Divalent metal ion complexes of S100B in the absence and presence of pentamidine

As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca²⁺-loaded and Zn²⁺,Ca²⁺-loaded S100B were determined by X-ray crystallography at 2.15 Å (R_free = 0.266) and 1.85 Å (R_free = 0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca²⁺-loaded S100B and Zn²⁺,Ca²⁺-loaded S100B determined here (1.88 Å; R_free = 0.267). In the presence and absence of Zn²⁺, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (± Zn²⁺), and the second Pnt molecule was mapped to the dimer interface (site 2; ± Zn²⁺) and in a pocket near residues that define the Zn²⁺ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn²⁺ to Ca²⁺-S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt-Zn²⁺,Ca²⁺-S100B when compared to Pnt-Ca²⁺-S100B. That Pnt can adapt to this Zn²⁺-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca²⁺- and Ca²⁺,Zn²⁺-bound S100B.     

Comparative genomic mapping between a 754 kb region flanking DREB1A in Arabidopsis thaliana and maize

Comparative mapping between model plant species for which the complete genome sequence is known and crop species has been suggested as a new strategy for the isolation of agronomically valuable genes. In this study, we tested whether comparative mapping between Arabidopsisand maize of a small region (754 kb) surrounding the DREB1A gene in Arabidopsis could lead to the identification of an orthologous region in maize containing the DREB1A homologue. The genomic sequence information available for Arabidopsis allowed for the selection of conserved, low-copy genes that were used for the identification of maize homologues in a large EST database. In total, 17 maize homologues were mapped. A second BLAST comparison of these genes to the recently completed Arabidopsis sequence revealed that 15 homologues are likely to be orthologous as the highest similarity score was obtained either with the original Arabidopsis gene or with a highly similar Arabidopsis gene localized on a duplication of the investigated region on chromosome 5. The map position of these genes showed a significant degree of orthology with the Arabidopsis region. Nevertheless, extensive duplications and rearrangements in the Arabidopsisand maize genomes as well as the evolutionary distance between Arabidopsis and maize make it unlikely that orthology and collinearity between these two species are sufficient to aid gene prediction and cloning in maize.     

Mapping QTLs for seed yield and drought susceptibility index in soy-bean（Glycine max L.） across different environments

Drought stress has long been a major constraint in maintaining yield stability of soybean （Glycine max （L.） Merr.） in rainfed ecosystems. The identification of consistent quantitative trait loci （QTL） involving seed yield per plant （YP） and drought susceptibility index （DSI） in a population across different environments would therefore be important in molecular marker-assisted breeding of soybean cultivars suitable for rainfed regions. The YP of a recombinant line population of 184 F2：7：11 lines from a cross of Kefengl and Nannong1138-2 was studied under water-stressed （WS） and well-watered （WW） conditions in field （F） and greenhouse （G） trials, and DSI for yield was calculated in two trials. Nineteen QTLs associated with YP-WS and YP-WW, and 10 QTLs associated with DSI, were identi- fied. Comparison of these QTL locations with previous findings showed that the majority of these regions control one or more traits re- lated to yield and other agronomic traits. One QTL on molecular linkage group （MLG） K for YP-F, and two QTLs on MLG C2 for YP-G, remained constant across different water regimes. The regions on MLG C2 for YP-WW-F and MLG H for YP-WS-F had a pleiotropic effect on DSI-F, and MLG A1 for YP-WS-G had a pleiotropic effect on DSI-G. The identification of consistent QTLs for YP and DSI across different environments will significantly improve the efficiency of selecting for drought tolerance in soybean.     

Molecular mapping of a major gene conferring resistance to maize mosaic virus

 The objective of this study was to determine the genetic basis of resistance to maize mosaic virus (MMV). Molecular markers were used to map resistance loci to MMV in a set of 91 maize (Zea mays L.) recombinant inbred lines (RILs), derived from the cross between Hi31 (a B68 conversion resistant to MMV) and Kil4 (a Thai inbred susceptible to MMV). The RILs were evaluated for MMV resistance in disease nurseries in Hawaii in the winter of 1993 and the summer of 1994. Twenty-eight highly susceptible RILs were chosen for gene mapping using the pooled-sampling approach. Initial evidence from the pooled DNA indicated that restriction fragment length polymorphism (RFLP) probes on chromosome 3 near the centromere were biased to the susceptible parent allele. Analysis of 91 RILs at 103 RFLP loci confirmed the presence of a major MMV resistance gene on chromosome 3. The resistant allele at this locus, previously named Mv1, is present in the resistant parent Hi31 and traces back to the Argentine parent used in conferring common rust resistance to B68. We conclude that resistance to MMV in B68 and Caribbean flints involves a major gene mv1 on chromosome 3 located between RFLP markers umc102 and php20508. Received: 12 June 1996 / Accepted: 5 July 1996     

Actin surface structure revealed by antibody imprints: evaluation of phage-display analysis of anti-actin antibodies

Phage-display peptide library analysis of an anti-F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X-ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage-display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti-actin immune serum. One hundred and forty-four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F-actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken beta-actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X-ray crystal structure suggested that within a 15 A radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root-mean-square deviation fits of 2.1-2.5 A. We conclude that phage-display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.     

A Simplified Mass-Transfer Model for Visual Pigments in Amphibian Retinal-Cone Outer Segments

When radiolabeled precursors and autoradiography are used to investigate turnover of protein components in photoreceptive cone outer segments (COSs), the labeled components—primarily visual pigment molecules (opsins)—are diffusely distributed along the COS. To further assess this COS labeling pattern, we derive a simplified mass-transfer model for quantifying the contributions of advective and diffusive mechanisms to the distribution of opsins within COSs of the frog retina. Two opsin-containing regions of the COS are evaluated: the core axial array of disks and the plasmalemma. Numerical solutions of the mass-transfer model indicate three distinct stages of system evolution. In the first stage, plasmalemma diffusion is dominant. In the second stage, the plasmalemma density reaches a metastable state and transfer between the plasmalemma and disk region occurs, which is followed by an increase in density that is qualitatively similar for both regions. The final stage consists of both regions slowly evolving to the steady-state solution. Our results indicate that autoradiographic and cognate approaches for tracking labeled opsins in the COS cannot be effective methodologies for assessing new disk formation at the base of the COS.     

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.     

Assignment of the Gene for Porcine Insulin-Like Growth Factor Binding Protein 1 to Chromosome 18 and Detection of Polymorphisms in Intron 2 by PCR–RFLP

We have obtained a partial cDNA and three BAC clones for the porcine insulin-like growth factor binding protein 1 gene (IGFBP-1). Results of fluorescence in situ and radiation hybrid (RH) mapping assigned this gene to porcine chromosome (SSC) 18q24-qter. We found two types of polymerase chain reaction–restriction-fragment-length polymorphisms (PCR–RFLP) in intron 2 by using FokI and AluI.     

Mass transfer limitation of sulfate in methanogenic aggregates

The role of mass transfer limitation of sulfate as a factor governing the competition between sulfate reducing and methane producing bacteria in methanogenic aggregates was theoretically evaluated by the calculation of steady-state sulfate microprofiles using a reference set of parameters obtained from the literature. The shooting method was used as a numerical technique for solving the mathematical model. The effect of the parameters on mass transport limitation was tested by varying each reference value of the parameters with a factor of 3. Sulfate limitation within granules prevailed at moderate (0.1 kg m(-3)) and low sulfate concentrations in the bulk liquid, at high maximum sulfate utilization rates (3.73 x 10(-5) kg SO(4) (2-) kg(-1) VSS S(-1) or biomass concentrations (40 KG VSS m(-3)), and in large aggregates (radius of 7.5 10(-4) m). The effective diffusion coefficient of sulfate and the affinity constant were less determinative for the penetration depth of sulfate within a granule. (c) 1994 John Wiley & Sons, Inc.     

N-glycan structures of a recombinant mouse soluble Fcγ receptor II

N-glycans of a recombinant mouse soluble Fc receptor II (sFcRII) expressed in baby hamster kidney cells were released from glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a three-dimensional high-performance liquid chromatography (HPLC) mapping technique on three kinds of HPLC columns [Takahashi, et al. (1995) Anal. Biochem. 226: 139–46]. Eighteen different major N-glycan structures were identified, of which six were neutral (45%), five mono-sialyl (49%), one di-sialyl (4.6%), five tri-sialyl (1.1%), and one tetra-sialyl (0.3%). All N-glycan structures determined were complex type with fucosylation at the N-acetylglucosamine residue of the reducing end, and N-acetylneuraminic acid, when present, was -(2,3)-linked. The existence of a unique structure containing both N-acetylgalactosamine and -(2,3)-N-acetylneuraminic acid residues at the reducing ends, as below, was confirmed by MALDI-TOF mass spectrometry.