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EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy has achieved favorable clinical outcomes in non–small cell lung cancer (NSCLC) patients with EGFR mutations. However, patients eventually develop resistance to EGFR-TKIs by several mechanisms. Adenine nucleotide translocase-2 (ANT2) is an oncogenic mitochondrial membrane–associated protein. We investigated the therapeutic potential of ANT2 inhibition to EGFR-TKI resistance in NSCLC using gefitinib-sensitive (PC9 and HCC827) and gefitinib-resistant (H1975 and HCC827/GR) NSCLC cell lines. ANT2 was inhibited by transfecting cells with an ANT2-specific shRNA. ANT2 expression was elevated in the H1975 and HCC827/GR cells compared with the PC9 and HCC827 cells. ANT2 upregulation in gefitinib-resistant cells was associated with increased SP1 binding to the ANT2 promoter. ANT2-specific shRNA decreased NSCLC cell viability. Moreover, ANT2-specific shRNA sensitized the H1975 and HCC827/GR cells to gefitinib, accompanied by HSP90 and EGFR downregulation. ANT2-specific shRNA also inactivated the PI3K/Akt signaling pathway in the H1975 and HCC827/GR cells, which was mediated by the suppression of miR-221/222 levels and by the subsequent restoration of PTEN. In EGFR-TKI–treated NSCLC patients, ANT2 expression was higher in patients exhibiting poor responses compared with patients showing excellent responses. Furthermore, ANT2 expression increased in tumor tissues biopsied after acquiring gefitinib resistance compared with tissues before gefitinib treatment. These findings suggest that ANT2 overexpression contributes to EGFR-TKI resistance in NSCLC and that ANT2 targeting may be considered a novel strategy for overcoming this resistance. Mol Cancer Ther; 15(6); 1387–96. ©2016 AACR.

PC9, H1975, HCC827, and HCC827/GR cells were treated with lonidamine, gefitinib or DMSO as a control and subsequently incubated for an additional 24 h. Then, cell viability was measured using CCK8 assays and presented as a relative value compared to that of DMSO-treated cells. The values presented in all of the histograms are the mean values +/- SD.

A549 cells were transfected with ANT2-specific shRNA or sc shRNA. At 24 hours of transfection, the expression levels of miR-221 and miR-222 were measured by qRT-PCR. The relative expression levels (2-ddCt) of these molecules were compared to those of cells transfected with sc shRNA.

At 24 h after transfection with ANT2-specific shRNA or sc shRNA, Western blotting was performed to determine the levels of ANT2 and actin as a loading control.

PC9, H1975, HCC827, and HCC827/GR cells were treated with gefitinib or DMSO as a control and subsequently incubated for an additional 24 h. Then, cell viability was measured using CCK8 assays and presented as a relative value compared to that of DMSO-treated cells. The values presented in all of the histograms are the mean values +/- SD.

H1975 cells were transfected with ANT siRNA, HSP90 siRNA or scramble (sc) siRNA. At 24 hours of transfection, expression levels of ANT2 and HSP90 were measured using qRT-PCR and the suppressive efficiency of siRNA was presented by relative expression levels (2-ddCt) of ANT2 and HSP90 compared with those of cells transfected with sc siRNA.

Prostate cancer is one of the major causes of cancer-related deaths among men globally. Medicinal plants have been explored as alternative treatment options. Herein, we assessed the in vitro cytotoxic effects of 70% ethanolic root extracts of six-month-old micropropagated Prunus africana (PIR) on PC-3 prostate cancer cells as an alternative to the traditionally used P. africana stem-bark extract (PWS) treatment. In vitro assays on PC-3 cells included annexin-V and propidium iodide staining, DAPI staining, and caspase-3 activity analysis through western blotting. PC-3 cells were exposed to PWS and PIR at different concentrations, and dose-dependent antiprostate cancer effects were observed. PC-3 cell viability was determined using CCK-8 assay, which yielded IC50 values of 52.30 and 82.40 μg/mL for PWS and PIR, respectively. Annexin-V and PI staining showed dose-dependent apoptosis of PC-3 cells. Significant ( p < 0.001 ) percent of DAPI-stained apoptotic PC-3 cells were observed in PWS, PIR, and doxorubicin treatment compared with the negative control. PWS treatment substantially elevated cleaved caspase-3 levels in PC-3 cells compared with the PIR treatment. These results provide evidence for the antiprostate cancer potential of PIR and sets a basis for further research to enhance future utilization of roots of young micropropagated P. africana for prostate cancer treatment as an alternative to stem bark. Moreover, micropropagation approach may help provide the required raw materials and hence reduce the demand for P. africana from endangered wild population.

Drought stress is one of the major constraints for soybean growth and productivity worldwide. The study was aimed to investigate drought-induced physiological and proteomic changes in soybeans, as well as drought relief using exogenous hydrogen peroxide (H2O2). In drought-stressed plants, H2O2 spray on the leaf surface improved relative water content (RWC), net photosynthetic rate (Pn), and stomatal conductance (Gs). Furthermore, exogenous H2O2 reduced drought stress-induced endogenous MDA and H2O2 levels, as well as increased the key antioxidant enzymes (SOD,CAT, APX and POD) activity and proline content in H2O2-treated soybean plants. These findings showed that H2O2 treatment significantly reduced drought stress by increasing the antioxidative defense system and osmotic adjustment. Furthermore, using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, a total of 27 differently expressed proteins was identified, wherein 23 were up-regulated and 4 were down-regulated under drought condition. These proteins were found to be involved in photosynthesis, energy and metabolism, plant defense and antioxidant, signaling and transport, and transcription regulation in response to H2O2 treatment in soybean under drought stress, according to in silico interactome analysis. These findings add to our understanding of H2O2-mediated drought stress alleviation, as well as the physiological and molecular responses of soybean to drought stress.

In this study, we developed a repeatable in vitro micropropagation protocol for the medicinal plant Asparagus cochinchinensis, based on indirect organogenesis using leaf segments cut from seedlings of in vitro-germinated seeds. We obtained 85% callus induction from the leaf segments within 4–5 weeks when grown on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP, 1.0 mg/L) and 1-naphthaleneacetic acid (NAA, 0.5 mg/L). We found that MS media supplemented with Kn (1.0 mg/L) in combination with NAA (1.0 mg/L) and BAP (1.0 mg/L) combined with NAA (0.5 mg/L) were the most effective in promoting shoot regeneration, yielding plantlets with 6.72 and 6.48 shoots per culture, respectively. When cultured on PGR-free half-strength MS medium, regenerated plants developed root systems with an average 11.0 roots per shoot cluster and an average length of 36.14 mm at 9 weeks. During acclimatization, regenerated plantlets showed 96.4% survival and exhibited normal growth characteristics and morphology. We also made an attempt to directly regenerate A. cochinchinensis from shoot apices but it was futile. Histological analyses revealed the presence of crystal idioblasts in young leaves from the early stages of leaf differentiation. The leaf-based plant regeneration technique developed herein could be employed for large-scale propagation of the plants over a short time period, thereby substantially contributing to the germplasm preservation and rapid propagation of A. cochinchinensis. A repeatable in vitro micropropagation protocol for Asparagus cochinchinensis was developed based on indirect organogenesis. Histological analysis revealed crystal idioblasts for the first time in leaf primordia of this species.

This present study evaluated the effects of processed P. multiflorum on osteogenesis using Sarcoma osteogenic (SaOS-2) cell lines and osteoclastogenesis of bone marrow-derived macrophage cells (BMM) and to elucidate differences in effect on the expression of bone-related proteins between commercially sold P. multiflorum and patented, in vitro-propagated Korea Institute of Oriental Medicine (KIOM) P. multiflorum. Raw P. multiflorum and P. multiflorum that were stir-baked and steamed in black bean juice were compared, and western blotting analysis was performed to investigate the expression of bone remodeling-related proteins in SaOS-2 cells. In the cells treated with P. multiflorum steamed in black bean juice, the expression of RANKL was decreased, whereas that of osteoprotegerin, alkaline phosphatase, Runx2, and osterix was increased. Owing to these results, we conclude that processed P. multiflorum can be used as an alternative treatment for bone diseases such as osteoporosis, osteopenia, periodontitis, and Paget’s disease.

Prostate cancer is one of the major causes of cancer-related deaths among men globally. Medicinal plants have been explored as alternative treatment options. Herein, we assessed the in vitro cytotoxic effects of 70% ethanolic root extracts of six-month-old micropropagated Prunus africana (PIR) on PC-3 prostate cancer cells as an alternative to the traditionally used P. africana stem-bark extract (PWS) treatment. In vitro assays on PC-3 cells included annexin-V and propidium iodide staining, DAPI staining, and caspase-3 activity analysis through western blotting. PC-3 cells were exposed to PWS and PIR at different concentrations, and dose-dependent antiprostate cancer effects were observed. PC-3 cell viability was determined using CCK-8 assay, which yielded IC50 values of 52.30 and 82.40 μg/mL for PWS and PIR, respectively. Annexin-V and PI staining showed dose-dependent apoptosis of PC-3 cells. Significant (p < 0.001) percent of DAPI-stained apoptotic PC-3 cells were observed in PWS, PIR, and doxorubicin treatment compared with the negative control. PWS treatment substantially elevated cleaved caspase-3 levels in PC-3 cells compared with the PIR treatment. These results provide evidence for the antiprostate cancer potential of PIR and sets a basis for further research to enhance future utilization of roots of young micropropagated P. africana for prostate cancer treatment as an alternative to stem bark. Moreover, micropropagation approach may help provide the required raw materials and hence reduce the demand for P. africana from endangered wild population.

Trichosanthes kirilowii Maxim. is a vital traditional herbal medicinal plant found in northeastern Asia. Its roots, fruits, and seeds are used as food and medicine. Roots harvested for medicinal use take over 3 yr to mature when the plant is grown in a traditional way through cultivation in the field. This coupled with uncertainty in identification of the plant when collected from the wild calls for a standard in vitro propagation system to meet the increasing demand for it. The purpose of this study was to develop a standard protocol for the in vitro micropropagation of T. kirilowii. Ten different media supplemented with different concentrations of plant growth regulators were evaluated. At 5 wk, De Greef and Jacobs medium supplemented with 0.1 mg L−1 kinetin led to optimal shoot growth, while the same medium supplemented with 0.5 mg L−1 indole 3-butyric acid induced optimal root growth, also at 5 wk. The micropropagated plants that were acclimatized for 8 wk in the greenhouse produced mature root tubers after planted in the field for 3 mo. Therefore, these findings provide a basis for future large-scale in vitro propagation of T. kirilowii.

Chronic inflammation is one of the causes of a number of non-infectious diseases in the world. Over the years, Tamarindus indica has played fundamental roles in traditional medicine as an anti-inflammatory and analgesic drug. It is a commercialized biocompatible medicinal plant species with a wide range of therapeutic window and with suggested LD50 greater than 5000 mg kg−1 body weight when administered to the Wistar rats. This review examined the anti-inflammatory and analgesic potential and mechanism of various extracts from T. indica pulp, leaves, seeds, stem bark, and roots. The preclinical studies provided strong pharmacological evidence for the anti-inflammatory and analgesic activities of the different parts of T. indica and this may be attributed to the various bioactive compounds in it including alkaloids, flavonoids, tannins, phenols, saponins, and steroids. The anti-inflammatory and analgesic effects of the extracts from the different parts of T. indica may be due to its ability to inhibit a number of biological processes including cyclooxygenase-2 (COX-2) expression, inducible nitric oxide synthase (iNOS), 5-lipoxygenase biosynthesis, and tumor necrosis factor-α. The analgesic activity of T. indica may also be through the activation of the opioidergic mechanism at both the peripheral and central levels. Although further pre-clinical studies still need to be conducted, these results demonstrated that T. indica has potent anti-inflammatory and analgesic activities and hence provides justification for its use in traditional medicine to treat body pain and other inflammatory related diseases including arthritis and offers a basis for future clinical studies and possible drug development.

Nitric oxide (NO) is a ubiquitous bimolecule that regulates various physiological processes in plants. Despite the widespread study of NO in plants, the involvement of NO in proteomic regulation has been poorly investigated compared to the physiological processes. To explore the NO-induced proteomic regulation in rice leaves, 2-week-old leaves were exposed to sodium nitroprusside for 12 h. Protein profiles were analyzed by two-dimensional gel electrophoresis. A total of 53 NO-regulated proteins were identified by the matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. Out of these proteins, 41 were up-regulated, 7 were down-regulated, and 5 were newly induced. The identified proteins were involved in photosynthesis, carbohydrate and energy metabolism, growth and development, metabolite biosynthesis, signal transduction, lignin modification, and defense response. Importantly, several key proteins including glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, ascorbate peroxidase, superoxide dismutase, catalase, 2-cysteine peroxiredoxin, peroxidase (newly induced), and glutathione S-transferase were significantly up-regulated in rice leaves and these proteins were associated with the ascorbate–glutathione cycle and ROS homeostasis system in plants. Thus, the identification of several key and newly induced proteins has provided a new insight into NO-mediated overall molecular response in plants.

Aspilia africana has been used for generations to treat many diseases in Africa. Its biological activities, including antioxidant and anti-inflammatory potential, are attributed to a number of secondary metabolites, including alkaloids and polyphenolics. The antioxidant activities of A. africana callus (CA), juvenile in vitro leaf (IL) and root (IR), ex vitro root (SR) and leaf (SL), and wild leaf (WL) dried samples were assessed based on their diphenylpicrylhydrazyl (DPPH) free radical scavenging abilities. The total phenolic and flavonoid content of different plant samples was compared. Further, high-pressure liquid chromatography (HPLC) was used to quantitatively determine chlorogenic acid content in the A. africana plant samples. Fourier transform near-infrared (FT-NIR) analysis was also carried out to compare the antioxidant phytochemical content in the A. africana plant tissues. Among the samples, IR, with the highest total phenolic content (167.84 ± 1.057 mg GAE/g), total flavonoid content (135.06 ± 0.786 mg RUE/g), and chlorogenic acid (5.23 ± 0.298 mg/g) content, had the most potent antioxidant activity (IC50 = 27.25 ± 5.028 μg/mL), followed by WL. The lowest polyphenolic content and antioxidant activity were observed in SR. The antioxidant activities of A. africana tissues were positively correlated with the total phenolic and flavonoid content in the samples. The differences in antioxidant activities of A. africana tissues could be attributed to the difference in their polyphenolic content. Our study reports, for the first time, the antioxidant activities of A. africana callus and roots (in vitro and ex vitro). The A. africana samples IR, CA, and WL could be valuable natural sources of antioxidants that could be further exploited for the development of useful pharmaceutical products.

Objective: Amniotic mesenchymal stromal cells (AMSCs) can be obtained from the mesoderm of human amniotic membrane. AMSCs derived from term baby show increased expression of genes associated with apoptosis and senescence. The objective of this study was to examine gene expression profiles of AMSCs derived from preterm (preterm AMSCs) and term labors (term AMSCs) and analyze common and different mechanisms. Materials and methods: We isolated and cultured AMSCs from 43 placentas: 27 from term labor and 16 from preterm labor. Microarray analysis and gene network analysis were performed to compare gene expression profile (GEP) of preterm (n = 6) with term AMSCs (n = 10). Senescence-associated gene (CDKN2A and CDKN2B) expression was also measured by reverse transcription quantitative PCR. Results: GEP demonstrated that preterm AMSCs showed upregulation of nicotinamide adenine dinucleotide biosynthetic process and downregulation of extracellular matrix, cholesterol import and transport, lipid storage, and maintenance of location. CDKN2A and CDKN2B genes showed similar expression levels between term and preterm AMSCs. CDKN2A gene expression was correlated with CDKN2B expression and population doubling time. Compared to term AMSCs, preterm AMSCs showed significantly different expression of genes associated with inflammatory response which could be one of the major players in labor events. Conclusion: Increased CDKN2A expression in AMSCs is associated with placental membrane aging which participates in both preterm and term labor. To the best of our knowledge, this is the first report to demonstrate the association of AMSCs with labor.

Polygonum multiflorum Thunb. is a perennial plant that belongs to Polygonaceae. Root tissues are the main plant parts used as medicinal herbs in Korean oriental medicine. The P. multiflorum tuber is well known for its medicinal properties in Korean oriental medicine, and it contains a number of useful substances (secondary metabolites of emodin, 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (TSG), etc.) that are increasing in demand, as several studies show that they have beneficial effects on the human body. In this study, the production volumes and useful material content differences between cultured P. multiflorum seedlings (culture seedlings: CSs), which had been grown using a tissue culture technique under optimized conditions, and existing varieties in circulation (seed seedlings: SSs) were determined using a long-term field test. The growth characteristics of the underground parts were investigated by harvesting the tuberous roots (medicinal parts) after 1 year, and the results showed that the fresh and dry weights of the CS tubers were higher than those of the SS tubers. However, the SS rootlets had higher fresh and dry weights than the CS rootlets. A liquid chromatography-mass spectrometry component analysis of the P. multiflorum tubers and a Fourier transform near-infrared spectrophotometer analysis of the roots were undertaken. The results showed that the levels of TSG, which is a medicinal substance produced by P. multiflorum, were higher in the CSs than in the SSs, but the differences were not significant. The CS results from this study will inform future studies on the mass production of P. multiflorum in the field because the medicinal area was greater in CSs than in SSs.

Osteoporosis is one of the main health problems in the world today characterized by low bone mass and deterioration in bone microarchitecture. In recent years, the use of natural products approach to treat it has been in the increase. In this study, in vitro antiosteoporosis activity and hepatotoxicity of P. jamasakura bark extracts were evaluated. Methods. Mouse bone marrow macrophage (BMM) cells were incubated with tartrate-resistant acid phosphate (TRAP) buffers and p-nitrophenyl phosphate and cultured with different P. jamasakura bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 μg/ml in the presence of the receptor activator of nuclear factor kappa-Β ligand (RANKL) for 6 days. The osteoclast TRAP activity and cell viability were measured. Nitric oxide (NO) assay was conducted using murine macrophage-like RAW 264.7 cells treated with P. jamasakura ethanolic and methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, 50, 100, and 200 μg/ml. For hepatotoxicity assessment, zebrafish larvae were exposed to P. jamasakura bark extracts, 0.05% dimethyl sulfoxide as a negative control, and 5 μM tamoxifen as a positive control. The surviving larvae were anesthetized and assessed for hepatocyte apoptosis. Results. TRAP activity was significantly inhibited (p P. jamasakura extracts compared to the control treatment. At 50 μg/ml, both ethanolic and methanolic extracts of P. jamasakura exhibited significant (p P. jamasakura ethanolic and methanolic extracts had significant inhibitory (p μg/ml and exhibited significant (p p Conclusion. The TRAP activity of P. jamasakura bark gives a foundation for further studies to enhance future development of antiosteoporosis drug.

Rehmannia glutinosa (Gaertn.) DC is a perennial plant belonging to the family Scropulariidae. The root of R. glutinosa is used in oriental medicine and mainly grown using rootstock rather than seed cultivation, which gives rise to several problems including root rot, and results in a low productivity and poor quality. To solve the challenges involved in R. glutinosa seed cultivation, our team previously used the formative features and genetic analysis of R. glutinosa to determine the optimal in vitro tissue culture conditions for producing sterile culture seedlings and rootstocks of R. glutinosa. The aim of the present study was to identify differences between R. glutinosa standard rootstock seedlings (SR), R. glutinosa culture rootstock seedlings (CR), and culture seedlings (CS) under field conditions. The reproductive characteristics of the aerial part were more robust while the area and length of leaves were smaller for SR than those for CR and CS. The characteristic that differed the most in SR was flowering, which did not occur in CR and CS. In addition, the fresh and dry weights of the subterranean parts of CR and CS were two-fold greater than those of SR. Fourier transform near-infrared (FT-NIR) analysis showed only slight differences between the chemical constituents of SR and its culture products, which was confirmed by measuring the content of catalpol, an indexing substance. Catalpol had a reduced content in the culture products compared to SR. However, this difference was not significant. Our findings will be useful for the identification of the best seedling type of R. glutinosa to enable its mass production.

This present study evaluated the effects of processed P. multiflorum on osteogenesis using Sarcoma osteogenic (SaOS-2) cell lines and osteoclastogenesis of bone marrow-derived macrophage cells (BMM) and to elucidate differences in effect on the expression of bone-related proteins between commercially sold P. multiflorum and patented, in vitro-propagated Korea Institute of Oriental Medicine (KIOM) P. multiflorum. Raw P. multiflorum and P. multiflorum that were stir-baked and steamed in black bean juice were compared, and western blotting analysis was performed to investigate the expression of bone remodeling-related proteins in SaOS-2 cells. In the cells treated with P. multiflorum steamed in black bean juice, the expression of RANKL was decreased, whereas that of osteoprotegerin, alkaline phosphatase, Runx2, and osterix was increased. Owing to these results, we conclude that processed P. multiflorum can be used as an alternative treatment for bone diseases such as osteoporosis, osteopenia, periodontitis, and Paget's disease.

Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) is an upstream signaling molecule that has been shown to induce stress tolerance in plants. In this study, the AtNDPK2 gene, under the control of a stress-inducible SWPA2 promoter, was introduced into the genome of tall fescue (Festuca arundinacea Schreb.) plants. The induction of the transgene expression mediated by methyl viologen (MV) and NaCl treatments were confirmed by RT-PCR and northern blot analysis, respectively. Under salt stress treatment, the transgenic tall fescue plants (SN) exhibited lower level of H2O2 and lipid peroxidation accumulations than the non-transgenic (NT) plants. The transgenic tall fescue plants also showed higher level of NDPK enzyme activity compared to NT plants. The SN plants were survived at 300 mM NaCl treatment, whereas the NT plants were severely affected. These results indicate that stress-inducible overexpression of AtNDPK2 might efficiently confer the salt stress tolerance in tall fescue plants.

In order to improve the transformation efficiency of zoysiagrass by increasing the frequency of callus formation from mature seeds and plant regeneration, the effect of pre-treatment with sea sand was examined. Mature zoysiagrass seeds were given 10 min of swelling time before sea sand treatment using a sea sand and seed ratio of 1 : 1 and a vortex shaking speed of 6 (1,000 rpm) for 10 min. The seeds showed increased callus formation that was more than 2 times the rate in the control. In addition, plant regeneration efficiency was also increased when embryogenic callus induced from sea sand-treated mature seeds was cultured in regeneration medium. These results will be very helpful for improving the genetic transformation frequency of zoysiagrass, a recalcitrant monocot grass. ( Key words : Callus, Pre-treatment, Sea sand, Zoysiagrass) Ⅰ. 서 론 우리나라를 중심으로 아시아 지역의 중국, 일본 등지에서 자생하는 것으로 알려져 있는 한국잔디(zoysiagrass)는 난지형 다년생 잔디로 알려져 있다(심 et al., 1998). 한국잔디는 지하경과 포복경에 의해 생육밀도를 높게 형성하는 특성이 있는 잔디이며, 또한 우리나라를 포함해 일본, 중국 등의 아시아 국가에서는 잔디 우점 초지를 이용하여 가축에게 방목용 초지로 활용한 예가 보고되고 있다(Dhital et al., 2010). 한국잔디로 조성된 초지는 목초생산성이 높을 뿐만 아니라 단위면적당 많은 수의 가축을 방목하는 고밀도의 방목강도에도 잘 견디는 특성이 있는 것으로 알려져 있다(Ito and Takatsuki, 2005). 잔디는 또한 다른 대부분의 C

As the needs of real-time visual object tracking are increasing in various kinds of application fields such as surveillance, entertainment, etc., kernel-based mean-shift tracking has received more interests. One of major issues in kernel-based mean-shift tracking is to be robust under partial or full occlusion status. This paper presents a real-time mean-shift tracking which is robust in partial occlusion by applying multi-mode local kernel weight. In the proposed method, a kernel is divided into multiple sub-kernels and each sub-kernel has a kernel weight to be determined according to the location of the sub-kernel. The experimental results show that the proposed method is more stable than the previous methods with multi-mode kernels in partial occlusion circumstance.

A comparative proteomic approach was carried out between two contrasting alfalfa cultivars, nonomu (NM-801; salt tolerant) and vernal (VN; salt intolerant) in terms of salt tolerance. Seedlings were subjected to salt stress (50 and 100 mM NaCl) for three days. Several physiological parameters (leaf water, chlorophyll, root Na(+), K(+), and Ca(2+)) and root proteome profile were analyzed. Comparison of physiological status revealed that NM-801 is more tolerant to salt than VN. Eighty three differentially expressed proteins were found on 2-DE maps, of which 50 were identified by MALDI-TOF or MALDI-TOF/TOF mass spectrometry. These proteins were involved in ion homeostasis, protein turnover and signaling, protein folding, cell wall components, carbohydrate and energy metabolism, reactive oxygen species regulation and detoxification, and purine and fatty acid metabolism. The comparative proteome analysis showed that 33 salt-responsive proteins were significantly changed in both cultivars, while 17 (14 in VN and 3 in NM-801) were cultivar-specific. Peroxidase, protein disulfide-isomerase, NAD synthetase, and isoflavone reductase were up-regulated significantly only in NM-801 in all salt concentrations. In addition, we identified novel proteins including NAD synthetase and biotin carboxylase-3 that were not reported previously as salt-responsive. Taken together, these results provide new insights of salt stress tolerance in alfalfa.

In order to reveal the aluminum (Al) stress tolerance mechanisms in alfalfa plant at low pH soil, a proteomic approach has been conducted. Alfalfa plants were exposed to Al stress for 5 days. The plant growth and total chlorophyll content are greatly affected by Al stress. The malondialdehyde (MDA) and H2O2 contents were increased in a low amount but free proline and soluble sugar contents, and the DPPH-radical scavenging activity were highly increased. These results indicate that antioxidant activity (DPPH activity) and osmoprotectants (proline and sugar) may involve in ROS (H2O2) homeostasis under Al stress. In proteomic analysis, over 500 protein spots were detected by 2-dimentional gel electrophoresis analysis. Total 17 Al stress-induced proteins were identified, of which 8 protein spots were up-regulated and 9 were down-regulated. The differential expression patterns of protein spots were selected and analyzed by the peptide mass fingerprinting (PMF) using MALDI-TOF MS analysis. Three protein spots corresponding to Rubisco were significantly down-regulated whereas peroxiredoxin and glutamine synthetase were up-regulated in response to Al stress. The different regulation patterns of identified proteins were involved in energy metabolism and antioxidant / ROS detoxification during Al stress in alfalfa. Taken together, these results provide new insight to understand the molecular mechanisms of alfalfa plant in terms of Al stress tolerance. (Key words : Alfalfa, Aluminum stress, Proteome)

We have previously investigated the proteome changes of rice leaves under heat stress (Lee et al. in Proteomics 2007a, 7:3369- 3383), wherein a group of antioxidant proteins and heat shock proteins (HSPs) were found to be regulated differently. The present study focuses on the biochemical changes and gene expression profiles of heat shock protein and antioxidant genes in rice leaves in response to heat stress (42°C) during a wide range of exposure times. The results show that hydrogen peroxide and proline contents increased significantly, suggesting an oxidative burst and osmotic imbalance under heat stress. The mRNA levels of chaperone 60, HSP70, HSP100, chloroplastic HSP26, and mitochondrial small HSP responded rapidly and showed maximum expression after 0.5 or 2 h under heat stress. Transcript levels of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and Cu-Zn superoxide dismutase (Cu-Zn SOD) showed a rapid and marked accumulation upon heat stress. While prolonged exposure to heat stress resulted in increased transcript levels of monodehydroascorbate reductase, peroxidase, glyoxalase 1, glutathione reductase, thioredoxin peroxidase, 2-Cysteine peroxiredoxin, and nucleoside diphosphate kinase 1, while the transcription of catalase was suppressed. Consistent with their changes in gene expression, the enzyme activities of APX and DHAR also increased significantly following exposure to heat stress. These results suggest that oxidative stress is usually caused by heat stress, and plants apply complex HSP- and antioxidant-mediated defense mechanisms to cope with heat stress.

Zoysiagrass (Zoysia japonica Steud.) is an important forage and turfgrass that spreads by stolons and rhizomes. Zoysiagrass stolon can be used directly for Agrobacterium-mediated genetic transformation by exploiting the potential of direct shoot formation. However, surface sterilization of field-grown stolons is difficult and remains to be explored. We developed an effective surface sterilization and culture method using the stolon explant for infection with Agrobacterium tumefaciens. Among various treatments, sequential disinfection in 30% bleach for 15 min followed by 0.1% mercuric chloride for 25 min resulted in the highest number of clean stolons. The efficacy of mercuric chloride was increased under vacuum conditions by incubating at 800 mbar for 5 min. The inclusion of 2.5 mg/l amphotericin B further prevents fungal growth in in vitro cultures. This protocol would speed up the development of transgenic plants by utilizing field-grown stolon nodes.

Glucose is a common medical analyte measuring in human serum or blood samples. The development of a primary method is necessary for the establishment of traceability in measurements. We have developed an isotope dilution liquid chromatography tandem mass spectrometry as a primary method for the measurement of glucose in human serum. Glucose and glucose- 13 C6 in sample were ionized in ESI negative mode and monitored at mass transfers of m/z 179/89 and 185/92 in MRM, respectively. Glucose was separated on NH2P50 2D column, and the mobile phase was 20 mM NH4OAc in 30% acetonitrile/70% water. Verification of this method was performed by the comparison with NIST SRMs. Our results agreed well with the SRM values. We have developed two levels of glucose serum certified reference material using this method and distributed them to the clinical laboratories in Korea as samples for proficiency testings. The expended uncertainty was about 1.2% on 95% confidence level. In proficiency testings, the results obtained from the clinical laboratories showed about 3.6% and 3.9% RSD to the certified values. Primary method can provide the traceability to the field laboratories through proficiency testings or certified reference materials.

Miscanthus sinensis is a promising bioenergy crop; however, its genome is poorly represented in sequence databases. As an initial step in the comprehensive analysis of the M. sinensis proteome, we report a reference 2-DE protein map of the leaf. A total of 316 protein spots were excised from the gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) or MALDI-TOF/TOF MS. Two hundred and thirty-two protein spots were identified, which are involved in a variety of cellular functions through distinct metabolic pathways. Functional annotation of the proteins revealed a nearly complete C3 and C4 cycle, starch and sugar synthesis pathway, glycolysis pathway, a significant portion of the pentose phosphate pathway, and many enzymes involved in secondary metabolism such as flavonoid/isoflavonoid, kaurene, chalcone, sesquiterpene and lignin biosynthesis. Other proteins belong to primary metabolism, transcription, protein synthesis, protein destination/storage, disease/defense, cell growth/division, transportation and signal transduction. To test the applicability of the constructed map, we studied the effect of heat stress on M. sinensis leaf proteome. Twenty-five protein spots were upregulated, five were newly induced and twenty-five spots were downregulated by heat treatment. The differentially accumulated proteins were involved in photosynthesis, energy metabolism, gene transcription, protein kinases and phosphatases, signal transduction, protein synthesis and heat shock responses. C4-specific pyruvate orthophosphate dikinase, Rubisco large subunit, Rubisco activase and some associated proteins were upregulated during heat stress and tend to restore upon recovery. Identification of these proteins provides some important clues regarding the way M. sinensis copes with hot climate. This work represents the first extensive proteomic description of M. sinensis and provides a reference map and heat-responsive candidates for future molecular and physiological studies of this bioenergy crop.