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PubMed2004

Crystal structure of the calcium-stabilized human factor IX Gla domain bound to a conformation-specific anti-factor IX antibody.

The binding of Factor IX to membranes during blood coagulation is mediated by the N-terminal gamma-carboxyglutamic acid-rich (Gla) domain, a membrane-anchoring domain found on vitamin K-dependent blood coagulation and regulatory proteins. Conformation-specific anti-Factor IX antibodies are directed at the calcium-stabilized Gla domain and interfere with Factor IX-membrane interaction. One such antibody, 10C12, recognizes the calcium-stabilized form of the Gla domain of Factor IX. We prepared the fully carboxylated Gla domain of Factor IX by solid phase peptide synthesis and crystallized Factor IX-(1-47) in complex with Fab fragments of the 10C12 antibody. The overall structure of the Gla domain in the Factor IX-(1-47)-antibody complex at 2.2 A is similar to the structure of the Factor IX Gla domain in the presence of calcium ions as determined by NMR spectroscopy (Freedman, S. J., Furie, B. C., Furie, B., and Baleja, J. D. (1995) Biochemistry 34, 12126-12137) and by x-ray crystallography (Shikamoto, Y., Morita, T., Fujimoto, Z., and Mizuno, H. (2003) J. Biol. Chem. 278, 24090-24094). The complex structure shows that the complementarity determining region loops of the 10C12 antibody form a hydrophobic pocket to accommodate the hydrophobic patch of the Gla domain consisting of Leu-6, Phe-9, and Val-10. Polar interactions also play an important role in the antibody-antigen recognition. Furthermore, the calcium coordination network of the Factor IX Gla domain is different than in Gla domain structures of other vitamin K-dependent proteins. We conclude that this antibody is directed at the membrane binding site in the omega loop of Factor IX and blocks Factor IX function by inhibiting its interaction with membranes.

Amino Acid Sequence↗

PubMed1992

Efficient production of a small peptide by expression as a multimeric form fused with the dihydrofolate reductase affinity handle.

A pentapeptide which potently inhibits primary IgE antibody formation, Asp-Ser-Asp-Gly-Lys (DSDGK), has been efficiently produced with the aid of the dihydrofolate reductase (DHFR) handle [M. Iwakura, et al. (1992) J. Biochem. 111, 37-45]. The genes coding fused proteins comprising DHFR and multimeric forms of DSDGK, namely, DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, were constructed and expressed in Escherichia coli. The C-terminal peptides attached to DHFR did not affect the expression or the function of the DHFR handle, even when the length of the C-terminal peptide was as long as 160 amino acid residues. The fused proteins were easily purified by methotrexate affinity chromatography, one of the major advantages of the DHFR handle. The fused proteins were digested with trypsin and the monomeric peptide, DSDGK, was purified by HPLC. The yields of the peptide were estimated to be 11, 43, and 99 mg per 1 gram of the total cell proteins from E. coli cells producing DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, respectively.

Amino Acid Sequence↗

PubMed2003

Scaling of impact fragmentation near the critical point.

We investigated two-dimensional brittle fragmentation with a flat impact experimentally, focusing on the low-impact-energy region near the fragmentation-critical point. We found that the universality class of fragmentation transition disagreed with that of percolation. However, the weighted mean mass of the fragments could be scaled using the pseudo-control-parameter multiplicity. The data for highly fragmented samples included a cumulative fragment mass distribution that clearly obeyed a power law. The exponent of this power law was 0.5 and it was independent of sample size. The fragment mass distributions in this regime seemed to collapse into a unified scaling function using weighted mean fragment mass scaling. We also examined the behavior of higher-order moments of the fragment mass distributions, and obtained multiscaling exponents that agreed with those of the simple biased cascade model.

Journal Article↗

PubMed2005

High specificity generally characterizes mycorrhizal association in rare lady's slipper orchids, genus Cypripedium.

Lady's slipper orchids (Cypripedium spp.) are rare terrestrial plants that grow throughout the temperate Northern Hemisphere. Like all orchids, they require mycorrhizal fungi for germination and seedling nutrition. The nutritional relationships of adult Cypripedium mycorrhizae are unclear; however, Cypripedium distribution may be limited by mycorrhizal specificity, whether this specificity occurs only during the seedling stage or carries on into adulthood. We attempted to identify the primary mycorrhizal symbionts for 100 Cypripedium plants, and successfully did so with two Cypripedium calceolus, 10 Cypripedium californicum, six Cypripedium candidum, 16 Cypripedium fasciculatum, two Cypripedium guttatum, 12 Cypripedium montanum, and 11 Cypripedium parviflorum plants from a total of 44 populations in Europe and North America, yielding fungal nuclear large subunit and mitochondrial large subunit sequence and RFLP (restriction fragment length polymorphism) data for 59 plants. Because orchid mycorrhizal fungi are typically observed without fruiting structures, we assessed fungal identity through direct PCR (polymerase chain reaction) amplification of fungal genes from mycorrhizally colonized root tissue. Phylogenetic analysis revealed that the great majority of Cypripedium mycorrhizal fungi are members of narrow clades within the fungal family Tulasnellaceae. Rarely occurring root endophytes include members of the Sebacinaceae, Ceratobasidiaceae, and the ascomycetous genus, Phialophora. C. californicum was the only orchid species with apparently low specificity, as it associated with tulasnelloid, ceratobasidioid, and sebacinoid fungi in roughly equal proportion. Our results add support to the growing literature showing that high specificity is not limited to nonphotosynthetic plants, but also occurs in photosynthetic ones.

Base Sequence↗

PubMed1980

Amplification of a mitochondrial DNA sequence in the cytoplasmically inherited 'ragged' mutant of Aspergillus amstelodami.

A comparison has been made between mtDNA of the cytoplasmically inherited 'ragged' mutant of Aspergillus amstelodami and that of the wild-type strain. Ragged mitochondria contain both the wild-type mitochondrial genome and several large DNA molecules which are not cleaved by the restriction endonucleases BamHI, HaeIII, HhaI, HindII, HindIII, PstI and MboI, but are converted by either EcoRI or HpaII into a single 820-840 base-pair fragment. Restriction analysis and molecular hybridization data indicate that this fragment contains sequences of wild-type mtDNA located within a 1200-base-pair segment of the 40,500-base-pair genome, for which a basic restriction map has been deduced. It is concluded that in the ragged mutant a small segment of wild-type mtDNA has been amplified as tandem repeats, which is reminiscent of the Rho- petite phenotype of yeast. The results are discussed in relation to the phenomenon of senescence in Podospora anserina.

Aspergillus↗

PubMed2004

Human monoclonal antibody fragment specific for glycoprotein G in herpes simplex virus type 2 with applications for serotype-specific diagnosis.

A combinatorial library was used to select a human monoclonal antibody fragment (Fab) with high affinity for G glycoprotein in herpes simplex virus type 2 (HSV-2). Tests with 112 clinical specimens demonstrated successful discrimination between HSV-2 and HSV-1, showing the potential of Fab as a low-cost tool for HSV subtyping in clinical diagnosis.

Amino Acid Sequence↗

PubMed1991

Genomic analysis of a virulent and a less virulent strain of the entomopathogenic fungus Beauveria bassiana, using restriction fragment length polymorphisms.

The genomic DNA of two strains of the entomopathogenic fungus Beauveria bassiana, strain GK2016, a "wild type" (virulent), and strain GK2051, a less virulent mutant to grasshoppers, was digested with 12 restriction endonucleases. Gel electrophoresis conditions were established to show restriction fragment length patterns visually in the digested DNA stained with ethidium bromide. The less virulent mutant was generated by ultraviolet illumination of conidiospores at a 95% lethal dose. Both strains of the fungi were identical in morphology as well as in 16 of 22 API-ZYM kit enzyme assays. Differences in levels of total enzyme activity were observed for esterase, esterase-lipase, beta-galactosidase, chitinase, and protease. A Neurospora crassa beta-tubulin gene (heterologous gene) and two homologous DNA probes (pJK16 and pJK18) hybridized to several specific DNA bands in B. bassiana strain GK2016 but not in strain GK2051. Strain GK2051 gave different restriction fragment length pattern when compared with its parent strain. Taken together, the data show restriction fragment length differences between the genomic DNA of the two strains, including the loss of some DNA sequences from the mutant strain, which may be involved in pathogenicity. Finally, B. bassiana GK2016 contains a beta-tubulin gene with at least partial homology to that of N. crassa.

Animals↗

PubMed2000

A human monoclonal IgE antibody defines a highly allergenic fragment of the major timothy grass pollen allergen, Phl p 5: molecular, immunological, and structural characterization of the epitope-containing domain.

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an alpha helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.

Allergens↗

PubMed2001

Cloning and sequence analysis of the endopolygalacturonase gene from the pitch canker fungus, Fusarium circinatum.

The fungus Fusarium circinatum causes pitch canker disease on mature pine trees and root rot and damping-off of pine seedlings. Endopolygalacturonases (endoPGs) play a major role during penetration of plants by fungi. Digestion of the pectic polysaccharides in the plant primary cell walls is one of the earliest functions of endoPGs during infection. The research objective was to clone and characterize the gene encoding endopolygalacturonase in F. circinatum. A 970-bp DNA fragment was cloned by using degenerate PCR amplification from F. circinatum DNA. Sequence data for this fragment were used to design specific primers for use in genome walking to amplify and sequence the remaining portion of the F. circinatum endoPG gene (Fcpg). The amino acid sequence predicted from this gene showed 90% and 87% similarity to Fusarium oxysporum and Fusarium moniliforme endoPGs, respectively.

Chromosome Walking↗

PubMed1999

Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme.

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U20618). The YLR328W gene was isolated, cloned into a T7-based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N-terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.

Amino Acid Sequence↗

PubMed1998

Monte Carlo simulation of the production of short DNA fragments by low-linear energy transfer radiation using higher-order DNA models.

A realistic DNA target model has been developed and implemented in the biophysical simulation code PARTRAC. It describes five levels of the B-DNA structure (nucleotides, DNA helices, nucleosomes, chromatin fiber structure and chromatin fiber loops) on an atomic level for the whole genome inside a mammalian cell nucleus. The model is capable of describing regular solenoidal, crossed-linker or zigzag structures as well as repeating stochastic arrangements of nucleosomes in the chromatin fiber. Electron tracks resulting from monoenergetic electrons with energies up to 100 keV and from 220 kVp X rays, starting at random positions in the cell, were superimposed on four DNA target models with different chromatin fiber structures. The yields of SSBs, DSBs and short single- and double-stranded DNA fragments were determined from spatial coincidences with strand atoms. Two parameters of the model-the energy necessary to create an SSB and the distance between two breaks that would be scored as a DSB-were adapted to equate simulated and measured strand break yields after X irradiation of human fibroblast cells. The integral fractions of short single- and double-stranded fragments were rather similar for all condensed chromatin fiber structures; they agreed with experimental data for DNA fragments below 2 kbp. The simulated fragment size distributions in the range from 0.1 to 1.5 kbp reflected the fiber structure irrespective of strandedness or electron energy. The distributions using a stochastic arrangement of nucleosomes in the chromatin fiber were found to be in better accordance with experimental data than those obtained with regular fiber structures.

DNA↗

PubMed1997

Molecular phylogenetic study of the Ranunculaceae: utility of the nuclear 26S ribosomal DNA in inferring intrafamilial relationships.

There are only a small number of molecular markers currently proven to be useful for phylogenetic inference within the flowering plants. We demonstrate that the 5' end of the 26S ribosomal DNA (ca. 1100 bp) is of great value for investigating generic to subfamilial relationships. We analyzed DNA sequences from 31 species of the Ranunculaceae and four species of the Berberidaceae to test phylogenetic relationships within the Ranunculaceae. The inferred phylogeny strongly supports the concept that the Thalictrum chromosome group is not monophyletic, but consists of three independent lineages: (1) Hydrastis, (2) Xanthorhiza and Coptis, and (3) Thalictrum, Aquilegia, and Enemion. Based on comparison with conventional taxonomic characters, we propose a hypothesis that the third group also includes the rest of the Thalictrum chromosome taxa that have a base chromosome number of seven. For the Ranunculus chromosome group, our study suggests several relationships that have not been recognized by conventional systematics. The inferred 26S rDNA topology is compared with results from two previously published molecular data sets: DNA sequences from rbcL, atpB, and 18S rDNA genes and restriction fragment length polymorphism data from chloroplast DNA. The three topologies are highly congruent and agree with karyological characters, but not with fruit type, both of which have often been used for the higher classification of the Ra- nunculaceae.

Base Sequence↗

PubMed2006

Specific primers for rapid detection of Microsporum audouinii by PCR in clinical samples.

This report describes application of PCR fingerprinting to identify common species of dermatophytes using the microsatellite primers M13, (GACA)4, and (GTG)5. The initial PCR analysis rendered a specific DNA fragment for Microsporum audouinii, which was cloned and sequenced. Based on the sequencing data of this fragment, forward (MA_1F) and reverse (MA_1R) primers were designed and verified by PCR to establish their reliability in the diagnosis of M. audouinii. These primers produced a singular PCR band of 431 bp specific only to strains and isolates of M. audouinii, based on a global test of 182 strains/isolates belonging to 11 species of dermatophytes. These findings indicate these primers are reliable for diagnostic purposes, and we recommend their use in laboratory analysis.

Adolescent↗

PubMed2003

A format for databasing and comparison of AFLP fingerprint profiles.

BACKGROUND: Amplified fragment length polymorphism (AFLP) is a PCR-based technique that involves restriction of genomic DNA followed by ligation of adaptors to the fragments generated and selective PCR amplification of a subset of these fragments. The amplified fragments are separated on a sequencing gel and visualized by autoradiography or fluorescent sequencing equipment. AFLP allows high-resolution genotyping but the lack of a format for databasing and comparison of AFLP fingerprint profiles limits its wider applications in profiling large numbers of biological samples. RESULTS: A scheme is described to represent a DNA fingerprint profile with a nucleotide sequence-like format in which the information line contains the minimal necessary details to interpret an AFLP DNA fingerprint profile. They include technique used, information on restriction enzymes, primer combination, biological source for DNA materials, fragment sizing and annotation. The bodylines contain information on size and relative intensity of DNA fragments by a string of defined alphabets or symbols. Algorithms for normalizing raw data, binning of fragments and comparing AFLP DNA fingerprint profiles are described. Firstly, the peak heights are normalized against their average and then represented by five symbols according to their relative intensities. Secondly, a binning algorithm based loosely on common springs and rubber bands is applied, which positions sequence fragments into their best possible integer approximations. A BLAST-like reward-penalty concept is used to compare AFLP fingerprint profiles by matching peaks using two metrics: score and percentage of similarity. A software package was developed based on our scheme and proposed algorithms. Example of use this software is given in evaluating novelty of a new tropical orchid cultivar by comparing its AFLP fingerprint profile against those of related commercial cultivars in a database. CONCLUSIONS: AFLP DNA fingerprint profiles can be databased and compared effectively with software developed based on our scheme and algorithms. It will facilitate wider use of this DNA fingerprinting technique in areas such as forensic study, intellectual property protection for biological materials and biodiversity management. Moreover, the same concepts can be applied to databasing and comparing DNA fingerprint profiles obtained with other DNA fingerprint techniques.

Algorithms↗

PubMed1992

Navigation by fragment fitting: a theory of hippocampal function.

This paper describes a computational theory of spatial learning and navigation and its possible realization in the hippocampus. In the theory, mammals store memories of their geographical environment as a large number of independent fragments. A typical fragment denotes a few prominent landmarks in some region, their geometric relations, and their nongeometric properties, such as smells and visual cues. Navigation involves piecing together current sense data and relevant fragments to form a local map of the animal's surroundings; this is like solving a jigsaw puzzle. This computational model has been implemented in a computer program, whose performance is broadly consistent with observed levels of animal performance, and laboratory results, in spatial learning. Possible realizations of the model in animal brains are discussed. Unlike some neural net models of spatial learning, the model is strongly geometric, and uses special neural structures to store and manipulate two-dimensional vectors and bearings. A possible neural architecture is described in which the hippocampus performs the geometric operations; this has a long-term memory for fragments (somewhere in the neocortex), which can associatively recall fragments into a number of parallel fragment fitters, in the dentate gyrus and CA3 regions. These vary the positions and orientations of their fragments, to optimize the fit of the fragments to each other and to the animal's recent sense data. A local map of the animal's surroundings is stored in CA1 and subicular regions, where matching of fragment positions and attributes takes place. Mismatches are passed back via the entorhinal cortex to improve the fit during the next hippocampal theta cycle. The model offers the potential for understanding current data on spatial learning, on the neuroanatomy of the hippocampus and on place cells in a coherent framework, as well as understanding the role of the hippocampus in nonpositional memory tasks. Comparisons with experimental data are given.

Animals↗

PubMed1992

Localization in the fibrinogen gamma-chain of a new site that is involved in the acceleration of the tissue-type plasminogen activator-catalysed activation of plasminogen.

In previous publications [e.g. Voskuilen, Vermond, Veeneman, Van Boom, Klasen, Zegers & Nieuwenhuizen (1987) J. Biol. Chem. 262, 5944-5946] we have shown that fibrin(ogen) chain fragment A alpha-(148-160) contains a site that contributes to the acceleration of Glu-plasminogen activation by tissue-type plasminogen activator (t-PA). In contrast with fibrin, this peptide, however, does not enhance the rate of mini-plasminogen activation. Therefore, possibly more stimulatory sites than A alpha-(148-160) are present in fibrin. In the present investigation we have localized a possible second type of stimulatory site in the fibrin(ogen) molecule. A whole CNBr digest of fibrinogen was applied to a Bio-Gel P-2 column run in water, pH 4. Two peaks with stimulatory activity were observed, one at the void volume and one between the void volume and the total volume. The former contained the previously described stimulating fragment FCB-2 [which comprises A alpha-(148-160)]; the latter had not been observed before and was characterized further. The stimulating material in the low-M(r) fraction of the Bio-Gel P-2 column was precipitated at pH 8.3 in a virtually pure form. It has a high tryptophan content, and an M(r) of 6500 as assessed by SDS/PAGE. On reduction, a main band of M(r) 2500 is seen, plus a weakly staining band of M(r) 4000. These properties plus the amino acid sequence data identify the fragment as FCB-5. FCB-5 consists of two chains, i.e. gamma-(311-336) and gamma-(337-379), linked by a single disulphide bond between Cys-gamma-326 and Cys-gamma-339. Both these chains and the disulphide bond appear to be essential for rate enhancement. FCB-5 enhances the activation rates of Glu-, mini- and micro-plasminogen, with all five kringles, only kringle V and without kringles respectively. FCB-5 binds t-PA, but none of the plasminogen forms binds to FCB-5. This indicates that the rate enhancements induced by FCB-5 are due to an effect on t-PA.

Amino Acid Sequence↗

PubMed2005

Life cycle studies of Myxobolus parviformis sp. n. (Myxozoa: Myxobolidae) from bream.

We experimentally followed the life cycle of Myxobolus parviformis sp. n., a myxozoan parasite from the gills of common bream Abramis brama L. Establishing the development of both stages (myxospore and actinospore) in 2 consecutive, full transmission trials, we were able to separate plasmodia of a common genotype from sympatric Myxobolus spp. occurring in naturally infected gill lamellae. Therefore, isolated gill plasmodia representing individual myxosporean 'clones' were used for subsequent infection of oligochaetes after molecular and morphological identification. The plasmodia of this species are located in median to distal regions of the primary gill filaments, forming small spherical pseudocysts. The comparatively small myxospores share some uniform characteristics: they taper posteriorly, have 2 inward inclining polar capsules that occupy half of the spores' interior space, and usually show 4 posterior sutural edge markings. The corresponding actinosporean has already been described morphologically and molecularly. The 18S rDNA sequence of the actinosporean stage was identical in all our samples, including myxospores. The triactinomyxon had a stout style, 32 sporozoites and short tapering caudal processes, and was shed by the tubificid oligochaete Limnodrilus hoffmeisteri. The ellipsoid sporoplasm was covered by a soft sheath which was emitted after valve shell opening upon stimulation by agitation or fish mucus. The molecular data (unique restriction fragment length polymorphism pattern and a 1586 bp 18S rDNA sequence) clearly differ from those for similar species and, together with the morphological data, justify the description of this parasite as a new species.

Animals↗

PubMedDate not supplied

[Development of rules for vaccine engineering based on the variability of peptide fragments in closely related proteins].

Based on the protein sequence data bank (PIR), the "variable fragment" bank, comprising pairs of closely related proteins, containing one or more strongly differing sites of primary structures was formed. The bank includes 465 "variable fragments" of 383 protein pairs. Amino acid residues composition of "variable fragments" was examined and indexes of potential amino acid residues variability was formed. An analysis of amino acid fragments replaceability was carried out by substituting the N-, C-terminal, or middle part of a chain), the fragments length differences and physico-chemical properties of residues, such as volume, hydrophobicity, polarity, isoelectric point, etc. Some general empirical rules of peptide insertions in carrier-proteins were created based on these analyses. The rules are directed for performing modifications maintaining the common structure and function of the carrier-protein molecule. The selection scheme for determining the regions suitable for modification and the criteria for defining the width of acceptable modifications in this regions were suggested. The use of potential variability profile for detecting regions suitable for peptide insertion was considered on the model of hepatitis B surface protein.

Amino Acid Sequence↗