• Biotechnology and Bioprocess Engineering:BBE
• /
• 제5권6호
• /
• pp.422-430
• /
• 2000

Mechanism of lignin biodegradation caused by basidiomycetes and the history of lignin biodegradation studies were briefly reviewed. The important roles of fungal extracellular ligninolytic enzymes such as lignin and manganese peroxidases (LiP and MnP) were also summarized. These enzymes were unique in their catalytic mechanisms and substrate specificities. Either LiP or MnP system is capable of oxidizing a variety of aromatic substrates via a one-electron oxidation. Extracellular fungal system for aromatic degradation is non-specific, which recently attracts many people working a bioremediation field. On the other hand, an intracellular degradation system for aromatic compounds is rather specific in the fungal cell. Structurally similar compounds were prepared and metabolized, indicating that an intracellular degradation strategy consisted of the cellular systems for substrate recognition and metabolic response. It was assumed that lignin-degrading fungi might be needed to develop multiple metabolic pathways for a variety of aromatic compounds caused by the action of non-specific ligninolytic enzymes on lignin. Our recent results on chemical stress responsible factors analyzed using mRNA differential display techniques were also mentioned.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2003년도 생물공학의 동향(XIII)
• /
• pp.787-788
• /
• 2003

• Asian-Australasian Journal of Animal Sciences
• /
• 제17권9호
• /
• pp.1192-1196
• /
• 2004

In order to detect the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performed to investigate the differences in gene expression of pig's Longissimus dorsi between the high-parent heterosis cross combination Landrace${\times}$Large White and the mid-parent heterosis cross combination Large White${\times}$Meishan. Three pig purebreds, Large White, Meishan, and Landrace and four types of reciprocal $F_1$ hybrids were analyzed using nine 3'-end anchored primers in combination with ten 5'-end arbitrary primers and nearly 7,000 reproducible bands were examined. The patterns of gene expression of each cross combination were analyzed and eight common patterns (fifteen kinds) were found. When the results from the two cross combinations were put together and compared, eight different typical expression patterns were observed, these indicated that the patterns of gene expression of these two cross combinations had obvious differences. Gene expression correlation and cluster analyses of the two cross combinations indicated that the gene expression of the mid-parent heterosis cross combination was correlated with maternal effect, but in the high-parent heterosis cross combination, paternal effect acted in the gene expression of the hybrids or the gene expression of the hybrids was biased towards one parent.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제13권1호
• /
• pp.31-35
• /
• 2006

The ubiquitin conjugating enzyme 2 (E2) is core component of ubiquitin proteasome pathway (UPP) which represents a selective mechanism for intracellular proteolysis in eukaryotic cells. The E2 has been implicated in the intracellular transfer of ubiquitin to target protein. We show here the involvement of E2 in antiviral immune of Bombyx mori to Bombyx mori nuclear polyhedrosis virus (BmNPV). In this study, mRNA fluorescent differential display PCR (FDD-PCR) was performed with BmNPV highly resistant silkworm strain NB and susceptible silkworm strain 306. At 24 h post BmNPV infection, FDD-PCR with the arbitrary primer AP34 showed that one cDNA band was down-regulated in the midgut of resistant strain, but highly expressed in susceptible strain. The deduced amino acid sequence of this cDNA clone share 99% identity with the recently published B. mori ubiquitin conjugating enzyme E2 (Genbank NO: DQ311351). Fluorescent quantitative PCR corroborated down regulation of E2 in resistant strain. We there conclude that BmNPV infection evokes strong response of susceptible strain including activation of UPP. BmNPV may evolve escape mechanisms that manipulate the UPP in order to persist in the infected host. In addition, the identification of down-regulation of E2 in resistant strain, as well as structure data, are essential to understanding how UPP operates in silkworm antiviral immune to BmNPV disease.

• Journal of Plant Biotechnology
• /
• 제30권4호
• /
• pp.323-328
• /
• 2003

토양 내의 고농도의 염은 심각한 환경스트레스 중의 하나로 농작물의 생산을 감소시킨다. 식물은 염 스트레스로부터 벗어나기 위하여 많은 단백질을 합성한다든지 유전자들의 발현을 조절하는 등 여러 가지 생리, 생화학적인 변화를 일으킨다. 퉁퉁마디는 우리나라에 자생하는 염생식물로 갯벌과 염전주위에서 생육한다. 퉁퉁마디의 생화학적, 분자생물학적 내염성 기구를 이해하기 위하여 differential display방법으로 NaCl에 의하여 발현이 증가되는 cDNA들을 분리하였다. 본 연구에서는 그 중 하나인 ShADL의 특성을 조사하였다. ShADL은fructose-1, 6-bisphosphate aldolase와 높은 유사성을 보였다. 이 유전자는 1293bp길이에 359개의 아미노산으로 구성된 open reading frame을 포함하고 있으며, 이로부터 추정되는 분자량은 39 kDa이었다. ShADL단백질은 애기장대의 aldolase와 86％의 높은 유사성을 나타내었으며 같은 염생식물인 com-mon ice plant의 adolase와는 78％의 유사성을 보였다. Northern 분석결과, ShADL 유전자는 NaCl의 농도가 증가함에 따라 발현량이 급격히 증가하는 것으로 나타났다.

• Molecules and Cells
• /
• 제25권1호
• /
• pp.78-85
• /
• 2008

In a search for new molecular pathways associated with asthma, we performed an mRNA differential display analysis using total RNA extracted from the tracheal tissues of ovalbumin (OVA)-challenged mice and sham controls. cDNAs corresponding to mRNAs for which expression levels were altered by OVA-challenge were isolate and sequenced. Twenty-eight genes differentially expressed in sham and OVA challenged mice were identified. A GenBank BLAST homology search revealed that they were related to cytoskeleton remodeling, transcription, protein synthesis and modification, energy production, and cell growth and differentiation. Two were selected for further characterization. Up-regulation of both the perinatal skeletal myosin heavy chain (skMHC) and fast skeletal muscle myosin light chain (skMLC) genes was confirmed by RT-PCR of trachea tissue from OVA challenged mice. Overexpression of skMLC protein was observed in the smooth muscle layers of OVA-challenged mice by immunohistochemistry, and the surface areas stained with skMLC antibody increased in the OVA-challenged mice. The overexpression of skMLC in murine asthma may be associated with the changes of bronchial smooth muscle.

• 대한의생명과학회지
• /
• 제15권4호
• /
• pp.301-307
• /
• 2009

Whole-body insulin resistance results largely from impaired insulin-stimulated glucose disposal in skeletal muscle. Our previous studies using differential display and quantitative real-time RT-PCR have shown that a novel cDNA band (DD23) had a higher level of expression in insulin resistant skeletal muscle and it was correlated with whole-body insulin action, independent of age, sex, and percent body fat. In this study, we cloned and characterized DD23. The DD23 sequence is part of the 3'UTR region of the RNA binding motif, single stranded interacting protein (RBMS3). We have cloned the full length cDNA for RBMS3 and identified two splice variants. These variants named DD23-L and DD23-S have 15 and 14 exons respectively and differ from RBMS3 in the 3'UTR significantly. Northern blot analyses showed that an ~8.8 kb mRNA transcript of DD23 was predominantly expressed in skeletal muscle and to a lesser extent in placenta, but not in heart, brain, lung, liver, or kidney, unlike RBMS3. Elevated expression levels of these novel alternatively spliced variants of RBMS3 in skeletal muscle may play a role in whole body insulin resistance.

• International Journal of Oral Biology
• /
• 제37권2호
• /
• pp.51-56
• /
• 2012

Tooth development involves bud, cap, bell and hard tissue formation stages, each of which is tightly controlled by regulatory molecules. The aim of this study was to identify genes that are differentially expressed during dental hard tissue differentiation. Sprague-Dawley rats at postnatal days 3, 6 and 9 were used in the analysis. Differential display RT-PCR (DD-PCR) was used to screen differentially expressed genes between the 2nd (root formation stage, during mineralization) and 3rd (cap stage, before mineralization) molar germs at postnatal day 9. The DNA detected in the 2nd molar germs showed homology to osteonectin only (GenBank accession no. NM_012656.1). The level of osteonectin mRNA expression was much higher in the 2nd molar germs than in the 3rd molar germs and was found to increase in a time-dependent manner from the early bell stage to the root formation stage in the 2nd molar germs. The pattern of osteonectin protein expression was consistent with these RT-PCR results. Osteonectin protein was found by immunofluorescent analysis to localize in odontoblasts and preodontoblasts rather than the dentin matrix itself. Further studies are needed to validate the involvement of osteonectin in mineralization and root formation.

• International Journal of Oral Biology
• /
• 제33권4호
• /
• pp.205-211
• /
• 2008

Gingival overgrowth can cause dental occlusion and seriously interfere with mastication, speech, and dental hygiene. It is observed in 25 to 81% of renal transplant patients treated with cyclosporine A (CsA). CsA-induced gingival overgrowth (CIGO) is caused by quantitative alteration of the extracellular matrix components, particularly collagen. However, the molecular mechanisms involved in the pathogenesis of CIGO remain poorly understood, despite intense clinical and laboratory investigations. The aim of the present work is to identify differentially expressed genes closely associated with CIGO. Human gingival fibroblasts were isolated by primary explant culture of gingival tissues from five healthy subjects (HGFs) and two patients with the CIGO (CIGO-HGFs). The proliferative activity of CsA-treated HGFs and CIGO-HGFs was examined using the MTT assay. The identification of differentially expressed genes in CsA-treated CIGO-HGF was performed by differential display reverse transcriptase-polymerase chain reaction (RT-PCR) followed by DNA sequencing. CsA significantly increased the proliferation of two HGFs and two CIGO-HGFs, whereas three HGFs were not affected. Seven genes, including the beta subunit of prolyl 4-hydroxylase (P4HB) and testican 1, were upregulated by CsA in a highly proliferative CIGO-HGF. The increased P4HB and testican-1 mRNA levels were confirmed in CsA-treated CIGO-HGFs by semiquantitative RT-PCR. Furthermore, CsA increased type I collagen mRNA levels and suppressed MMP-2 mRNA levels, which are regulated by P4HB and testican-1, respectively. These results suggest that CsA may induce gingival overgrowth through the upregulation of P4HB and testican-1, resulting in the accumulation of extracellular matrix components.

• 생명과학회지
• /
• 제17권12호
• /
• pp.1641-1648
• /
• 2007

Yippee 패밀리를 구성하는 yippee-유사 단백질들은 한 개의 zinc-finger 도메인을 지닌 Drosophila yippee 단백질의 homolog로서 모든 진핵생물에 존재하는 것으로 알려졌으나 그 기능은 밝혀진 바가 없다. 인체 T 림프구의 활성화과정 중 발현수준이 변화하는 유전자들을 선별하기 위해 인체 말초혈액에서 분리한 resting T 세포, immobilized anti-CD3에 의해 26시간 혹은 30시간 동안 활성화시킨 T 세포로부터 각각 정제한 total RNA를 이용하여 ODD-PCR을 수행한 결과, resting T 세포에서는 발현되지만 immobilized anti-CD3 활성화에 의해 세포주기를 개시하여 $G_1/S$ boundary에 도달한 T 세포들로부터는 전혀 발현되지 않는 흥미로운 유전자로서 Drosophila yippee 단백질 유전자의 인체 homolog인 YPEL5 유전자를 분리하였다. 노던 블로팅법으로 T 세포 활성화에 뒤이은 YPEL5 mRNA의 발현 변화를 조사한 결과, ${\sim}2.2kb$ 크기의 YPEL5 mRNA는 resting T 세포를 비롯하여 immobilize anti-CD3에 의한 활성화 후 1.5시간까지는 확인되었으나 활성화 후 5시간 이후부터 48시간에 이르는 시간에는 전혀 확인되지 않았다. YPEL5 단백질을 GFP-fusion 단백질로서 인체 암세포주인 HeLa 세포에 transfection하여 발현시킨 결과, GFP-YPEL5 단백질이 모두 핵에 위치하는 것으로 나타나 YPEL5 단백질이 핵단백질임을 확인하였다. 또한 YPEL5의 기능을 규명하기 위해 YPEL5 발현벡터를 HeLa 세포에 transfection 하고 발현시켜 HeLa 세포의 증식에 미치는 YPEL5의 영향을 MTT assay로 분석한 결과, vector plasmid를 transfection시킨 대조구의 47% 수준으로 세포증식이 감소하는 것으로 나타났다. 이러한 결과들은 YPEL5 mRNA의 발현이 T 세포 수용체를 통한 T 세포 활성화의 초기단계에 현저히 감소됨을 보여주며, 또한 YPEL5가 핵단백질로서 세포증식에 대해 저해효과를 미칠 수 있음을 시사한다.