Synthesis of phospholipase A2 inhibitory biflavonoids

A series of C-C biflavones was designed to investigate the relationship between structural array of different flavone-flavone subunit linkage and the inhibitory activity against phospholipase A2 (PLA2). Among six classes of C-C biflavones designed, four classes of C-C biflavones, which have flavone-flavone subunit linkages at A ring-A ring, A ring-B ring, B ring-B ring, and B ring-C ring, were synthesized. The synthetic biflavones exhibited somewhat different inhibitory activities against sPLA2-IIA. Among them, the biflavone a having a C-C 4'-4' linkage showed comparable inhibitory activity with that of the natural biflavonoid, ochnaflavone, and 7-fold stronger activity than that of amentoflavone. Further chemical modification is being carried out in order to obtain the chemically optimized biflavonoids.     

A molecular modeling study of the interaction of 2'-fluoro-substituted analogues of dUMP/FdUMP with thymidylate synthase

Molecular dynamics simulations and free energy calculations are presented, exploring previously described experimentally studied interactions of a series of 2'-fluoro-substituted dUMP/FdUMP analogues with thymidylate synthase (TS). The results show the inhibitory behaviors of 2'-F-ara-UMP, 2',2'-diF-dUMP and 2',5-diF-ara-UMP to be dependent upon the binding positions and orientations adopted by the molecules of these compounds in the active site of TS. The binding mode of 2',5-diF-ara-UMP suggests a novel role of the active site residue Trp 80, stabilizing through hydrophobic stacking the binding position of the pyrimidine ring in 2',5-diF-ara-UMP.     

A urokinase-type plasminogen activator-inhibiting cyclic peptide with an unusual P2 residue and an extended protease binding surface demonstrates new modalities for enzyme inhibition

To find new principles for inhibiting serine proteases, we screened phage-displayed random peptide repertoires with urokinase-type plasminogen activator (uPA) as the target. The most frequent of the isolated phage clones contained the disulfide bridge-constrained sequence CSWRGLENHRMC, which we designated upain-1. When expressed recombinantly with a protein fusion partner, upain-1 inhibited the enzymatic activity of uPA competitively with a temperature and pH-dependent K(i), which at 25 degrees C and pH 7.4 was approximately 500 nm. At the same conditions, the equilibrium dissociation constant K(D), monitored by displacement of p-aminobenzamidine from the specificity pocket of uPA, was approximately 400 nm. By an inhibitory screen against other serine proteases, including trypsin, upain-1 was found to be highly selective for uPA. The cyclical structure of upain-1 was indispensable for uPA binding. Alanine-scanning mutagenesis identified Arg(4) of upain-1 as the P(1) residue and indicated an extended binding interaction including the specificity pocket and the 37-, 60-, and 97-loops of uPA and the P(1), P(2), P(3)', P(4)', and the P(5)' residues of upain-1. Substitution with alanine of the P(2) residue, Trp(3), converted upain-1 into a distinct, although poor, uPA substrate. Upain-1 represents a new type of uPA inhibitor that achieves selectivity by targeting uPA-specific surface loops. Most likely, the inhibitory activity depends on its cyclical structure and the unusual P(2) residue preventing the scissile bond from assuming a tetrahedral geometry and thus from undergoing hydrolysis. Peptide-derived inhibitors such as upain-1 may provide novel mechanistic information about enzyme-inhibitor interactions and alternative methodologies for designing effective protease inhibitors.     

Caspase-3 binds diverse P4 residues in peptides as revealed by crystallography and structural modeling

Caspase-3 recognition of various P4 residues in its numerous protein substrates was investigated by crystallography, kinetics, and calculations on model complexes. Asp is the most frequent P4 residue in peptide substrates, although a wide variety of P4 residues are found in the cellular proteins cleaved by caspase-3. The binding of peptidic inhibitors with hydrophobic P4 residues, or no P4 residue, is illustrated by crystal structures of caspase-3 complexes with Ac-IEPD-Cho, Ac-WEHD-Cho, Ac-YVAD-Cho, and Boc-D(OMe)-Fmk at resolutions of 1.9–2.6 Å. The P4 residues formed favorable hydrophobic interactions in two separate hydrophobic regions of the binding site. The side chains of P4 Ile and Tyr form hydrophobic interactions with caspase-3 residues Trp206 and Trp214 within a non-polar pocket of the S4 subsite, while P4 Trp interacts with Phe250 and Phe252 that can also form the S5 subsite. These interactions of hydrophobic P4 residues are distinct from those for polar P4 Asp, which indicates the adaptability of caspase-3 for binding diverse P4 residues. The predicted trends in peptide binding from molecular models had high correlation with experimental values for peptide inhibitors. Analysis of structural models for the binding of 20 different amino acids at P4 in the aldehyde peptide Ac-XEVD-Cho suggested that the majority of hydrophilic P4 residues interact with Phe250, while hydrophobic residues interact with Trp206, Phe250, and Trp214. Overall, the S4 pocket of caspase-3 exhibits flexible adaptation for different residues and the new structures and models, especially for hydrophobic P4 residues, will be helpful for the design of caspase-3 based drugs.     

Protein kinase C-alpha and -delta are required for NADPH oxidase activation in WKYMVm-stimulated IMR90 human fibroblasts

Gene duplications in rodents have given rise to a family of proteases that are expressed exclusively in placenta. To define the biological role of these enzymes specific inhibitors are needed to differentiate their activities from other more ubiquitously expressed proteases, such as cathepsins B and L. Libraries of peptidyl inhibitors based upon a 4-cyclohexanone pharmacophore were screened for inhibition of cathepsins P, L, and B. The tightest binding dipeptidyl inhibitor for cathepsin P contained Tyr in P(2) and Trp in P(2)('), consistent with the specificity of this enzyme for hydrophobic amino acids at these sites in synthetic substrates. An inhibitor containing Trp in both P(2) and P(2)(') provided better discrimination between cathepsin P and cathepsins B and L. Extension of the inhibitors to include P(3), and P(3)(') amino acids identified an inhibitor with Trp in P(2), P(2)('), and P(3), and Phe in P(3)(') that bound to cathepsin P with a K(i) of 32 nM. This specificity for inhibitors with hydrophobic aromatic amino acids in these four positions is unique among the lysosomal cysteine proteases. This inhibitor bound to cathepsin P an order of magnitude tighter than to mouse and human cathepsin L and two orders of magnitude tighter than to human cathepsin B. Cbz-Trp-Trp-4-cyclohexanone-Trp-Phe-OMe can discriminate cathepsin P from cathepsins B and L and consequently can be used to specifically inhibit and identify cathepsin P in cellular systems.     

西南桦幼苗接种丛枝菌根真菌的生长与光合生理响应

为了解丛枝菌根真菌(arbuscular mycorrhiza,AMF)对西南桦幼苗生长和光合生理的影响,对西南桦(Betula alnoides)优良无性系接种AMF菌株后的生长、光合参数、叶绿素含量和荧光参数进行了研究。结果表明,6个AMF菌株均能与西南桦无性系幼苗形成共生体,接种根内球囊霉(Glomus intraradices)菌株(AMF5)和摩西球囊霉(G.mosseae)HUN03B菌株(AMF3)显著提高了幼苗生长量、净光合速率、水分利用效率、叶绿素含量和荧光参数(P0.05),显示出AMF5、AMF3与幼苗的亲和力明显优于其他菌株。西南桦4个无性系间的菌根侵染率差异不显著(P0.05),但AMF对无性系FB4、BY1的促生效应显著优于FB4+和A5。因此,适合与西南桦共生的优良菌株为AMF5和AMF3,这为西南桦菌根化育苗提供理论依据。     

Inhibition mechanism of cytokine activity of human autocrine motility factor examined by crystal structure analyses and site-directed mutagenesis studies

Autocrine motility factor (AMF), a tumor-secreted cytokine, stimulates cell migration in vitro and metastasis in vivo. AMF is genetically identical with the extracellular cytokines neuroleukin (NLK) and maturation factor (MF) and, interestingly, the intracellular enzyme phosphohexose isomerase (PHI). The crystal structures of the inhibitor-free open form and the inhibitor (erythrose 4-phosphate, E4P, a strong inhibitor of AMF's cytokine activity)-bound closed form of human AMF have been determined at 1.9 A and 2.4 A resolution, respectively. Upon E4P binding, local conformation changes (open to closed) occur around the inhibitor-binding site. The E4P-bound structure shows that the location of the inhibitor (of cytokine activity) binding site of human AMF is very similar to those of the inhibitor (of enzymatic activity) binding sites of PHIs. The present study shows clearly that there is structural overlap of the regions responsible for the enzymatic and cytokine functions of AMF and PHI and suggests two scenarios for the inhibition mechanism of cytokine activity of AMF by the carbohydrate phosphate group. One likely scenario is that the compound could compete for AMF binding with the carbohydrate moiety of the AMF receptor (AMFR), which is a glycosylated seven-transmembrane helix protein. The other scenario is that the local conformation changes upon inhibitor binding may affect the AMF-AMFR interactions. To examine roles of the residues in the inhibitor-binding site, two mutant AMFs were prepared. Replacements of His389, which is hydrogen-bonded to the hydroxyl group of E4P by Phe, and Thr215, which is hydrogen-bonded to the phosphate group of E4P by Asp, result in mutant AMFs that are impaired in cytokine activity. These results suggest a role for these amino acids in recognition of a carbohydrate moiety of the AMFR. Since the E4P is one of the smallest compounds having AMF inhibitor activity, knowledge of the present crystal structure would provide an insight into the lead compound design of more effective AMF inhibitors.     

Sequence-specific 1H NMR assignments and structural characterization of bovine seminal fluid protein PDC-109 domain b

Sequence-specific resonance assignments for the isolated second or b domain of the bovine seminal fluid protein PDC-109 have been obtained from analysis of two-dimensional 1H NMR experiments recorded at 500 MHz. These assignments include the identification of all aromatic and most aliphatic amino acid resonances. Stereospecific assignment of resonances stemming from the Val2 CH3 gamma,gamma' groups and from seven CH beta,beta' geminal pairs has been accomplished by analysis of 3J alpha beta coupling constants in conjunction with patterns of cross-peak intensities observed in two-dimensional nuclear Overhauser effect (NOESY) spectra. Analysis of NOESY and 3J alpha NH data reveals a small antiparallel beta-sheet involving stretches containing residues 25-28 and 39-42, a cis-proline residue (Pro4), antiparallel strands consisting of residues 1-3, 5-7, and 10-13, and an aromatic cluster composed of Tyr7, Trp26, and Tyr33. The results of distance geometry and restrained molecular dynamics calculations indicate that the global fold of the PDC-109 b domain, a type II module related to those found in fibronectin, is somewhat different from that predicted by modeling the structure on the basis of homology between type II and kringle units. A shallow depression in the molecular surface which presents a solvent-exposed hydrophobic area--a potential ligand-binding site-is identified in the NMR-based models.     

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket.

Serine endoproteases such as trypsins and subtilisins are known to have an extended substrate binding region that interacts with residues P6 to P3' of a substrate. In order to investigate the structural and functional effects of replacing residues at the S4 substrate binding pocket, the serine protease from the alkalophilic Bacillus strain PB92, which shows homology with the subtilisins, was mutated at positions 102 and 126-128. Substitution of Val102 by Trp results in a 12-fold increase in activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpNA). An X-ray structure analysis of the V102W mutant shows that the Trp side chain occupies a hydrophobic pocket at the surface of the molecule leaving a narrow crevice for the P4 residue of a substrate. Better binding of sAAPFpNA by the mutant compared with the wild type protein as indicated by the kinetic data might be due to the hydrophobic interaction of Ala P4 of the substrate with the introduced Trp102 side chain. The observed difference in binding of sAAPFpNA by protease PB92 and thermitase, both of which possess a Trp at position 102, is probably related to the amino acid substitutions at positions 105 and 126 (in the protease PB92 numbering). Kinetic data for the variants obtained by random mutation of residues Ser126, Pro127 and Ser128 reveal that the activity towards sAAPFpNA increases when a hydrophobic residue is introduced at position 126.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical replacement of P1' arginine residue at the first reactive site of peanut protease inhibitor B-III

The contribution of the P1' residue at the first reactive site of peanut protease inhibitor B-III to the inhibition was analyzed by replacement of the P1' Arg(11) with other amino acids (Arg, Ser, Ala, Leu, Phe, Asp) after selective modification of the second reactive site. The Arg derivative had the same trypsin inhibitory activity as the native inhibitor (Ki = 2 X 10(-9) M). The Ser derivative inhibited more weakly (Ki = 2 X 10(-8) M). The Ala and Leu derivatives inhibited trypsin very weakly (Ki = 2 X 10(-7) M and 4 X 10(-7) M, respectively), and the Phe and Asp derivatives not at all. These results suggest that the P1' arginine residue is best for inhibitory activity at the first reactive site of B-III, although it has been suggested that a P1' serine residue at the reactive site is best for inhibitory activity of Bowman-Birk type inhibitors.     

Extended binding inhibitors of chymotrypsin that interact with leaving group subsites S1'-S3'

We have synthesized inhibitors of chymotrypsin, based on fluoromethyl ketones, that bind at S and S' subsites. "Small" inhibitors of serine proteases, which have previously been synthesized, only interact with S subsites. The parent compound is Ac-Leu-ambo-Phe-CF2H (1) (Ki = 25 X 10(-6) M). This inhibitor was modified by successively replacing H of the -CF2H group by -CH2CH2CONHCH3, (4), -CH2CH2CONH-Leu-NHMe (5), -CH2CH2CONH-Leu-Val-OEt (6), and -CH2CH2CONH-Leu-Arg-OMe (7). Corresponding Ki values are 7.8 (4), 0.23 (5), 0.21 (6), and 0.014 (7) microM. Extending 5 to 6 by addition of Val-OEt at P3' does not decrease Ki. In contrast, extension of 5 to 7 by incorporating Arg-OMe at P3' decreases Ki approximately 15-fold, suggesting interaction between Arg and the S3' subsite but no corresponding interaction at that subsite with Val. These results are in accordance with results obtained with the homologous family of avian ovomucoid third domain proteins. Proteins with Arg at the P3' position show highly favorable interactions with the protease at the S3' subsite [Park, S. J. (1985) Ph.D. Thesis, Purdue University; M. Laskowski, Jr., personal communication]. These results establish that incorporation of residues which interact with S' subsites significantly increases the efficacy of inhibitors and that valuable information concerning the most effective amino acid composition of small inhibitors can be obtained from the amino acid sequence of protein inhibitors.     

Substrate-related potent inhibitors of brain metalloendopeptidase

Rat brain metalloendopeptidase (EC 3.4.24.15) generates Leu- and Met-enkephalin from several larger opioid peptides and is capable of degrading a number of neuropeptides. Substrate-related N-(1-carboxy-3-phenylpropyl) peptide derivatives were synthesized and tested for enzyme inhibition. The best of these derivatives, N-[1(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, inhibited the enzyme in a competitive manner with a Ki of 16 nM. The data indicate that the carboxyl group of the N-(1-carboxy-3-phenylpropyl) moiety coordinates with the active site zinc atom and that the remaining part of the inhibitor is necessary for interaction with the substrate recognition site of the enzyme. Replacement of the 1-carboxy-3-phenylpropyl group by a carboxymethyl group decreased the inhibitory potency by more than 3 orders of magnitude, emphasizing the importance of the hydrophobic phenyl group for inhibitor binding to a hydrophobic pocket at the S1 subsite. Replacement of the Tyr residue by an Ala residue decreased the inhibitory potency by more than 20-fold. Changes in the structure of the residue interacting with the S1' subsite could cause a more than 60-fold change in inhibition. The inhibitors were either ineffective or only weakly inhibitory against membrane-bound metalloendopeptidase ("enkephalinase", EC 3.4.24.11), an enzyme highly active in rabbit kidney but also present in brain. The data indicate the presence of an extended binding site in the enzyme with residues interacting with S1, S1', and S3' subsites largely determining inhibitor binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biflavonoids from Torreya nucifera displaying SARS-CoV 3CL(pro) inhibition

As part of our search for botanical sources of SARS-CoV 3CL(pro) inhibitors, we selected Torreya nucifera, which is traditionally used as a medicinal plant in Asia. The ethanol extract of T. nucifera leaves exhibited good SARS-CoV 3CL(pro) inhibitory activity (62% at 100μg/mL). Following bioactivity-guided fractionation, eight diterpenoids (1-8) and four biflavonoids (9-12) were isolated and evaluated for SARS-CoV 3CL(pro) inhibition using fluorescence resonance energy transfer analysis. Of these compounds, the biflavone amentoflavone (9) (IC(50)=8.3μM) showed most potent 3CL(pro) inhibitory effect. Three additional authentic flavones (apigenin, luteolin and quercetin) were tested to establish the basic structure-activity relationship of biflavones. Apigenin, luteolin, and quercetin inhibited 3CL(pro) activity with IC(50) values of 280.8, 20.2, and 23.8μM, respectively. Values of binding energy obtained in a molecular docking study supported the results of enzymatic assays. More potent activity appeared to be associated with the presence of an apigenin moiety at position C-3' of flavones, as biflavone had an effect on 3CL(pro) inhibitory activity.     

Inhibition of Mycobacterium tuberculosis tyrosine phosphatase PtpA by synthetic chalcones: Kinetics,molecular modeling,toxicity and effect on growth

Tuberculosis (TB) is a major cause of morbidity and mortality throughout the world, and it is estimated that one-third of the world’s population is infected with Mycobacterium tuberculosis. Among a series of tested compounds, we have recently identified five synthetic chalcones which inhibit the activity of M. tuberculosis protein tyrosine phosphatase A (PtpA), an enzyme associated with M. tuberculosis infectivity. Kinetic studies demonstrated that these compounds are reversible competitive inhibitors. In this work we also carried out the analysis of the molecular recognition of these inhibitors on their macromolecular target, PtpA, through molecular modeling. We observed that the predominant determinants responsible for the inhibitory activity of the chalcones are the positions of the two methoxyl groups at the A-ring, that establish hydrogen bonds with the amino acid residues Arg17, His49, and Thr12 in the active site of PtpA, and the substitution of the phenyl ring for a 2-naphthyl group as B-ring, that undergoes π stacking hydrophobic interaction with the Trp48 residue from PtpA. Interestingly, reduction of mycobacterial survival in human macrophages upon inhibitor treatment suggests their potential use as novel therapeutics. The biological activity, synthetic versatility, and low cost are clear advantages of this new class of potential tuberculostatic agents.     

Site-directed mutagenesis of alanine-382 of human antithrombin III

Antithrombin III Hamilton is a structural variant of antithrombin III (AT-III) with normal heparin affinity but impaired serine protease inhibitory activity. The molecular defect of AT-III-Hamilton is a substitution of threonine for alanine at amino acid residue 382. Recently it has been shown that both plasma-derived and cell-free-derived AT-III-Hamilton polypeptides act as substrates rather than inhibitors of thrombin and factor Xa. In the present study, the cell-free expression phagemid vector pGEM-3Zf(+)-AT-III1-432 was mutated at amino acid residue 382 of AT-III to generate 7 cell-free-derived variants. All these cell-free-derived AT-III variants were able to bind heparin as effectively as cell-free-derived normal AT-III. In terms of alpha-thrombin inhibitory activity each variant reacted differently. Variants could be grouped into 3 categories with respect to thrombin-AT-III complex formation: (1) near normal activity (glycine, isoleucine, leucine, valine); (2) low activity (threonine, glutamine); (3) no detectable activity (lysine). These data suggest that mutations at position 382 of AT-III may have a variable effect on protease inhibitory activity, depending on either the stability of the P12-P9 region of the exposed loop of AT-III, or the inability of the amino acid residue at position 382 to interact with a conserved hydrophobic pocket consisting of phenylalanine (at positions 77, 221 and 422) and isoleucine (position 412) residues.     

Discovery of (S)-6-methoxy-chroman-3-carboxylic acid (4-pyridin-4-yl-phenyl)-amide as potent and isoform selective ROCK2 inhibitors

ROCK1 and ROCK2 are highly homologous isoforms. Accumulated studies indicate that they have distinct different functions, and the development of isoform selective ROCK inhibitors will pave new roads for the treatment of various diseases. In this work, a series of amide-chroman derivatives were synthesized and biologically evaluated in order to develop potent and isoform selective ROCK2 inhibitors. Remarkably, (S)-6-methoxy-chroman-3-carboxylic acid (4-pyridin-4-yl-phenyl)-amide ((S)-7c) possessed ROCK2 inhibitory activity with an IC₅₀ value of 3?nM and 22.7-fold isoform selectivity (vs. ROCK1). Molecular docking indicated that hydrophobic interactions were the key element for the high potency and isoform selectivity of (S)-7c. The binding free energies predicted by MM/GBSA were in good agreement with the experimental bioactivities, and the analysis of individual energy terms suggested that residue Lys105 in ROCK1 or Lys121 in ROCK2 was the key residue for the isoform selectivity of (S)-7c.     

Ab initio electronic structure of the progestogen norethisterone and its 5 alpha-derivatives

The steroid 17 alpha-ethynyl-19-nor-4-androsten-17 beta-ol, 3-one (Norethisterone; NET) and its 5 alpha-dihydro (5 alpha-NET), 3 alpha- and 3 beta-tetrahydro derivatives (3 alpha,5 alpha- and 3 beta,5 alpha-NET), were comparatively studied by the ab initio quantum mechanics theory. Additionally, 5 alpha-androstan-3 beta,17 beta-diol (ADIOL) was also studied. The Hartree-Fock method and the 6-31G(*) basis set were used to obtain the lowest energy conformation, geometries, electronic structure and physicochemical properties of the steroids. The results showed bond distances and valence angles similar among all steroids, but some differences in dihedral angles in the A-B-ring system were observed. The electronic structure analysis showed that NET has both frontier orbitals that is, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) located at the C4-C5 pi-bond. In A-ring reduced derivatives, the HOMO was found at the 17 beta-OH and ethynyl groups. In the case of 5 alpha-NET, the LUMO was confined to the A-ring and its C3 carbonyl group while the two NET tetrahydro-reduced derivatives showed the LUMO at the 17 beta-OH and ethynyl groups. The energy changes of the rotational barrier of the 17 beta-OH group suggest that its movement is somewhat restricted by the 17 alpha-ethynyl group. Interestingly both groups at C17 form a single electrostatic potential with high electronic density. On the other side, the 19-nor condition increases the A-ring mobility. However, the 3 beta-OH group of 3 beta,5 alpha-NET may rotate without significant energy differences as compared to the same group in ADIOL. The electronic structure of NET and its A-ring reduced derivatives explains in some extent their interaction with androgen and progesterone receptors as well as their selectivity for the estrogen alpha-receptor.     

Design of serine proteinase inhibitors by combinatorial chemistry using trypsin inhibitor SFTI-1 as a starting structure.

A small peptide library of monocyclic SFTI-1 trypsin inhibitor from sunflower seeds modified in positions P(1) and P(4)' was synthesized using a portioning-mixing method. The peptide library was deconvoluted by the iterative approach in solution. Two trypsin ([Met(9)]-SFTI-1 and [Arg(5), Abu(9)]-SFTI-1), one chymotrypsin ([Phe(5)]-SFTI-1) and one human elastase ([Leu(5), Trp(9)]-SFTI-1) inhibitors were selected and resynthesized. The values of their association equilibrium constants (K(a)) with target enzymes indicate that they are potent inhibitors. In addition, the last two analoges belong to the most active inhibitors of this size. The results obtained show that the conserved Pro(9) residue in the Bowman-Birk inhibitor (BBI)s is not essential for inhibitory activity.     

The network structure of plant-arbuscular mycorrhizal fungi

Ecological network theory predicts that in mutualistic systems specialists tend to interact with a subset of species with which generalists interact (i.e. nestedness). Approaching plant-arbuscular mycorrhizal fungi (AMF) association using network analyses will allow the generality of this pattern to be expanded to the ubiquitous plant-AMF mutualism. Based on certain plant-AMF specificity recently suggested, networks are expected to be nested as a result of their mutualistic nature, and modular, with certain species interacting more tightly than others. Network analyses were used to test for nestedness and modularity and to compare the different contribution of plant and AMF to the overall nestedness. Plant-AMF networks share general network properties with other mutualisms. Plant species with few AMFs in their roots tend to associate with those AMFs recorded in most plant species. AMFs present in a few plant species occur in plant species sheltering most AMF (i.e. nestedness). This plant-AMF network presents weakly interlinked subsets of species, strongly connected internally (i.e. modularity). Both plants and AMF show a nested structure, although AMFs have lower nestedness than plants. The plant-AMF interaction pattern is interpreted in the context of how plant-AMF associations can be underlying mechanisms shaping plant community assemblages.     

慈菇蛋白酶抑制剂A和B中Trp残基周围构象与酶抑制专一性的关系

通过定点诱变结合荧光光谱学方法研究了慈菇蛋白酶抑制剂A和B(APIA和APIB)Trp残基周围构象与酶抑制专一性之间的关系。研究表明APIB中的两个Trp残基 (93和 12 2位 )所处环境的疏水性要比APIA中的强。Trp定点诱变研究表明 ,在APIB中 ,Trp12 2 周围环境的疏水性要比Trp93 强。用Ser和Leu分别替代 82位Leu和 87位Arg ,使APIB中色氨酸荧光特性变得与APIA的基本相同 ,同时还发现其酶的抑制专一性也变得趋近APIA的 ,暗示Trp周围的构象与酶抑制剂的抑制专一性有关。