu}M$ ABA, the production of ${\alpha}-amylase$ was completely inhibited by both $5{\mu}M$ and $10{\mu}M$ ABA within 4 hours. Regardless of concentration of GA, the addition of $5{\mu}M$ ABA in midcourse completely inhibited the production of ${\alpha}-amylase$ 3) ABA treatment gave no effect on the secretion of ${\alpha}-amylase$. 4) There were no differences in RNA fractions between GA treatment and concomitant treatment with GA and ABA in the barlye embryoless half seeds. 5) While GA treatment increased the r-RNA fraction, ABA treatment decreased it and increased the s-RNA fraction in the coleoptile sections. 6) GA treatment increased RNA-DNA fraction best ABA treatment decreased it in the coleoptile sections. 7) While GA treatment suppressed RNase activity, ABA treatment increased it in the coleoptile sections. 8) GA treatment gave no great effect on the total RNA but ABA treatment remarkably diminished it in the coleoptile sections. 9) While GA treatment increased the growth and chlorophyll content, ABA treatment decreased them in the coleoptile sections. 10) GA treatment increased the protein synthesis and polysome formation but ABA treatment decreased them in the coleoptile sections. 11) The inhibition effect of ABA on polysome formation seemed to be resulted from the inhibition of r-RNA synthesis by ABA.

• Preventive Nutrition and Food Science
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• v.1 no.2
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• pp.269-277
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• 1996

Bacteriocins are proteins produced by a heterogeneous group of bacteria that have a bactericidal effect on closely related organisms. Recently, bacteriocins from lactic acid bacteria and other food-related organisms have been the subject of much research because of their potential as food biopreservatives. Various modifications of agar plate diffusion assays are the most widely used methods even though the limitations of such assays are generally recognized. The ability to obtain a concentrated crude preparation on bacteriocin by optimizing production parameters greatly simplifies recovery of bacteriocin on subsequent purification steps. Some studies performed to optimize bacteriocins have been purified to homogeneity, and the amino acid sequences of many of these purified bacteriocins have been determined. Obtaining characterization data on purified bacteriocin will minimize the risk of overlapping of research and confusion on identification of these compounds. Several me-chanisms leading to cell death have been hypothesized. These include depletion of the proton motive force(PMF) across the cell membrane: RNase and/or DNase activity within the sensitive cell; and pore formation and lysis of sensitive cells at the cell membrane.

• BMB Reports
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• v.29 no.2
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• pp.133-136
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• 1996

The affinity cleavage reagent Methidiumpropyl-EDTA-Iron(II) is applied to the structural analysis of 5S rRNA. Analysis of cleavage sites induced by MPE-Fe(II) on 5S rRNA shows that MPE intercalates easily between the unstable base pairs or into the bulges, thereby it strongly cuts the nucleosides nearby. The stable helical stems A, B, D and E as well as loop d are weakly cut. Most of the single-stranded loops are not cleaved. Based on the cleavage pattern of the 5S rRNA by MPE-Fe(II) and RNase V1, we suggest that MPE-Fe(II) may be used as a potential chemical probe in searching for the unstable helical regions of RNA, and for the sequences that appear to be involved in folding and distorting 5S rRNA.

• Molecules and Cells
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• v.40 no.12
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• pp.889-896
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• 2017

Nuclear bodies are subnuclear, spheroidal, and membraneless compartments that concentrate specific proteins and/or RNAs. They serve as sites of biogenesis, storage, and sequestration of specific RNAs, proteins, or ribonucleoprotein complexes. Recent studies reveal that a subset of nuclear bodies in various eukaryotic organisms is constructed using architectural long noncoding RNAs (arcRNAs). Here, we describe the unifying mechanistic principles of the construction and function of these bodies, especially focusing on liquid-liquid phase separation induced by architectural molecules that form multiple weakly adhesive interactions. We also discuss three possible advantages of using arcRNAs rather than architectural proteins to build the bodies: position-specificity, rapidity, and economy in sequestering nucleic acid-binding proteins. Moreover, we introduce two recently devised methods to discover novel arcRNA-constructed bodies; one that focuses on the RNase-sensitivity of these bodies, and another that focuses on "semi-extractability" of arcRNAs.

• BMB Reports
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• v.30 no.4
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• pp.258-261
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• 1997

A simple quantitative assay method for ribonuclease activity has been developed. This method is based on the decrease of fluorescence intensity emitted by the ethidium bromide bound to RNA due to the degradation of RNA by ribonuclease. The substrate RNA was reacted with ribonuclease A and the fluorescence intensity was measured after the addition of ethidium bromide. The intensity difference was calculated using a blank reaction mixture containing no RNase. Whole cellular RNA substrate produced a significant error and was not suitable for this assay method possibly because of local microheterogeniety caused by high molecular weight rRNA. but satisfying results were obtained with tRNA substrate. The intensity difference increased linearly by raising enzyme concentration up to $2{\times}10^{-4}$ Kunitz Units of ribonuclease A. More refined and reliable results were obtained by use of initial reaction velocities which were calculated from the plots of intensity difference vs time. A linear relationship between initial velocities and enzyme concentrations was observed up to 0.01 Kunitz Units of enzyme.