• BMB Reports
• /
• 제57권5호
• /
• pp.232-237
• /
• 2024

This study investigated how adipose tissue-derived mesenchymal stem cells (AT-MSCs) respond to chondrogenic induction using droplet-based single-cell RNA sequencing (scRNA-seq). We analyzed 37,219 high-quality transcripts from control cells and cells induced for 1 week (1W) and 2 weeks (2W). Four distinct cell clusters (0-3), undetectable by bulk analysis, exhibited varying proportions. Cluster 1 dominated in control and 1W cells, whereas clusters (3, 2, and 0) exclusively dominated in control, 1W, and 2W cells, respectively. Furthermore, heterogeneous chondrogenic markers expression within clusters emerged. Gene ontology (GO) enrichment analysis of differentially expressed genes unveiled cluster-specific variations in key biological processes (BP): (1) Cluster 1 exhibited up-regulation of GO-BP terms related to ribosome biogenesis and translational control, crucial for maintaining stem cell properties and homeostasis; (2) Additionally, cluster 1 showed up-regulation of GO-BP terms associated with mitochondrial oxidative metabolism; (3) Cluster 3 displayed up-regulation of GO-BP terms related to cell proliferation; (4) Clusters 0 and 2 demonstrated similar up-regulation of GO-BP terms linked to collagen fibril organization and supramolecular fiber organization. However, only cluster 0 showed a significant decrease in GO-BP terms related to ribosome production, implying a potential correlation between ribosome regulation and the differentiation stages of AT-MSCs. Overall, our findings highlight heterogeneous cell clusters with varying balances between proliferation and differentiation before, and after, chondrogenic stimulation. This provides enhanced insights into the single-cell dynamics of AT-MSCs during chondrogenic differentiation.

• Journal of Microbiology
• /
• 제42권4호
• /
• pp.361-364
• /
• 2004

Internal ribosome entry site (IRES) of the hepatitis C virus (HCV) is known to be essential for HCV replication and most conserved among HCV variants. Hence, IRES RNA is a good therapeutic target for RNA-based inhibitors, such as ribozymes. We previously proposed a new anti-HCV modulation strategy based on trans-splicing ribozymes, which can selectively replace HCV transcripts with a new RNA that exerts anti-HCV activity. To explore this procedure, sites which are accessible to ribozymes in HCV IRES were previously determined by employing an RNA mapping method in vitro. In this study, we evaluate the intracellular accessibility of the ribozymes by comparing the trans-splicing activ­ities in cells of several ribozymes targeting different sites of the HCV IRES RNA. We assessed the intra­cellular activities of the ribozymes by monitoring their target-specific induction degree of both reporter gene activity and cytotoxin expression. The ribozyme capable of targeting the most accessible site iden­tified by the mapping studies then harbored the most active trans-splicing activity in cells. These results suggest that the target sites predicted to be accessible are truly the most accessible in the cells, and thus, could be applied to the development of various RNA-based anti-HCV therapies.

• Journal of Microbiology and Biotechnology
• /
• 제25권7호
• /
• pp.963-977
• /
• 2015

The well-characterized gram-positive bacterium Bacillus subtilis is an outstanding industrial candidate for protein expression owing to its single membrane and high capacity of secretion, simplifying the downstream processing of secretory proteins. During the last few years, there has been continuous progress in the illustration of secretion mechanisms and application of this robust host in various fields of life science, such as enzyme production, feed additives, and food and pharmaceutical industries. Here, we review the developments of Bacillus subtilis as a highly promising expression system illuminating strong chemical- and temperatureinducible and other types of promoters, strategies for ribosome-binding-site utilization, and the novel approach of signal peptide selection. Furthermore, we outline the main steps of the Sec pathway and the relevant elements as well as their interactions. In addition, we introduce the latest discoveries of Tat-related complex structures and functions and the countless applications of this full-folded protein secretion pathway. This review also lists some of the current understandings of ATP-binding cassette transporters. According to the extensive knowledge on the genetic modification strategies and molecular biology of Bacillus subtilis, we propose some suggestions and strategies for improving the yield of intended productions. We expect this to promote striking future developments in the optimization and application of this bacterium.

• 한국자기공명학회논문지
• /
• 제17권1호
• /
• pp.54-58
• /
• 2013

SAV0506 is an 87 residue hypothetical protein from Staphylococcus aureus strain Mu50 and also predicted to have similar function to ribosome associated heat shock protein, Hsp 15. Hsp15 is thought to be involved in the repair mechanism of erroneously produced 50S ribosome subunit. In this report, we present the sequence specific backbone resonance assignment of SAV0506. About 82.5% of all resonances could be assigned unambiguously. By analyzing deviations of the $C{\alpha}$ and $C{\beta}$ chemical shift values, we could predict the secondary structure of SAV0506. This study is an essential step towards the structural characterization of SAV0506.

• 미생물학회지
• /
• 제30권6호
• /
• pp.472-478
• /
• 1992

HTLV-1 이 pol 유전자산물을 합성하기 위해서는 genome-size mRNA 를 번역해 나아가는 ribosome 이 -1 방향으로 두차례 frame 을 바꾸어야 한다. 우리는 단 한차계의 frameshifting 만으로도 많은 양의 Gag-Pro-Pol polyprotein 의 합성어 가능하도록 gag 와 pro 유전자의 frame 을 연결시킨 mutant RNA 를 제작하였다. 이 돌연변이를 이용하여 ribosome 의 shift site 하류영역내에 형성이 예상되는 RNA 의 이차구조 또는 삼차 구조가 -1 frameshifting 을 결정하는 인자로서 작용하는지의 여부를 조사하였다. 결손변이주의를 해석한 결과 pro-pol 중첩영역에서 효율적으로 frameshifting 이 일어나기 위해서는 stem-loop 가 필수적으로 형성되어야 하지만 pseudoknot 의 형성은 그다지 중요하지 않다는 사실을 알았다.

• BMB Reports
• /
• 제49권4호
• /
• pp.199-200
• /
• 2016

Gene regulation in the brain is essential for long-term plasticity and memory formation. Despite this established notion, the quantitative translational map in the brain during memory formation has not been reported. To systematically probe the changes in protein synthesis during memory formation, our recent study exploited ribosome profiling using the mouse hippocampal tissues at multiple time points after a learning event. Analysis of the resulting database revealed novel types of gene regulation after learning. First, the translation of a group of genes was rapidly suppressed without change in mRNA levels. At later time points, the expression of another group of genes was downregulated through reduction in mRNA levels. This reduction was predicted to be downstream of inhibition of ESR1 (Estrogen Receptor 1) signaling. Overexpressing Nrsn1, one of the genes whose translation was suppressed, or activating ESR1 by injecting an agonist interfered with memory formation, suggesting the functional importance of these findings. Moreover, the translation of genes encoding the translational machineries was found to be suppressed, among other genes in the mouse hippocampus. Together, this unbiased approach has revealed previously unidentified characteristics of gene regulation in the brain and highlighted the importance of repressive controls.

• BMB Reports
• /
• 제36권6호
• /
• pp.538-544
• /
• 2003

The hepatitis C virus (HCV) is a major causative agent of chronic hepatitis and hepatocellular carcinoma. The development of alternative antiviral therapies is warranted because current treatments for the HCV infection affect only a limited number of patients and lead to significant toxicities. The HCV genome is exclusively present in the RNA form; therefore, ribozyme strategies to target certain HCV sequences have been proposed as anti-HCV treatments. In this study, we determined which regions of the internal ribosome entry site (IRES) of HCV are accessible to ribozymes by employing an RNA mapping strategy that is based on a trans-splicing ribozyme library. We then discovered that the loop regions of the domain IIIb of HCV IRES appeared to be particularly accessible. Moreover, to verify if the target sites that were predicted to be accessible are truly the most accessible, we assessed the ribozyme activities by comparing not only the trans-splicing activities in vitro but also the trans-cleavage activities in cells of several ribozymes that targeted different sites. The ribozyme that could target the most accessible site identified by mapping studies was then the most active with high fidelity in cells as well as in vitro. These results demonstrate that the RNA mapping strategy represents an effective method to determine the accessible regions of target RNAs and have important implications for the development of various antiviral therapies which are based on RNA such as ribozyme, antisense, or siRNA.

• 대한수의학회지
• /
• 제39권4호
• /
• pp.772-777
• /
• 1999

The uncaped genomic RNA of bovine viral diarrhea virus (BVDV) initiates translation by recruitment of eukaryotic translation initiation factors at the internal ribosome entry site (IRES). N-terminal protease ($N^{pro}$) is the first translation product of the open reading frame (ORF). By using the vaccinia virus SP6 RNA polymerase transient expression system, we showed previously that deletion of $N^{pro}$ region reduced translation by 21%. To better understand the biological significance of $N^{pro}$ for translation, we carried out a mutational analysis of the $N^{pro}$ region of BVDV cloned in the intercistronic region of a bicistronic reporter plasmid. We constructed a bicistronic expression vector in which the entire 5 UTR and the mutated $N^{pro}$ region (${\Delta}386-901$, ${\Delta}415-901$ and ${\Delta}657-901$) was cloned between two reporter genes, chloramphenicol acetyltransferase (CAT) and luciferase (LUC). In vivo translation analyses showed that $N^{pro}$ region was dispensible for efficient translation. The results indicate that the $N^{pro}$ region is not essential for BVDV RNA translation and the 3' boundary of BVDV IRES is expanded into $N^{pro}$ region, suggesting that $N^{pro}$ may not play a major role in BVDV replication.

• Applied Microscopy
• /
• 제1권1호
• /
• pp.11-17
• /
• 1969

Conidio spores of Aspergillus niger (strain No. NRRL 330) cultured on potato dextrose agar media were studied by electron microscopy, using the thin sectioning techniques. Conidio spores to be sectioned were fixed by triple methods with $K_2Cr_2O_7$, Glutaraldehyde and $OsO_4$. After dehydrated with alcohol, the specimens were embedded in metacrylate and epon resin media, and thinly sectioned by Porter-Blum MT-2. After sectioned these specimens were negative-stained with uranyl acetate and observed. by Hitachi HS-6 electron microscope. The results of this experiment were summarized as follows. 1. The structures of spore ,wall system seem to be formed 4 layers; exosporium, basal layer, spore coat and unit cell membrane. The protuberance of spore surface that was looked like hair appears to be protrusived from the basal layer. 2. The 3 layers of unit cell membrane was constituted outer layer membrane, inner layer membrane and inter-mediate light layer. 3. The structures of intra cytoplasmic membrane appear as spiral form which was consisted of 3 layers membrane system; outer membrane, inner membrane, and intermediate layer, which has pits. 4. The cement substance of spore coat and cortex may be changed quantitatively by physiological state in cell. 5. In some cases, we observed that the ribosome was transformed into poly ribosome group, and the storage materials and the protein crystals were changed variously. It. has been suggested that the morphological change of some cytoplasmic materials may be caused by some specialized function of the physiological stage.

• Advances in Traditional Medicine
• /
• 제3권3호
• /
• pp.151-156
• /
• 2003

The leaves of Mirabilis jalapa L contains protein fraction presumed ribosome-inactivating protein (RIP). RIP is a group of protein that has RNA N-glycosidase activity that is capable to inhibit protein synthesis. Protein fraction of the plant was shown to be cytotoxic on HeLa cell-line, however, the mechanism by which the protein kill the cells is not identified yet, whether trough apoptosis, necrosis, or other mechanism. This research aim to study the mechanism of cell death caused by the protein fraction isolated from the leaves of this plant on HeLa and Raji cell-line, as representative of different kind of cancer cells. Results showed that protein fraction isolated from the leaves of Mirabilis jalapa L was more cytotoxic to HeLa cell-line (LC50: 0.65 mg/ml) than to Raji cell-line (1.815 mg/ml) on 48 hours incubation time. Moreover, it was demonstrated that the death of HeLa cells caused by the protein fraction was due to induction of apoptosis, while on Raji cell-line was due to non-apoptosis way, presumably via necrosis.