Bowman-birk proteinase inhibitor confers heavy metal and multiple drug tolerance in yeast

Cultured Coptis japonica cells show tolerance to various toxic compounds. By yeast functional screening of cadmium (Cd) plates with its cDNA library, we isolated a gene encoding Bowman-Birk proteinase inhibitor (CjBBI). The yeast transformant of CjBBI showed multiple tolerance to various drugs adding to Cd, and revealed reduced Cd accumulation in cells. Preferential organs for Cjbbi expression were aerial parts of intact plants, and the subcellular localization of CjBBI was shown, using its green fluorescent protein fusion, to be the apoplast. Induction of Cjbbi expression by Cd treatment suggested that CjBBI was responsible for the tolerance to Cd observed in C. japonica cells.     

Iron chelation and a free radical scavenger suppress angiotensin II-induced upregulation of TGF-beta1 in the heart

Long-term administration of angiotensin II causes myocardial loss and cardiac fibrosis. We previously found iron deposition in the heart of the angiotensin II-infused rat, which may promote angiotensin II-induced cardiac damage. In the present study, we have investigated whether an iron chelator (deferoxamine) and a free radical scavenger (T-0970) affect the angiotensin II-induced upregulation of transforming growth factor-beta1 (TGF-beta1). Angiotensin II infusion for 7 days caused a robust increase in TGF-beta1 mRNA expression in vascular smooth muscle cells, myofibroblast-like cells, and migrated monocytes/macrophages. T-0970 and deferoxamine suppressed the upregulation of TGF-beta1 mRNA and reduced the extent of cardiac fibrosis in the heart of rats treated with angiotensin II. These agents blocked the angiotensin II-induced upregulation of heme oxygenase-1, a potent oxidative and cellular stress-responsive gene, but they did not significantly affect systolic blood pressure or plasma levels of aldosterone. In addition, T-0970 and deferoxamine suppressed the angiotensin II-induced upregulation of monocyte chemoattractant protein-1 in the heart. These results collectively suggest that iron and the iron-mediated generation of reactive oxygen species may contribute to angiotensin II-induced upregulation of profibrotic and proinflammatory genes, such as TGF-beta1 and monocyte chemoattractant protein-1.     

Applications of biotope mapping for spatial environmental planning and policy: case studies in urban ecosystems in Korea

The purpose of this paper is to investigate the current status and method of biotope mapping for practical use for landscape planning and environmental policy in urban ecosystem in Korea. We examine current ecological mapping of Seoul, Seongnam, Daegu, and Yongin. Ecological mapping is examined closely in terms of investigation methodology, investigation subject, classification of urban landscape, and the present condition of application. Biotope mapping in Seoul and Seongnam were carried out by the city governments concerned with the pre-set budgets earmarked for mapping. In order to promote the utilization of biotope maps for city planning in Korea, the following actions should be considered. First, the survey method should be standardized by introducing a uniform standard with respect to the scope of survey, the quality of primary data used, the survey method, and the level of the survey. Second, it is necessary to identify a basic category of biotope for each area by consolidating the outcome of the previous surveys. Third, it is highly desirable to minimize the differences between the evaluation criteria and the assessment factors. Fourth, it is ideal to apply the results of the biotope evaluation to city planning in an indirect manner through reflecting the results first in the landscape plans. In order to facilitate this alternative utilization, it is necessary to strengthen the control provisions contained in the ordinances of the city concerned or to enact a set of new provisions in the ordinances so that biotope mapping could be used more widely as a criterion for the spatial environmental impact assessment.     

Interaction analysis between tmRNA and SmpB from Thermus thermophilus

Small protein B, SmpB, is a tmRNA-specific binding protein essential for trans-translation. We examined the interaction between SmpB and tmRNA from Thermus thermophilus, using biochemical and NMR methods. Chemical footprinting analyses using full-length tmRNA demonstrated that the sites protected upon SmpB binding are located exclusively in the tRNA-like domain (TLD) of tmRNA. To clarify the SmpB binding sites, we constructed several segments derived from TLD. Optical biosensor interaction analyses and melting profile analyses with mutational studies showed that SmpB efficiently binds to only a 30-nt segment that forms a stem and loop, with the 5' and 3' extensions composed of the D-loop and variable-loop analogues. The conserved sequences, 16UCGA and 319GAC, in the extensions are responsible for the SmpB binding. These results agree with the those visualized by the cocrystal structure of TLD and SmpB from Aquifex aeolicus. In addition, NMR chemical shift mapping analyses, using the 30-nt segment and (15)N-labeled SmpB, revealed the characteristic RNA binding mode. The hydrogen bond pattern around beta2 changes, with the Gly in beta2, which acts as a hinge, showing the largest chemical shift change. It appears that SmpB undergoes structural changes indicating an induced fit upon binding to the specific region of TLD.     

Molecular characterization of N-acylethanolamine-hydrolyzing acid amidase, a novel member of the choloylglycine hydrolase family with structural and functional similarity to acid ceramidase

Bioactive N-acylethanolamines, including anandamide (an endocannabinoid) and N-palmitoylethanolamine (an anti-inflammatory and neuroprotective substance), are hydrolyzed to fatty acids and ethanolamine by fatty acid amide hydrolase. Moreover, we found another amidohydrolase catalyzing the same reaction only at acidic pH, and we purified it from rat lung (Ueda, N., Yamanaka, K., and Yamamoto, S. (2001) J. Biol. Chem. 276, 35552-35557). Here we report complementary DNA cloning and functional expression of the enzyme termed "N-acylethanolamine-hydrolyzing acid amidase (NAAA)" from human, rat, and mouse. The deduced primary structures revealed that NAAA had no homology to fatty acid amide hydrolase but belonged to the choloylglycine hydrolase family. Human NAAA was essentially identical to a gene product that had been noted to resemble acid ceramidase but lacked ceramide hydrolyzing activity. The recombinant human NAAA overexpressed in HEK293 cells hydrolyzed various N-acylethanolamines with N-palmitoylethanolamine as the most reactive substrate. Most interestingly, a very low ceramide hydrolyzing activity was also detected with NAAA, and N-lauroylethanolamine hydrolyzing activity was observed with acid ceramidase. By the use of tunicamycin and endoglycosidase, NAAA was found to be a glycoprotein. Furthermore, the enzyme was proteolytically processed to a shorter form at pH 4.5 but not at pH 7.4. Expression analysis of a green fluorescent protein-NAAA fusion protein showed a lysosome-like distribution in HEK293 cells. The organ distribution of the messenger RNA in rats revealed its wide distribution with the highest expression in lung. These results demonstrated that NAAA is a novel N-acylethanolamine-hydrolyzing enzyme that shows structural and functional similarity to acid ceramidase.     

Changes in patch mosaics and vegetation structure of rural forested landscapes under shifting human impacts in South Korea

Landscape change in rural ecosystems is a major global issue because they are an important ecological and socio-cultural resource. The aim of this study was to demonstrate the driving forces behind landscape changes. Changes in the patterns and processes of landscape mosaics and vegetation community in two rural areas (Teokdong-ri, TD and Teokseong-ri, TS) in Korea were studied between 1988 and 2002. Quantitative data of vegetation community, landscape mosaics, and local statistics of the two study areas were analyzed. Agricultural efficiency increased even decreasing household population and the agricultural area. In both areas, as the human dependence on natural resources decreased, the patterns and processes of landscape mosaics and forest structure were changed due to changing human impacts on land use. Owing to the abandonment of forest management for 14 years, the spatial heterogeneity of TD significantly increased more than that of TS. In detrended correspondence analysis ordination, floristic compositions of forest patches of two areas were unevenly located on the ordination axes. Pinus densiflora communities of both areas in 1988 were separately located in 2002 as two directions according to management intensity. The species composition of P. densiflora in one direction of 1988 became similar to those of Quercus forests in 2002. This may be because species composition of P. densiflora has been influenced due to changed habitat environments. Finally, we concluded that the development of forest communities and vegetation succession predicted the spatial pattern of future landscape mosaics and due to the vegetation dynamics of rural ecosystems in Korea.Nomenclature: Miyawaki et al. (1994), Lee (1993), and Lee (1998) for identifying plant species.     

A randomized phase II trial of personalized peptide vaccine plus low dose estramustine phosphate (EMP) versus standard dose EMP in patients with castration resistant prostate cancer

Personalized peptide vaccination (PPV) combined with chemotherapy could be a novel approach for many cancer patients. In this randomized study, we evaluated the anti-tumor effect and safety of PPV plus low-dose estramustine phosphate (EMP) as compared to standard-dose EMP for HLA-A2- or -A24-positive patients with castration resistant prostate cancer. Patients were randomized into groups receiving either PPV plus low-dose EMP (280 mg/day) or standard-dose EMP (560 mg/day). After disease progression, patients were switched to the opposite regime. The primary end point was progression-free survival (PFS). We randomly assigned 28 patients to receive PPV plus low-dose EMP and 29 patients to receive standard-dose EMP. Nineteen events in the PPV group and 20 events in the EMP group occurred during the first treatment. Median PFS for the first treatment was 8.5 months in the PPV group and 2.8 months in the EMP group with a hazard ratio (HR) of 0.28 (95% CI, 0.14–0.61; log-rank P = 0.0012), while there was no difference for median PFS for the second treatment. The HR for overall survival was 0.3 (95% CI, 0.1–0.91) in favor of the PPV plus low-dose EMP group (log-rank, P = 0.0328). The PPV plus low-dose EMP was well tolerated without major adverse effects and with increased levels of IgG and cytotoxic-T cell responses to the vaccinated peptides. PPV plus low-dose EMP was associated with an improvement in PSA-based PFS as compared to the standard-dose EMP alone.