• BMB Reports
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• v.40 no.1
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• pp.107-112
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• 2007

To study the functioning of HSP70 in Escherichia coli, we selected NtHSP70-2 (AY372070) from among three genomic clones isolated in Nicotiana tabacum. Recombinant NtHSP70-2, containing a hexahistidine tag at the amino-terminus, was constructed, expressed in E. coli, and purified by $Ni^{2+}$ affinity chromatography and Q Sepharose Fast Flow anion exchange chromatography. The expressed fusion protein, $H_6NtHSP70$-2 (hexahistidine-tagged Nicotiana tabacum heat shock protein 70-2), maintained the stability of E. coli proteins up to 90$^{\circ}C$. Measuring the light scattering of luciferase (luc) revealed that NtHSP70-2 prevents the aggregation of luc without ATP during high-temperature stress. In a functional bioassay (1 h at 50$^{\circ}C$) for recombinant $H_6NtHSP70$-2, E. coli cells overexpressing $H_6NtHSP70$-2 survived about seven times longer than those lacking $H_6NtHSP70$-2. After 2 h at 50$^{\circ}C$, only the E. coli overexpressing $H_6NtHSP70$-2 survived under such conditions. Our NtHSP70-2 bioassays, as well as in vitro studies, strongly suggest that HSP70 confers thermo-tolerance to E. coli.

• Clinical and Experimental Pediatrics
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• v.49 no.5
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• pp.539-544
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• 2006

Purpose : The purpose of this study was to determine whether N-terminal fragment of B-type natriuretic peptide(NT-proBNP) may be used to differentiate acute Kawasaki disease(KD) from other clinically similar diseases. Methods : Using electrochemiluminescence immunoassay, NT-proBNP concentrations were measured in the acute phase within 10 days after the onset of KD(n=58) and in the convalescent phase, 60 to 81 days after the onset(n=51), and also in patients with acute febrile disease as a control(n=34). Echocardiography was performed to detect pericardial effusion(PE) and coronary artery lesions(CAL), and to measure the left ventricular dimension at diastole(LVIDd) and ejection fraction(LVEF). The cutoff value of NT-proBNP for separating KD from other diseases was determined. Results : NT-proBNP concentration in the acute phases of KD was significantly higher than that in the control group($1,501.6{\pm}2,132.6$ vs. $139.0{\pm}88.8pg/mL$, P<0.0001). In KD patients, NT-proBNP was elevated in the acute phase and was lowered in the convalescent phase($1,466.0{\pm}2,173.2$ vs. $117.5{\pm}95.5pg/mL$, P<0.0001). The cutoff value of 260 pg/mL discriminated KD patients from other patients, with a sensitivity of 93 percent and a specificity of 88 percent. The NT-proBNP was higher in patients with PE(n=17) compared with those without PE(n=41)($1,784.2{\pm}1,903.1$ vs. $1,384.4{\pm}2,232.6pg/mL$, P=0.52). Comparison of NT-proBNP could not be done between patients with CAL and those without, owing to a small number of patients with CAL(n=3). There was no correlation between NT-proBNP and LVEF index(r=0.104, P=0.46) or LVIDd index(r=0.171, P=0.22). Conclusion : NT-proBNP increases in the acute phase of KD and decreases to within normal range in the convalescent phase. NT-proBNP >260 pg/mL may be highly suggestive of acute KD. NT-proBNP may be used as a diagnostic tool for KD.

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.253-256
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• 2004

Porcine fibroblasts were transferred into enucleated bovine oocytes for the interspecies nuclear transfer (NT). After NT, the embryos were cultured in three different culture systems. The media used for the experiment were CR1aa and NCSU23. The culture systems used for the experiment were: 1. Culture in CR1aa for 7 days (CR). 2. Culture in CR1aa for 2 days and subsequently in NCSU23 for 5 days (CR-NC). 3. Culture in NCSU23 for 7 days (NC). Bovine (intraspecies) NT group was used as a control. The oocytes in bovine NT group were treated the same as interspecies NT embryos except using bovine fibroblasts as nuclear donors. Regardless of their nuclear origin (interspecies vs bovine), the embryos in CR (68.4% vs 77.2%) and CR-NC (67.8% vs 70.5%) showed better developmental competence to the 2-cell stage (p<0.05) than those in NC (41.0% vs 10.0%). Bovine NT embryos in CR-NC did not develop over the 4-cell stage after the medium replacement, while interspecies NT embryos in CR-NC continued to develop and could reach over the 8-cell stage (12.2%). Blastocysts were only found in bovine NT group (17.4%), but no blastocyst was found in interspecies NT group. This study suggests that the development of interspecies NT embryos mostly depends on their recipient cytoplasm during the culture in vitro.

• Journal of Plant Biotechnology
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• v.2 no.2
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• pp.79-82
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• 2000

We have isolated a cDNA encoding a calcium-dependent protein kinase (CDPK) in Nicotiana tabacum, which was designated NtCDPK1. Accumulation of the NtCDPK1 mRNA was stimulated by various stimuli, including phytohormones, CaCl$_2$ wounding, fungal elicitors, chitin and methyl jasmonate. The NtCDPK1 gene encodes a functional Ser/Thr protein kinase of which phosphorylation activity is strongly induced by calcium. By analyzing expression of the NtCDPK1-GFP fusion protein and by immunoblotting with antibody which reacts with NtCDPK1, we found that NtCDPK1 is localized in membrane and nucleus in plant cells. Silencing expression of the NtCDPK1 transgene resulted in marked decrease of lateral root development in the transgenic tobacco plants. Yeast two hybrid screening using NtCDPK1 as a bait identified a tobacco homologue of proteasome regulatory subunit 21D7, designated Nt21D7. The 21D7 mRNA has been shown to be predominantly expressed in proliferating tissues in the cell cycledependent manner in carrot. The recombinant NtCDPK1 protein associated with Nt21D7 in vitro, and could phosphorylate the Nt21D7 protein in vitro in the presence of calcium, suggesting that Nt21D7 protein is a natural substrate of NtCDPK1 in tobacco. These results suggest that NtCDPK1 may regulate tell proliferation processes, such as lateral root formation, by regulating specificity and/or activity of proteasome-mediated protein degradation pathway.

• Journal of Embryo Transfer
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• v.23 no.2
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• pp.101-108
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• 2008

This study investigated the developmental ability and gene expression of somatic cell nuclear transfer embryos using ear skin fibroblast cells derived from miniature pig. When miniature pig (m) and landrace pig (p) were used as donor cells, there were no differences in cleavage (79.2 vs. 78.2%) and blastocyst rates (27.4 vs. 29.7%). However, mNT blastocysts showed significantly higher apoptosis rate than that of pNT blastocysts (6.1 vs. 1.7%) (p<0.05). The number of nuclei in pNT blastosysts was significantly higher than that of mNT (35.8 vs. 29.3) (p<0.05). Blastocysts were analyzed using Realtime RT-PCR to determine the expression of Bax-${\alpha}$, Bcl-xl, H19, IGF2, IGF2r and Xist. Bax-${\alpha}$ was higher in mNT blastocyst than pNT blastocyst (p<0.05). There was no difference in Bcl-xl between two NT groups. Bax-${\alpha}$/Bcl-xl was, however, significantly higher in mNT blastocyst compared to pNT. The expression of imprinting genes were aberrant in blastocysts derived from NT compared to in vivo blastocysts. H19 and IGF2r were significantly lower in mNT blastocysts (p<0.05). The expression of IGF2 and Xist was similar in two NT groups. However, imprinting genes were expressed aberrantly in mNT compared to pNT blastocysts. The present results suggest that the NT between donor cells derived from miniature pig and recipient oocytes derived from crossbred pig might affect reprogramming of donor cell, resulting in high apoptosis and aberrant expression patterns of imprinting genes.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.47 no.5
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• pp.384-389
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• 2002

No-tillage (NT) practice for corn production has advantages of reduction of soil erosion and energy conservation. Research on effects of NT for sweet corn or super sweet corn is very limited. Hybrids of sugary (su) and shrunken-2 (sh2) were tested under NT and conventional tillage (CT) practices to investigate plant characters, ear characters, fresh yield, and grain yield. Sugary hybrids were Golden Cross Bantam 70 (GCB70), Sprint, Geumdanok, and Danok3. Shrunken-2 hybrids were BSS9472, Cambella90, GSS9299, Jubilee, KS-Y-65, and Chodangok1. Emergence rates under NT were lower than those under CT for su, while there was no difference between tillage systems for sh2. There were no differences between CT and NT for days to tasseling and silking, plant height, and ear height for both su and sh2. Ear characters such as ear length, number of kernel rows, number of kernels per row, and t100-kernel weight under NT were not significantly different from those under CT. There were no differences between two tillage practice for fresh and grain yield, rather they showed trend of increases under NT practices. Results from this trial indicate that NT practice for both su and sh2 cultivation may be possible to recommend to farmers.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.3
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• pp.227-234
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• 2000

Many reports suggest that neurotensin (NT) in the gastrointestinal tract may play a possible role as a neurotransmitter, a circulating hormone, or a modulator of motor activity. NT exerts various actions in the intestine; it produces contractile and relaxant responses in intestinal smooth muscle. This study was designed to investigate the effect of NT on motility of antral circular muscle strips in guinea-pig stomach. To assess the role of $Ca^{2+}$ influx in underlying mechanism, slow waves were simultaneously recorded with spontaneous contractions using conventional intracellular microelectrode technique. At the concentration of $10^{-7}$ M, where NT showed maximum response, NT enhanced the magnitude $(863{\pm}198%,\;mean\;SEM,\;n=13)$ and the frequency $(154{\pm}10.3%,\;n=11)$ of spontaneous contractions. NT evoked a slight hyperpolarization of membrane potential, tall and steep slow waves with abortive spikes $(278{\pm}50%,\;n=4).$ These effects were not affected by atropine $(2\;{\mu}M),$ guanethidine $(2\;{\mu}M)$ and tetrodotoxin (0.2μM). NT-induced contractile responses were abolished in $Ca^{2+}-free$ solution and reduced greatly to near abolition by $10\;{\mu}M$ of verapamil or 0.2 mM of $CdCl_2.$ Verapamil attenuated the effects of NT on frequency and amplitude of the slow waves. Taken together, these results indicate that NT enhances contractility in guinea-pig gastric antral circular muscle and $Ca^{2+}$ influx through the voltage-operated $Ca^{2+}$ channel appears to play an important role in the NT-induced contractile mechanism.

• Journal of Life Science
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• v.18 no.8
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• pp.1023-1030
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• 2008

NtMEK2, which is the tobacco MAPK kinase that is upstream of SIPK and WIPK, was identified using the dexamethasone (DEX)-inducible gain-of-function transgenic system. Expression of $NtNEK2^{DD}$, a constitutively active mutant of NtNEK2, leads to HR-like cell death, which indicates that the NtMEK2-SIPK/WIPK cascade controls defense responses in tobacco. However, little is known about the downstream target substrates or defense-related genes that are regulated by the NtMEK2-SIPK/ WIPK cascade. In this study, ACP-based differential display RT-PCR was used to isolate the downstream effectors mediated by the NtMEK2-SIPK/WIPK cascade in $NtNEK2^{DD}$ transgenic plants. The results identified 6 novel differentially expressed genes (DEGs). These included pathogen induced protein 2-4 (pI2-4), monoterpene synthase 2 (MTS2), seven in absentia protein (SINA), cell death marker protein 1 (CDM1), hydroxyproline-rich glycoprotein (HRGP) and unknown genes (DEG45). The induction of these genes was confirmed by RT-PCR of samples obtained from $NtNEK2^{DD}$ plants. Additionally, when compared with other isolated DEGs, the pI2-4, CDM1 and HRGP genes were significantly up-regulated in response to treatment with salicylic acid and tobacco mosaic virus. Taken together, these results suggest that three novel DEGs were regulated by the NtMEK2-SIPK/WIPK cascade involved in disease resistance in tobacco.

• Journal of Veterinary Clinics
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• v.35 no.2
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• pp.35-38
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• 2018

Recently assessment of suppression of tumorigenicity 2 (ST2) level has become a useful cardiac biomarker in human medicine. This study compared serum ST2 levels and N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels between healthy dogs and dogs with chronic mitral valve disease. Twenty client-owned dogs were investigated. Dogs were divided into normal, asymptomatic, and symptomatic groups. Serum samples were used to measure levels of NT-proBNP and ST2. Samples for NT-proBNP were sent to IDEXX laboratory for analysis while ST2 levels were measured by using a canine interleukin 33 receptor ELISA kit. There was a significant difference in NT-proBNP levels between asymptomatic and symptomatic groups (P < 0.01), and between normal and symptomatic groups (P < 0.01). In contrast, ST2 levels were not relatively different between asymptomatic and symptomatic groups (P > 0.05). There was no significant difference was observed among all groups in ST2 study. The usefulness of measuring NT-proBNP level as a cardiac biomarker in dogs with chronic mitral valve disease was confirmed, but usefulness of the ST2 level was not observed. Further investigations are needed to evaluate the potential usefulness of ST2 level as a cardiac biomarker in canines.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.515-523
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• 2001

The dinoflagellates have some primitive nuclear features and are evolutionarily intermediate between prokaryotes and eukaryotes. The small subunit ribosomal RAN gene, the 5.8S ribosomal RNA gene, and the internal transcribed spacer (ITS) of Gonyaulax polyedra were cloned, and their sequences were analyzed to better understand their evolutionary position. The small subunit ribosomal RNA gene was 1,794 nt long, the large subunit ribosomal RNA gene was approximately 3,500 nt long, and the 5.8S ribosomal RNA gene was 159 nt long. The first internal transcribed spacer (ITS1) was 191 nt long, and the second internal transcribed spacer (ITS2) was 185 nt long. The intergenic spacer of the ribosomal RNA gene (IGS) was about 2,200 nt long, indicating that 5,800 nt of transcribed sequences were separated by roughly 2,200 nt of intergenic spacer. The ribosomal RNA genes were repeated many times and arranged in a head-to-tail, tandemly repeated manner. The repeating unit of ribosomal RNA gene of G. polyedra was proposed to be 8,000 nt long. Based on the lengths of ribosomal RNA, sequence alignments with representative organisms, and phylogenetic analysis on ribosomal RNA, G. polyedra appears to be one of the alveolates branched from the eukaryotic crown and, among dinoflagellates, it seems to not have emerged early.