NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)-III of the squash family and comparison with those of counterparts of CMTI-V of the potato I family.

Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd, is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; Mr 3 kDa) of the squash family and rCMTI-V (Mr approximately 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different Khyd values--2.4 (CMTI-III) and 9 (CMTI-V)--at 25 degrees C. The reactive-site peptide bond (P1-P1') is that between Arg5 and Ile6 in CMTI-III, and that between Lys44 and Asp45 in CMTI-V. The order parameters (S2) of backbone NHs of uniformly 15N-labeled rCMTI-III and rCMTI-III* were determined from measurements of 15N spin-lattice and spin-spin relaxation rates, and [1H]-15N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI-III [(S2) = 0.71] and rCMTI-III* [(S2) = 0.63] are more flexible than those of rCMTI-V [(S2) = 0.83] and rCMTI-V* [(S2) = 0.85]. The binding loop residues, P4-P1, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P1'-P4' residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys3-Cys20 crosslink. In contrast, the newly formed N-terminal (Pn' residues) becomes more flexible only in rCMTI-III*, most likely due to lack of an interaction between the P1' residue and the scaffold in rCMTI-III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn' residues seem to facilitate the resynthesis of the P1-P1' bond, leading to a lower Khyd value.     

林隙（gap）更新动态研究进展

林隙（ｇａｐ）更新动态研究进展臧润国（中国林业科学研究院生态环境研究所,北京１０００９３）ＲｅｓｅａｒｃｈＡｄｖａｎｃｅｓｏｆＧａｐＲｅｇｅｎｅｒａｔｉｏｎＤｙｎａｍｉｃｓ．ＺａｎｇＲｕｎｇｕｏ（ＩｎｓｔｉｔｕｔｅｏｆＥｃｏｌｏｇｙａｎｄＥｎｖｉｒ...     

气候因子和非气候因子对白三叶草叶片生长的影响

本文采用裂区试验设计,研究了刈割频率（强度）和品种对永久性混播草地组分白三叶草叶片生长的影响,并通过回归分析探讨了气候因子与白三叶草叶片生长的相关性。刈割对叶片出生率和每个葡萄茎现存叶片数的处理效应具季节依赖性特性,在夏季生长期,高频率刈割 处理。三叶草品种对叶片出生率和每葡萄茎观存叶片数均无显著的处理效应,但刈割与品种对秋初生长期叶片的出生率有显著的互作效应,对秋季生长期每葡萄茎现存叶片数有微弱     

Short-term and long-term effects of mowing on the vegetation of two calcareous fens

Abstract. Short-term field experiments are often used to predict and evaluate long-term management effects. Based on a mowing experiment in two calcareous fens near Lake Neuchâtel, Switzerland, we investigated whether short-term treatment effects (i.e. during the first four years) were confirmed by long-term results (13 - 14 yr). Plots were mown in summer or in winter or left unmown. The main long-term trends in overall species composition (based on percentage cover estimates) were already observable in the first four years: mown and unmown plots diverged, whereas summer-cut and winter-cut plots remained similar. At the individual species level, however, short-term and long-term treatment effects differed considerably: many species whose abundance seemed affected by treatments during the first four years showed no response in the long term, and vice versa. These discrepancies were similar when based on cover estimates or on counts of shoots. Species responses did actually depend on the time scale considered. Short-term and long-term treatment effects on species richness were similar (i.e. a decrease in unmown plots), although only long-term effects were significant. Treatment effects on the above-ground biomass varied considerably, and short-term trends (lower biomass in unmown plots) differed from long-term trends (higher biomass in unmown plots). Our sites showed little overall change in species composition during the period investigated, and treatment effects were low compared with other similar experiments. If study sites are less stable or treatment effects more drastic, a short-term evaluation is expected to be even less reliable. Knowledge on species dynamics at a site may help to choose the adequate spatial and temporal scale of investigation, and thus increase the efficiency of management experiments.     

Computerized classification of Mediterranean vegetation using panchromatic aerial photographs

Abstract. Historical aerial photographs are an important source for data on medium- to long-term (10 - 50 yr) vegetation changes. Older photographs are panchromatic, and manual interpretation has traditionally been used to derive vegetation data from such photographs. We present a method for computerized analysis of panchromatic aerial photographs, which enables one to create high resolution, accurate vegetation maps. Our approach is exemplified using two aerial photographs (from 1964 and 1992) of a test area on Mt. Meron, Israel. Spatial resolution (pixel size) of the geo-rectified photos was 0.30 m and spatial accuracy (RMS error) ca. 1 m. An illumination adjustment prior to classification was found to be essential in reducing misclassification error rates. Two classification approaches were employed: a standard maximum-likelihood supervised classifier, and a modification of a supervised classification, which takes into account spectral properties of individual pixels as well as their neighbourhood characteristics. Accuracy of the maximum likelihood classification was 81 % in the 1992 image and 54 % in the 1964 image. The neighbour classifier increased accuracy to 89 % and 82 % respectively. The overall results suggest that computerized analysis of sequences of panchromatic aerial photographs may serve as a valuable tool for the quantification of medium-term vegetation changes.     

The anatomy of the interspecific abundance–range size relationship for the British avifauna: I. Spatial patterns

Data from the British Trust for Ornithology Common Birds Census and two atlases of breeding birds were used to examine the form of the interspecific abundance–range size relationship for the British avifauna. The relationship is positive for both farmland and woodland habitats and over two different periods, with some evidence of curvilinearity, using either proportion of occupied sites or numbers of occupied 10 × 10 km squares as measures of range size, and mean density at occupied sites as a measure of abundance. A log-linear plot gives the highest correlation. The relationship is stronger if based on maximum local densities than if based on average densities, but there is no relationship using minimum local densities. Relationships based on abundances at individual sites are uniformly positive for all sites, although the relationships for many sites also show evidence of curvilinearity, especially when range size is measured as the proportion of occupied sites. Species show significant concordance in their rank abundances across sites. We discuss some implications of these results.     

Travelling wave dynamics and self-organization in a spatio-temporally structured population

We analyse spatial population dynamics showing that periodic or period-like chaotic dynamics produce self-organization structures, such as travelling waves. We suggest that self-organized patterns are associated with spatial synchrony patterns that often depend on geographical distance between subpopulations. The population dynamics also show statistical spatial autocorrelation patterns. We contrast our theoretical simulations with empirical data on annual damages in young sapling stands caused by voles. We conclude, on the basis of the periodicity, synchrony, and spatial autocorrelation patterns, and our simulation results, that vole dynamics represent travelling waves in population dynamics. We suggest that because such synchrony patterns are frequently observed in natural populations, spatial self-organization may be more common in population dynamics than reported in the literature.     

Computer modeling of δ opioid receptor and interaction with ligands

Summary Human σ opioid receptor (σOR), a G-protein-coupled receptor, has been modeled using the helix axes as revealed by the crystallographic structure of bacteriorhodopsin and ligand binding profiles of single-point mutants of σOR. The model revealed feasibility of existence of a second disulfide bridge between the transmembrane helices (TMHs) 6 and 7, Cys²⁷³-Cys³⁰³. A common binding site has been suggested for high-affinity selective agonists DPDPE, DPLPE, DTLET, BW373U86 and antagonist Naltrindole. Docking calculations have shown that the amino group of the ligands forms a hydrogen bond with the imidazole ring of His³⁰¹ (TMH7) rather than with Asp¹²⁸ (TMH3) and is not a cation counterpart of this highly conserved aspartyl residue. All the findings and the model shed light on the putative structure and functioning of opioid receptors and can be used for designing further mutagenesis experiments.     

α-Bn-o-AMPA as a cis peptide bond mimic in somatostatin analogues

Summary In a previous communication we reported the racemic synthesis of the cis peptide bond mimic α-benzyl-o-aminomethylphenylacetic acid and its incorporation in the cyclic somatostatin analoguesc[α(R andS)Bn-o-AMPA-Phe⁷-d-Trp⁸-Lys⁹-Thr¹⁰]. Since the epimeric peptides exhibit different binding affinities, we completed the structure-activity study with an asymmetric synthesis. A model for the solution conformation ofc[α(R andS)Bn-o-AMPA-Phe⁷-d-Trp⁸-Lys⁹-Thr¹⁰] is proposed on the basis of a 2D NMR study in CD₃OH and restrained molecular dynamics.     

Molecular Modeling of the NPY Binding Site on the Y1 Receptor

A three-dimensional model of the neuropeptide Y (NPY) - rat Y₁ (rY₁) receptor complex and of the NPY _13-36 - rY₁ receptor complex was constructed by molecular modeling based on the electron density projection map of rhodopsin and on site-directed mutagenesis studies of neuropeptide receptors. In order to further guide the modeling, the nucleotide sequences encoding Trp287, Cys295 and His297 in the third extracellular loop of the rY₁ receptor, were altered by site-directed mutagenesis experiments. Single-point mutated receptors were expressed in COS-7 cells, and tested for their ability to bind radio labelled NPY (³H-NPY). Mutations of Trp287 and His297 completely abolished binding of ³H-NPY. The Cys295Ser mutation only slightly decreased the binding of ³H-NPY, suggesting that the involvement of Cys295 in a disulphide bond is not essential for maintaining the correct three-dimensional structure of the binding site for NPY. Molecular dynamics simulations of NPY-rY₁ receptor interactions suggested that Asp199, Asp103 and Asp286 in the receptor interact, respectively, with Lys4, Arg33 and Arg35 of NPY. The simulations also suggested that His297 acts as a hydrogen acceptor from Arg35 in NPY, and that Tyr1 of NPY interacts with a binding pocket on the receptor formed by Asn115, Asp286, Trp287 and His297. Tyr36 in NPY interacted both with Thr41 and Tyr99 via hydrogen bonds, and also with Asn296, His297 and Phe301. The present study suggests that amino acid residues at the extracellular end of the transmembrane helices and in the extracellular loops are strongly involved in binding to NPY and NPY_13-36.Electronic Supplementary Material available.