Preclinical Evaluation of a Novel Polymer-free Everolimus-eluting Stent in a Mid-term Porcine Coronary Restenosis Model.

BACKGROUND: Titanium dioxide films exhibit good biocompatibility and may be effective as drug-binding matrices for drug-eluting stents. We conducted a mid-term evaluation of a novel polymer-free everolimus-eluting stent using nitrogen-doped titanium dioxide film deposition (TIGEREVOLUTION®) in comparison with a commercial durable polymer everolimus-eluting stent (XIENCE Alpine®) in a porcine coronary restenosis model. METHODS: Twenty-eight coronary arteries from 14 mini-pigs were randomly allocated to TIGEREVOLUTION® stent and XIENCE Alpine® stent groups. The stents were implanted in the coronary artery at a 1.1-1.2:1 stent-to-artery ratio. Eleven stented coronary arteries in each group were finally analyzed using coronary angiography, optical coherence tomography, and histopathologic evaluation 6 months after stenting. RESULTS: Quantitative coronary analysis showed no significant differences in the pre-procedural, post-procedural, and 6-month lumen diameters between the groups. In the volumetric analysis of optical coherence tomography at 6 months, no significant differences were observed in stent volume, lumen volume, and percent area stenosis between the groups. There were no significant differences in injury score, inflammation score, or fibrin score between the groups, although the fibrin score was zero in the TIGEREVOLUTION® stent group (0 vs. 0.07 ± 0.11, P = 0.180). CONCLUSION: Preclinical evaluation, including optical coherence tomographic findings 6 months after stenting, demonstrated that the TIGEREVOLUTION® stent exhibited efficacy and safety comparable with the XIENCE Alpine® stent, supporting the need for further clinical studies on the TIGEREVOLUTION® stent.

Alteration of the Soil Microbiota in Ginseng Rusty Roots: Application of Machine Learning Algorithm to Explore Potential Biomarkers for Diagnostic and Predictive Analytics.

Conceptualization to utilize microbial composition as a prediction tool has been widely applied in human cohorts, yet the potential capacity of soil microbiota as a diagnostic tool to predict plant phenotype remains unknown. Here, we collected 130 soil samples which are 54 healthy controls and 76 ginseng rusty roots (GRRs). Alpha diversities including Shannon, Simpson, Chao1, and phylogenetic diversity were significantly decreased in GRR (P < 0.05). Moreover, we identified 30 potential biomarkers. The optimized markers were obtained through fivefold cross-validation on a support vector machine and yielded a robust area under the curve of 0.856. Notably, evaluation of multi-index classification performance including accuracy, F1-score, and Kappa coefficient also showed robust discriminative capability (90.99%, 0.903, and 0.808). Taken together, our results suggest that the disease affects the microbial community and offers the potential ability of soil microbiota to identifying farms at the risk of GRR.

Effect of a bioconverted product of Lotus corniculatus seed on the axillary microbiome and body odor.

The skin microbiome, especially the axillary microbiome, consists of odor-causing bacteria that decompose odorless sweat into malodor compounds, which contributes to the formation of body odor. Plant-derived products are a cheap source of bioactive compounds that are common ingredients in cosmetics. Microbial bioconversion of natural products is an ecofriendly and economical method for production of new or improved biologically active compounds. Therefore, in this study, we tested the potential of a Lactobacillus acidophilus KNU-02-mediated bioconverted product (BLC) of Lotus corniculatus seed to reduce axillary malodor and its effect on the associated axillary microbiota. A chemical profile analysis revealed that benzoic acid was the most abundant chemical compound in BLC, which increased following bioconversion. Moreover, BLC treatment was found to reduce the intensity of axillary malodor. We tested the axillary microbiome of 18 study participants, divided equally into BLC and placebo groups, and revealed through 16S rRNA gene sequencing that Staphylococcus, Corynebacterium, and Anaerococcus were the dominant taxa, and some of these taxa were significantly associated with axillary malodor. After one week of BLC treatment, the abundance of Corynebacterium and Anaerococcus, which are associated with well-known odor-related genes that produce volatile fatty acids, had significantly reduced. Likewise, the identified odor-related genes decreased after the application of BLC. BLC treatment enhanced the richness and network density of the axillary microbial community. The placebo group, on the other hand, showed no difference in the microbial richness, odor associated taxa, and predicted functional genes after a week. The results demonstrated that BLC has the potential to reduce the axillary malodor and the associated odor-causing bacteria, which makes BLC a viable deodorant material in cosmetic products.

Tpeak-Tend interval during therapeutic hypothermia can predict upcoming ventricular fibrillation in subjects with aborted arrhythmic sudden cardiac death: 3-years follow-up results.

AIMS: Prolonged Tpeak-Tend interval has been shown to be markers of arrhythmogenesis in various cardiac disorders. However, its dynamicity is one of the obstacles to predict fatal ventricular arrhythmia. This study investigated whether Tpeak-Tend interval during therapeutic hypothermia (TH) is associated with ventricular fibrillation (VF) inducibility and clinical arrhythmia in subjects with aborted arrhythmic sudden cardiac death (SCD). METHODS AND RESULTS: The study group included 31 patients (24 males, age 39.1 ± 17.6 years) presenting with arrhythmic SCD in whom Tpeak-Tend interval and J-wave amplitude were measured in electrocardiogram (ECG) of the earliest medical contact and during TH; these patients underwent programmed ventricular stimulation. The summation of J-wave amplitude and QTc interval increased during TH. However, it was not associated with VF inducibility. Patients with inducible VF showed a small Tpeak-Tend interval dispersion in the baseline 12-lead ECG (68.8 ± 24.7 vs. 94.0 ± 55.6 ms, P = 0.044) and a marked increase of the dispersion during the TH (36.2 ± 51.2 vs. -6.1 ± 45.5 ms, P = 0.039). Twenty-four patients underwent implantable cardioverter defibrillator (ICD) implantation. Among them, the patients with long QTc, Tpeak-Tend, and precordial Tpeak-Tend during the TH developed VF more frequently (QTc, 511.9 ± 53.71 ms vs. 566.5 ± 56.08 ms, P = 0.038; Tpeak-Tend interval, 145.6 ± 38.4 ms vs. 185.7 ± 49.95 ms, P = 0.048; precordial Tpeak-Tend interval, 139.3 ± 35.11 ms vs. 185.7 ± 49.95 ms, P = 0.018). The initial VF inducibility was not related with the VF development in follow-up. CONCLUSION: In patients with aborted arrhythmic SCD, long Tpeak-Tend interval and QTc interval during TH could predict VF development in their follow-up.

Elucidating how bamboo salt interacts with supported lipid membranes: influence of alkalinity on membrane fluidity.

Bamboo salt is a traditional medicine produced from sea salt. It is widely used in Oriental medicine and is an alkalizing agent with reported antiinflammatory, antimicrobial and chemotherapeutic properties. Notwithstanding, linking specific molecular mechanisms with these properties has been challenging to establish in biological systems. In part, this issue may be related to bamboo salt eliciting nonspecific effects on components found within these systems. Herein, we investigated the effects of bamboo salt solution on supported lipid bilayers as a model system to characterize the interaction between lipid membranes and bamboo salt. The atomic composition of unprocessed and processed bamboo salts was first analyzed by mass spectrometry, and we identified several elements that have not been previously reported in other bamboo salt preparations. The alkalinity of hydrated samples was also measured and determined to be between pH 10 and 11 for bamboo salts. The effect of processed bamboo salt solutions on the fluidic properties of a supported lipid bilayer on glass was next investigated by fluorescence recovery after photobleaching (FRAP) analysis. It was demonstrated that, with increasing ionic strength of the bamboo salt solution, the fluidity of a lipid bilayer increased. On the contrary, increasing the ionic strength of near-neutral buffer solutions with sodium chloride salt diminished fluidity. To reconcile these two observations, we identified that solution alkalinity is critical for the effects of bamboo salt on membrane fluidity, as confirmed using three additional commercial bamboo salt preparations. Extended-DLVO model calculations support that the effects of bamboo salt on lipid membranes are due to the alkalinity imparting a stronger hydration force. Collectively, the results of this work demonstrate that processing of bamboo salt strongly affects its atomic composition and that the alkalinity of bamboo salt solutions contributes to its effect on membrane fluidity.

Ginseng protects against respiratory syncytial virus by modulating multiple immune cells and inhibiting viral replication.

Ginseng has been used in humans for thousands of years but its effects on viral infection have not been well understood. We investigated the effects of red ginseng extract (RGE) on respiratory syncytial virus (RSV) infection using in vitro cell culture and in vivo mouse models. RGE partially protected human epithelial (HEp2) cells from RSV-induced cell death and viral replication. In addition, RGE significantly inhibited the production of RSV-induced pro-inflammatory cytokine (TNF-α) in murine dendritic and macrophage-like cells. More importantly, RGE intranasal pre-treatment prevented loss of mouse body weight after RSV infection. RGE treatment improved lung viral clearance and enhanced the production of interferon (IFN-γ) in bronchoalveolar lavage cells upon RSV infection of mice. Analysis of cellular phenotypes in bronchoalveolar lavage fluids showed that RGE treatment increased the populations of CD8+ T cells and CD11c+ dendritic cells upon RSV infection of mice. Taken together, these results provide evidence that ginseng has protective effects against RSV infection through multiple mechanisms, which include improving cell survival, partial inhibition of viral replication and modulation of cytokine production and types of immune cells migrating into the lung.

Ginseng diminishes lung disease in mice immunized with formalin-inactivated respiratory syncytial virus after challenge by modulating host immune responses.

Formalin-inactivated respiratory syncytial virus (FI-RSV) immunization is known to cause severe pulmonary inflammatory disease after subsequent RSV infection. Ginseng has been used in humans for thousands of years due to its potential health benefits. We investigated whether ginseng would have immune modulating effects on RSV infection in mice previously immunized with FI-RSV. Oral administration of mice with ginseng increased IgG2a isotype antibody responses to FI-RSV immunization, indicating T-helper type 1 (Th1) immune responses. Ginseng-treated mice that were nonimmunized or previously immunized with FI-RSV showed improved protection against RSV challenge compared with control mice without ginseng treatment. Ginseng-mediated improved clinical outcomes after live RSV infection were evidenced by diminished weight losses, decreased interleukin-4 cytokine production but increased interferon-γ production, modulation of CD3 T-cell populations toward a Th1 response, and reduced inflammatory response. Ginseng-mediated protective host immune modulation against RSV pulmonary inflammation was observed in different strains of wild-type and mutant mice. These results indicate that ginseng can modulate host immune responses to FI-RSV immunization and RSV infection, resulting in protective effects against pulmonary inflammatory disease.

Antiviral activity of ginseng extract against respiratory syncytial virus infection.

Panax ginseng has been known to have a number of immuno-modulatory effects. In this study, we investigated whether Panax Korean red ginseng extract (KRGE) has in vitro and in vivo antiviral effects on respiratory syncytial virus (RSV) infection. KRGE improved the survival of human lung epithelial cells against RSV infection and inhibited RSV replication. In addition, KRGE treatment suppressed the expression of RSV-induced inflammatory cytokine genes (IL-6 and IL-8) and the formation of reactive oxygen species in epithelial cell cultures. Oral administration of mice with KRGE resulted in lowering lung viral loads after RSV infection. Additionally, the in vivo effects of KRGE showed an enhanced level of interferon-γ (IFN-γ) producing dendritic cells subsequent to RSV infection. Taken together, these results suggested that KRGE has antiviral activity against RSV infection.

Immunomodulatory activity of red ginseng against influenza A virus infection.

Ginseng herbal medicine has been known to have beneficial effects on improving human health. We investigated whether red ginseng extract (RGE) has preventive effects on influenza A virus infection in vivo and in vitro. RGE was found to improve survival of human lung epithelial cells upon influenza virus infection. Also, RGE treatment reduced the expression of pro-inflammatory genes (IL-6, IL-8) probably in part through interference with the formation of reactive oxygen species by influenza A virus infection. Long-term oral administration of mice with RGE showed multiple immunomodulatory effects such as stimulating antiviral cytokine IFN-γ production after influenza A virus infection. In addition, RGE administration in mice inhibited the infiltration of inflammatory cells into the bronchial lumens. Therefore, RGE might have the potential beneficial effects on preventing influenza A virus infections via its multiple immunomodulatory functions.

Effects of anti-inflammatory and Rehmanniae radix pharmacopuncture on atopic dermatitis in NC/Nga mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by pruritic and erythematous skin lesions. The purpose of this study was to investigate the suppressive effects of anti-inflammatory and Rehmanniae radix pharmacopuncture on the development of AD-like skin lesions in NC/Nga mice. The AD was induced on the mice's back skin by using biostir AD. The experimental groups were divided into three groups, PPI (anti-inflammatory pharmacopuncture), PPII (Rehmanniae radix pharmacopuncture, hydrodistillation extraction) and PPIII (Rehmanniae radix pharmacopuncture, MeOH extraction). All mice were treated using a 1-mL syringe to inject 0.1 mL of pharmacopuncture at right and left acupoints (BL13) on alternate days. In the control group, normal saline was used instead of pharmacopuncture. The following factors were investigated: (1) optical observations made with a handscope and clinical skin scores were evaluated; (2) tissue (general/immune) mast cells and CCR3(+) eosinophils, as well as vascular endothelial growth factor, fibroblast growth factor, and epidermal growth factor immunoreactive changes were evaluated; (3) CD4(+) and CD8(+) cells in the spleen were immunohistochemically examined; and, (4) the serum immunoglobulin (Ig)E level and lymphokines [interleukin (IL)-2, IL-4] were measured. In the PPI and the PPIII groups, the clinical skin score, total number of mast cells, CCR3(+) eosinophils immunoreaction, and total serum IgE, IL-2, and IL-4 levels were lower than the control group. The PPI and the PPIII groups also showed strong immunohistochemical reactions for vascular endothelial growth factor and fibroblast growth factor. The PPI group particularly showed a very strong immunohistochemical reaction for epidermal growth factor. All groups showed strong immune activity for CD8(+). The PPIII group showed strong immunity for both CD4(+) and CD8(+). From the above results, Rehmanniae radix pharmacopuncture (MeOH extraction) and anti-inflammatory pharmacopuncture exerted anti-allergic and anti-inflammatory effects, suggesting that they are promising agents for improving AD-related symptoms.

Buxus Microphylla var. Koreana Nakai Extract for the Treatment of Gastric Cancer.

OBJECTIVES: Buxus Microphylla var. Koreana Nakai Extract (BMKNE) is used as a folk remedy for malaria and veneral disease. In the present study, we investigated the effects of BMKNE in the growth and the survival of AGS cells, the most common human gastric adenocarcinoma cell lines. METHODS: The AGS cells were treated with varying concentrations of BMKNE. Analyses of the sub G1 peak, the caspase-3 and -9 activities, and the mitochondrial depolarization were conducted to determine whether AGS cell death occured by apoptosis. Also, to identify the role of transient receptor potential melastatin (TRPM) 7 channels in AGS cell growth and survival, we used human embryonic kidney (HEK) 293 cells overexpressed with TRPM7 channels. RESULTS: Experimental results showed that the sub G1 peak, the caspase-3 and -9 activities, and the mitochondrial depolarization were increased. Therefore, BMKNE was found to induce the apoptosis of these cells, and this apoptosis was inhibited by SB203580 (a p38 mitogen-activated protein kinase (MAPK) inhibitor), and by a c-jun NH2-terminal kinase (JNK) II inhibitor. Furthermore, BMKNE inhibited TRPM7 currents and TRPM7 channel over-expressions in HEK 293 cells, exacerbating BMKNE-induced cell death. CONCLUSIONS: These findings indicate that BMKNE inhibits the growth and the survival of gastric cancer cells due to a blockade of the TRPM7 channel's activity and MAPK signaling. Therefore, BMKNE is a potential drug for treatment of gastric cancer, and both the TRPM7 channel and MAPK signaling may play an important role in survival in gastric cancer cells.

Overexpression of Arabidopsis YUCCA6 in potato results in high-auxin developmental phenotypes and enhanced resistance to water deficit.

Indole-3-acetic acid (IAA), a major plant auxin, is produced in both tryptophan-dependent and tryptophan-independent pathways. A major pathway in Arabidopsis thaliana generates IAA in two reactions from tryptophan. Step one converts tryptophan to indole-3-pyruvic acid (IPA) by tryptophan aminotransferases followed by a rate-limiting step converting IPA to IAA catalyzed by YUCCA proteins. We identified eight putative StYUC (Solanum tuberosum YUCCA) genes whose deduced amino acid sequences share 50%-70% identity with those of Arabidopsis YUCCA proteins. All include canonical, conserved YUCCA sequences: FATGY motif, FMO signature sequence, and FAD-binding and NADP-binding sequences. In addition, five genes were found with ~50% amino acid sequence identity to Arabidopsis tryptophan aminotransferases. Transgenic potato (Solanum tuberosum cv. Jowon) constitutively overexpressing Arabidopsis AtYUC6 displayed high-auxin phenotypes such as narrow downward-curled leaves, increased height, erect stature, and longevity. Transgenic potato plants overexpressing AtYUC6 showed enhanced drought tolerance based on reduced water loss. The phenotype was correlated with reduced levels of reactive oxygen species in leaves. The results suggest a functional YUCCA pathway of auxin biosynthesis in potato that may be exploited to alter plant responses to the environment.

Protective efficacy of baculovirus-derived influenza virus-like particles bearing H5 HA alone or in combination with M1 in chickens.

Since 2003, the highly pathogenic avian influenza (HPAI) H5N1 has become a serious problem in animals and an increasing threat to public health. To develop effective vaccines for H5 HPAI in chickens, virus-like particles (VLP) were produced using a baculovirus expression system. The particles comprised hemagglutinin (HA) alone (HA-VLP) or HA in combination with a matrix protein (M1; HAM-VLP) derived from a recent clade 2.3.2.1 H5N1 HPAI virus. To compare the immunogenicity and protective efficacy of these VLPs, 10 µg HAM-VLP, the equivalent amounts of HA incorporated HA-VLP or whole inactivated virus (WIV), were emulsified with mineral oil and used to immunize chickens. The serum hemagglutination inhibition antibody levels induced by HA-VLP and HAM-VLP were comparable to WIV. Antibodies to nucleoprotein were detected only in the WIV group. Immunized chickens in each group survived and were protected against a lethal homologous virus challenge, showing no clinical signs of infection. The challenge virus was detected intermittently in some oropharyngeal swabs, but not in cloacal swabs or various organs, which means that VLPs and WIV provide protection against systemic but not local virus replication in chickens. After the challenge, the HA-VLP group showed significantly increased serum antibody levels compared to the HAM-VLP and WIV groups, and some chickens in the HA-VLP group seroconverted with respect to nucleoprotein. Taken together, these results suggest that VLPs may be an effective method for controlling HPAI in chickens. They could be applied to a differentiating infected from vaccinated animals (DIVA) strategy. In addition, it is likely that HAM-VLP is more efficacious than HA-VLP in chickens.

Protective effect of Korean red ginseng extract on the infections by H1N1 and H3N2 influenza viruses in mice.

Ginseng has been used in humans for thousands of years and is known to have multiple biological and immunomodulatory effects. In this study, we investigated whether Korean red ginseng extract would have preventive and antiviral effects on influenza virus infection. Oral administration to mice of red ginseng extract prior to infection significantly increased survival after infection with the 2009 pandemic H1N1 virus. Daily oral treatment of vaccinated mice with red ginseng extract provided enhanced cross-protection against antigenically distinct H1N1 and H3N2 influenza viruses. Naive mice that were infected with virus mixed with red ginseng extract showed significantly enhanced protection, lower levels of lung viral titers and interleukin-6, but higher levels of interferon-γ compared with control mice having virus infections without red ginseng extract, indicating an antiviral effect of ginseng. In addition, ginseng extract exhibited inhibitory effects on the growth of influenza virus in vitro. This study provides evidence that intake of ginseng extract will have beneficial effects on preventing lethal infection with newly emerging influenza viruses.

Protective effect of ginseng polysaccharides on influenza viral infection.

Ginseng polysaccharide has been known to have multiple immunomodulatory effects. In this study, we investigated whether Panax ginseng polysaccharide (GP) would have a preventive effect on influenza infection. Administration of mice with GP prior to infection was found to confer a survival benefit against infection with H1N1 (A/PR/8/34) and H3N2 (A/Philippines/82) influenza viruses. Mice infected with the 2009 H1N1 virus suspended in GP solution showed moderately enhanced survival rates and lower levels of lung viral titers and the inflammatory cytokine (IL-6). Daily treatment of vaccinated mice with GP improved their survival against heterosubtypic lethal challenge. This study demonstrates the first evidence that GP can be used as a remedy against influenza viral infection.

The effect of needle-embedding therapy and pharmacopuncture therapy on patients with urinary incontinence.

OBJECTIVES: This study was designed to evaluate the effect of traditional Korean medical therapy, consisting of needle-embedding therapy and pharmacopuncture therapy, on patients with urinary incontinence. METHODS: Twenty-nine patients with urinary incontinence underwent two sessions of traditional Korean medical therapy in a month. The pressure and the duration of pelvic muscle contraction were measured and compared. The primary endpoint of the study was improvement in the strength of pelvic floor muscle contraction. The paired t-test was used for the statistical analysis. RESULTS: Before treatment, a maximum pressure of 16.03 ± 6.28 mmHg and an average pressure of 9.62 ± 4.98 mmHg were measured, and the duration was 11.82 ± 12.08 seconds. After the first treatment, the pressures were 27.41 ± 10.46 mmHg (maximum) and 18.62 ± 9.72 mmHg (average), and the duration was 40.75 ± 60.02 seconds. After the second treatment, the pressures were 29 ± 14.86 mmHg (maximum) and 20.31 ± 11.51 mmHg (average), and the duration was 34.62 ± 42.02 seconds. Comparisons between before treatment and first treatment results and between before treatment and second treatment results showed statistically significant changes but the difference between the first treatment result and the second treatment result was not statistically significant. CONCLUSIONS: Patients receiving traditional Korean medical therapy showed improved pelvic muscle contraction ability after a single treatment. If strength of pelvic floor muscle contraction is improved, symptoms of urinary incontinence also get better. Traditional Korean medical therapy, with a focus on needle-embedding therapy and pharmacopuncture therapy, may be effective for treating urinary incontinence.

AtCML8, a calmodulin-like protein, differentially activating CaM-dependent enzymes in Arabidopsis thaliana.

Plants express many calmodulins (CaMs) and calmodulin-like (CML) proteins that sense and transduce different Ca(2+) signals. Previously, we reported divergent soybean (Glycine max) CaM isoforms (GmCaM4/5) with differential abilities to activate CaM-dependent enzymes. To elucidate biological functions of divergent CaM proteins, we isolated a cDNA encoding a CML protein, AtCML8, from Arabidopsis. AtCML8 shows highest identity with GmCaM4 at the protein sequence level. Expression of AtCML8 was high in roots, leaves, and flowers but low in stems. In addition, the expression of AtCML8 was induced by exposure to salicylic acid or NaCl. AtCML8 showed typical characteristics of CaM such as Ca(2+)-dependent electrophoretic mobility shift and Ca(2+) binding ability. In immunoblot analyses, AtCML8 was recognized only by antiserum against GmCaM4 but not by GmCaM1 antibodies. Interestingly, AtCML8 was able to activate phosphodiesterase (PDE) but did not activate NAD kinase. These results suggest that AtCML8 acts as a CML protein in Arabidopsis with characteristics similar to soybean divergent GmCaM4 at the biochemical levels.

Isolation and characterization of a novel calcium/calmodulin-dependent protein kinase, AtCK, from arabidopsis.

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin (Ca(2+)/CaM)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about Ca(2+)/CaM-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative Ca(2+)-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a Ca(2+)-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM Mn(2+). The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other Ca(2+)/CaM-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and Ca(2+)/CaM-dependent protein kinase), increasing the concentration of calmodulin to more than 3 microgram suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis Ca(2+)/CaM-dependent protein kinase which is presumably involved in CaM-mediated signaling.

Isolation of a calmodulin-binding transcription factor from rice (Oryza sativa L.).

Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horse-radish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca(2+)-dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5'-TWCG(C/T)GTKKKKTKCG-3' (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate beta-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.