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PubMed1987

The structure of bovine secretory component.

Bovine secretory component (SC) has been cleaved with trypsin into a series of fragments and their N-terminal amino acid sequences have been determined. The close homology with the known sequence of human SC has enabled the sequential order of the fragments to be deduced. The results indicate that bovine SC consists of a single glycosylated polypeptide chain (Mr 74,000) folded into five globular immunoglobulin-like domains. A protein (Mr 94,000) has been isolated from detergent solubilised bovine epithelial membranes from liver, intestine and mammary gland. This membrane protein is specific for the binding of J-chain linked IgM and IgA dimers. It can be proteolytically cleaved into a water soluble SC-like portion and a detergent soluble hydrophobic portion. Bovine SC is therefore most likely to be the extracellular part of an epithelial receptor which mediates the transport of IgA dimers to mucosal surfaces. The various tryptic fragments from bovine SC have been shown to differ in their relative binding affinities for IgM and IgA dimers. The results imply that the first three domains of bovine SC are most involved in binding and domains 4 and 5 play subsidiary roles. Computerized prediction and modelling methods have been used to deduce possible tertiary and quaternary structures for SC. There are good indications that the molecule has an elonaged "zig-zag" structure stabilized by longitudinal inter-domain contacts. A model of SC bound to IgA dimer is presented.

Amino Acid Sequence↗

PubMed1991

Comparison of human interleukin-1 beta and its 163-171 peptide in bone resorption and the immune response.

Human interleukin-1 beta (IL-1 beta) caused a dose- and time-dependent enhancement of the release of 45Ca from prelabeled mouse calvaria in organ culture. In addition, IL-1 beta dose-dependently stimulated the formation of prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha in the calvarial bones. However, IL-1 beta-induced 45Ca release was only partially inhibited by blocking the PGE2 response with indomethacin, suggesting that enhanced PGE2 formation in response to IL-1 beta is not necessary to obtain a bone resorptive effect, but that prostaglandins potentiate the action of IL-1 beta. The synthetic nonapeptide VQGEESNDK, corresponding to the fragment 163-171 of human IL-1 beta, administered simultaneously with antigen (SRBC) to C3H/HeN male mice, induced a dose-dependent enhancement of specific antibody-producing cells in the spleen (PFC). The degree of PFC stimulation was comparable to that caused by native human IL-1 beta. In mouse bone cultures, neither 45Ca release nor prostanoid formation was stimulated by fragment 163-171. These data indicate that (1) IL-1 beta-induced stimulation of bone resorption is dissociable from IL-1 beta-induced increase of prostanoid biosynthesis and (2) the epitope of the IL-1 beta molecule involved in the immunostimulatory effects may be different from that involved in the stimulatory effects on bone resorption.

Amino Acid Sequence↗

PubMed1998

Folding, calcium binding, and structural characterization of a concatemer of the first and second ligand-binding modules of the low-density lipoprotein receptor.

The ligand-binding domain of the low-density lipoprotein (LDL) receptor is comprised of seven tandemly repeated ligand-binding modules, each being approximately 40 amino acids long and containing six conserved cysteine residues. We have expressed and characterized a concatemer of the first two modules (LB1 and LB2) of the human LDL receptor. Oxidative folding of the recombinant concatemer (rLB1-2), in the presence of calcium ions, gave a single dominant isomer with six disulfide bonds. Peptic cleavage of the short linker region that connects the last cysteine residue of LB1 and the first cysteine residue of LB2 yielded two discrete fragments, thus excluding the presence of intermodule disulfide bonds. The N-terminal module, LB1, reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDL receptor. From this, we concluded that the first module was correctly folded, with the same set of disulfide bonds as LB1 of the LDL receptor. The disulfide bond connections of LB2 were identified from mass spectral analysis of fragments formed by digestion of the C-terminal peptic fragment with elastase. These data showed that the disulfide bonds of LB2 connected Cys(I) and Cys(III), Cys(II) and Cys(V), and Cys(IV) and Cys(VI). This pattern is identical to that found for recombinant LB1 and LB2. The concatemer has two high-affinity calcium-binding sites, one per module. An analysis of the secondary chemical shifts of Calpha protons shows that the conformations of LB1 and LB2 in the concatemer are very similar to those of the individual modules, with no evidence for strong interactions between the two modules.

Amino Acid Sequence↗

PubMed1999

Conformational changes of gp120 in epitopes near the CCR5 binding site are induced by CD4 and a CD4 miniprotein mimetic.

Binding of the T-cell antigen CD4 to human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has been reported to induce conformational rearrangements in the envelope complex that facilitate recognition of the CCR5 coreceptor and consequent viral entry into cells. To better understand the mechanism of virus docking and cell fusion, we developed a three-component gp120-CD4-17b optical biosensor assay to visualize the CD4-induced conformational change of gp120 as seen through envelope binding to a neutralizing human antibody, 17b, which binds to epitopes overlapping the CCR5 binding site. The 17b Fab fragment was immobilized on a dextran sensor surface, and kinetics of gp120 binding were evaluated by both global and linear transformation analyses. Adding soluble CD4 (sCD4) increased the association rate of full-length JR-FL gp120 by 25-fold. This change is consistent with greater exposure of the 17b binding epitope on gp120 when CD4 is bound and correlates with CD4-induced conformational changes in gp120 leading to higher affinity binding to coreceptor. A smaller enhancement of 17b binding by sCD4 was observed with a mutant of gp120, DeltaJR-FL protein, which lacks V1 and V2 variable loops and N- and C-termini. Biosensor results for JR-FL and DeltaJR-FL argue that CD4-induced conformational changes in the equilibrium state of gp120 lead both to movement of V1/V2 loops and to conformational rearrangement in the gp120 core structure and that both of these lead to greater exposure of the coreceptor-binding epitope in gp120. A 17b binding enhancement effect on JR-FL also was observed with a 32-amino acid charybdotoxin miniprotein construct that contains an epitope predicted to mimic the Phe 43/Arg 59 region of CD4 and that competes with CD4 for gp120 binding. Results with this construct argue that CD4-mimicking molecules with surrogate structural elements for the Phe 43/Arg 59 components of CD4 are sufficient to elicit a similar gp120 conformational isomerization as expressed by CD4 itself.

Amino Acid Sequence↗

PubMed1989

Evidence that glutathione S-transferases B1B1 and B2B2 are the products of separate genes and that their expression in human liver is subject to inter-individual variation. Molecular relationships between the B1 and B2 subunits and other Alpha class glutathione S-transferases.

The Alpha class glutathione S-transferases (GSTs) in human liver are composed of polypeptides of Mr 25,900. These enzymes are dimeric, and two immunochemically distinct subunits, B1 and B2, have been described that combine to form GSTs B1B1, B1B2 and B2B2 [Stockman, Beckett & Hayes (1985) Biochem. J. 227, 457-465]. Gradient affinity elution from GSH-Sepharose has been used to resolve the three Alpha class GSTs, and this method has been applied to demonstrate marked inter-individual differences in the hepatic content of GSTs B1B1, B1B2 and B2B2. The B1 and B2 subunits can be resolved by reverse-phase h.p.l.c., and their elution positions suggest that they are equivalent to the alpha chi and alpha y h.p.l.c. peaks described by Ketterer and his colleagues [Ostlund Farrants, Meyer, Coles, Southan, Aitken, Johnson & Ketterer (1987) Biochem. J. 245, 423-428]. The B1 and B2 subunits have now been cleaved with CNBr and the fragments subjected to automated amino acid sequence analysis. The sequence data show that B1 and B2 subunits do not arise from post-translational modification, as had been previously believed for the hepatic Alpha class GSTs, but are instead the products of separate genes; B1 and B2 subunits were found to contain different amino acid residues at positions 88, 110, 111, 112, 116, 124 and 127. The relationship between the B1 and B2 subunits and the cloned GTH1 and GTH2 cDNA sequences [Rhoads, Zarlengo & Tu (1987) Biochem. Biophys. Res. Commun. 145, 474-481] is discussed.

Adult↗

PubMed2001

The structure of a human type III Fcgamma receptor in complex with Fc.

Fcgamma receptors mediate antibody-dependent inflammatory responses and cytotoxicity as well as certain autoimmune dysfunctions. Here we report the crystal structure of a human Fc receptor (FcgammaRIIIB) in complex with an Fc fragment of human IgG1 determined from orthorhombic and hexagonal crystal forms at 3.0- and 3.5-A resolution, respectively. The refined structures from the two crystal forms are nearly identical with no significant discrepancies between the coordinates. Regions of the C-terminal domain of FcgammaRIII, including the BC, C'E, FG loops, and the C' beta-strand, bind asymmetrically to the lower hinge region, residues Leu(234)-Pro(238), of both Fc chains creating a 1:1 receptor-ligand stoichiometry. Minor conformational changes are observed in both the receptor and Fc upon complex formation. Hydrophobic residues, hydrogen bonds, and salt bridges are distributed throughout the receptor.Fc interface. Sequence comparisons of the receptor-ligand interface residues suggest a conserved binding mode common to all members of immunoglobulin-like Fc receptors. Structural comparison between FcgammaRIII.Fc and FcepsilonRI.Fc complexes highlights the differences in ligand recognition between the high and low affinity receptors. Although not in direct contact with the receptor, the carbohydrate attached to the conserved glycosylation residue Asn(297) on Fc may stabilize the conformation of the receptor-binding epitope on Fc. An antibody-FcgammaRIII model suggests two possible ligand-induced receptor aggregations.

Amino Acid Sequence↗

PubMed2006

The phylogeny of Staphylococcus aureus - which genes make the best intra-species markers?

The ability to make informed decisions on the suitability of alternative marker loci is central for population and epidemiological investigations. This issue was addressed using Staphylococcus aureus as a model population by generating nucleotide sequence data from 33 gene fragments in a representative sample of 30 strains. Supplementing the data with pre-existing multilocus sequence typing data, an intra-species tree based on approximately 17.8 kb of sequence was reconstructed and the goodness of fit of each individual gene tree was computed. No strong association was noted between gene function per se and phylogenetic reliability, but it is suggested that candidate loci should possess at least the average degree of nucleotide diversity for all genes in the genome. In the case of S. aureus this threshold is >1 % mean pairwise diversity.

DNA, Bacterial↗

PubMed1986

Polymorphic restriction sites in the horse beta-globin gene cluster.

Horse DNA samples digested with PstI and probed with the rabbit beta 1 globin gene show three phenotypes determined by one fragment of variable length (about 5.1 or 3.3 kb). Family data demonstrate that these fragments segregate as Mendelian alleles. The frequencies of the two alleles are 0.66 for the 3.3-kb fragment and 0.34 for the 5.1-kb one. Another polymorphism has been detected with BamHI. Again three phenotypes determined by two alleles (fragments of 7.5 and 3.8 kb) have been observed. Allelic frequencies of the 7.5- and 3.8-kb fragments are 0.24 and 0.76 respectively. The two polymorphic sites are non-randomly associated.

Animals↗

PubMed1993

DNA sequence variation and phylogenetic relationships among strains of Pseudomonas syringae pv. syringae inferred from restriction site maps and restriction fragment length polymorphism.

We evaluated the restriction fragment length polymorphism of genomic DNA among 53 strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Twenty-nine strains were isolated from beans, and the rest were isolated from 11 other hosts. Southern blots of DNA digested with EcoRI or HindIII were hybridized to two random probes from a cosmid library of P. syringae pv. syringae and a hrp (hypersensitive reaction and pathogenicity) cluster cloned from P. syringae pv. syringae. The size of hybridizing fragments was determined, and a similarity matrix was constructed by comparing strains on a pairwise basis for the presence or absence of fragments. The proportion of shared fragments was then used to estimate sequence divergence. Dendrograms were produced by using the unweighted pair group method with averages and the neighbor-joining method. For the hrp region, BamHI, EcoRI, EcoRV, and HindIII restriction sites were mapped for six representative bean strains and used to construct EcoRI and HindIII restriction maps for all 30 strains pathogenic on beans. Restriction mapping revealed the presence of a 3-kb insertion in nine bean strains and a probable second insertion or deletion event on the left-hand side of the hrp cluster that biased estimates of nucleotide sequence divergence from fragment comparisons. This demonstrated that the determination of phylogenetic relationships among bacteria by using restriction fragment length polymorphism data requires mapping restriction sites to remove the effect of insertion or deletion events on the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Bacterial↗

PubMed1989

Identification of integrin-like matrix receptors with affinity for interstitial collagens.

Antibodies to a rat liver membrane glycoprotein with an Mr of 115,000 (nonreduced) inhibited the attachment of rat hepatocytes and primary rat heart fibroblasts to both collagen and fibronectin. The Mr 115,000 glycoprotein cross-reacted immunologically with the beta 1-chain of the rat hepatocyte fibronectin receptor (HFNR), and the two proteins showed identical peptide maps after proteolytic cleavage. It was concluded that the Mr 115,000 protein was similar or identical to the beta 1-chain of Arg-Gly-Asp (RGD)-directed matrix receptors. Although collagen type I contains several RGD sequences, the attachment of hepatocytes and fibroblasts to collagen type I was not inhibited by the synthetic peptide GRGDTP in concentrations that blocked adhesion to fibronectin. Furthermore, hepatocytes adhered equally well to collagen fragments, generated by cyanogen bromide cleavage, lacking RGD sequences as to fragments containing this sequence. Antibodies to the Mr 115,000 protein inhibited the adhesion of hepatocytes to both types of collagen fragments. Taken together, these data indicate the presence of collagen receptors that share the beta-subunit with the HFNR but that are not directed to RGD sequences. Tentative alpha-chains of the collagen matrix receptor complex were isolated by immunoprecipitation of surface 125I-labeled fibroblast membrane proteins purified by affinity chromatography on immobilized collagen type I. Data are presented indicating that proteins with Mr around 145,000 and 170,000 (nonreduced) are associated in noncovalently linked complexes with the Mr 115,000 protein. These complexes have affinity for collagen and thus have properties expected for integrin-like collagen receptors.

Amino Acid Sequence↗

PubMed1995

DNA sequence assembly and genetic algorithms new results and puzzling insights.

Applying genetic algorithms to DNA sequence assembly is not a straightforward process. Significantly improved results in terms of performance, quality of results, and the scaling of applicability have been realized through non-standard and even counter-intuitive parameter settings. Specifically, the solution time for a 10kb data set was reduced by an order of magnitude, and a 20kb data set that was previously unsolved by the genetic algorithm was solved in a time that represents only a linear increase from the 10kb data set. Additionally, significant progress has been made on a 35kb data set representing real biological data. A single contig solution was found for a 752 fragment subset of the data set, and a 15 contig solution was found for the full data set. This paper discusses the new results, the modifications to the previous genetic algorithm used in this study, the experimental design process by which the new results were obtained, the questions raised by these results, and some preliminary attempts to explain these results.

Algorithms↗

PubMed1995

[Fragments 183-198 and 125-145 of the alpha-subunits of the Torpedo californica nicotinic acetylcholinergic receptor binds alpha-bungarotoxin and neurotoxin II from Naja naja oxiana].

Interaction of the mono[125I]iodinated alpha-bungarotoxin and neurotoxin II Naja naja oxiana with the synthetic peptides corresponding to the fragments of the alpha-subunit of nicotinic acetylcholine receptor from Torpedo californica was studied. It was found that both toxins bind to the fragments alpha 186-198, alpha 183-198 and alpha 125-145 adsorbed to the 96-well P.E.T.G. assay plates (COSTAR). Acm-groups on Cys residues did not prevent toxin binding by the peptides studied. Determination of the binding parameters showed that alpha-bungarotoxin interacts with fragment alpha 125-145 less effectively than neurotoxin II. The data obtained demonstrate the presence of different toxin-binding sites on alpha-subunit and confirm the model of multipoint neurotoxin-receptor interaction.

Amino Acid Sequence↗

PubMed2006

Comparative analyses of population structure in two subspecies of Nigella degenii: evidence for diversifying selection on pollen-color dimorphisms.

Flower color can be a major determinant of plant fitness, not only because of preferential visitation by pollinators but also because of pleiotropic relationships between the expression of floral pigments and biochemically related compounds that influence vegetative performance variables. Different environments may therefore favor different pigmentation phenotypes. We examined whether spatially varying selection has played a major role in shaping large-scale patterns of differentiation in two subspecies of Nigella degenii (Ranunculaceae), with particular emphasis on pollen color. The two subspecies appear to have been genetically isolated for substantial periods of time and, therefore, provide a "replicated test" for the effect of natural selection. Estimates of population structure based on a suite of floral and vegetative characters were compared with the corresponding data for amplified fragment length polymorphism (AFLP) markers, which were assumed to be selectively neutral. We found low levels of genetic structure within the subspecies using both the AFLP markers (FST < or = 0.05) and quantitative characters (QST < or = 0.15), with no statistically significant differences between the two measures. There is, therefore, no evidence of diversifying selection being important in structuring variation in quantitative characters within each of the two subspecies. In contrast, estimates of differentiation in pollen color (FST > or = 0.10) significantly exceeded the neutral expectations (estimated from AFLP data), suggesting that local adaptation has played a key role in the evolution of this monogenic character.

Analysis of Variance↗

PubMedDate not supplied

Mining a chemical database for fragment co-occurrence: discovery of "chemical clichés".

Nowadays millions of different compounds are known, their structures stored in electronic databases. Analysis of these data could yield valuable insights into the laws of chemistry and the habits of chemists. We have therefore explored the public database of the National Cancer Institute (>250,000 compounds) by pattern searching. We split the molecules of this database into fragments to find out which fragments exist, how frequent they are, and whether the occurrence of one fragment in a molecule is related to the occurrence of another, nonoverlapping fragment. It turns out that some fragments and combinations of fragments are so frequent that they can be called "chemical clichés". We believe that the fragment data can give insight into the chemical space explored so far by synthesis. The lists of fragments and their (co-)occurrences can help create novel chemical compounds by (i) systematically listing the most popular and therefore most easily used substituents and ring systems for synthesizing new compounds, (ii) being an easily accessible repository for rarer fragments suitable for lead compound optimization, and (iii) pointing out some of the yet unexplored parts of chemical space.

Antineoplastic Agents↗

PubMed1994

Pathological and Molecular Characterization of Xanthomonas campestris Strains Causing Diseases of Cassava (Manihot esculenta).

Fifty-one strains representing Xanthomonas campestris pv. manihotis and cassavae and different pathovars occurring on plants of the family Euphorbiaceae were characterized by ribotyping with a 16S+23S rRNA probe of Escherichia coli and by restriction fragment length polymorphism analysis with a plasmid probe from X. campestris pv. manihotis. Pathogenicity tests were performed on cassava (Manihot esculenta). Histological comparative studies were conducted on strains of two pathovars of X. campestris (vascular and mesophyllic) that attack cassava. Our results indicated that X. campestris pv. manihotis and cassavae have different modes of action in the host and supplemented the taxonomic data on restriction fragment length polymorphism that clearly separate the two pathovars. The plasmid probe could detect multiple restriction fragment length polymorphisms among strains of the pathovar studied. Ribotyping provides a useful tool for rapid identification of X. campestris pathovars on cassava.

Journal Article↗

PubMed2001

Induction and rejoining of DNA double-strand breaks in normal human skin fibroblasts after exposure to radiation of different linear energy transfer: possible roles of track structure and chromatin organization.

DNA double-strand breaks are nonrandomly induced by high-LET radiation. Differences in the induction and rejoining of DSBs after irradiation with ions having different LET were detected by fragment analysis. The data obtained indicate that the track structure of the traversing particle and its interaction with the different chromatin structures of the cellular DNA influence the yield as well as the distribution of the induced damage. The induction and rejoining of clustered DSBs induced by the same nitrogen ion fluence at LETs of 80-225 keV/microm were investigated by a detailed analysis of the DNA fragmentation patterns in normal human fibroblasts. The DSBs in the cells were allowed to rejoin during incubations for 0-20 h. Two separate pulsed-field gel electrophoresis protocols were used, optimized for separation of fragments in the size ranges 1-6 Mbp and 5 kbp-1.5 Mbp. A strong influence of LET on the level of DSB induction was evident. The DSB yield increased from 4.5 +/- 0.2 to 10.0 +/- 0.3 DSBs per particle traversal through the cell nucleus when LET increased from 80 to 225 keV/microm. Further, the size distribution of the DNA fragments showed a significant dependence on radiation quality, with an excess of fragments at 50-200 kbp and around 1 Mbp. Differences in repair kinetics were also evident, with slower rejoining for increasing LET, and the initial nonrandom fragment distributions were still present after 1 h of repair.

Chromatin↗

PubMed2006

A Consensus Data Mining secondary structure prediction by combining GOR V and Fragment Database Mining.

The major aim of tertiary structure prediction is to obtain protein models with the highest possible accuracy. Fold recognition, homology modeling, and de novo prediction methods typically use predicted secondary structures as input, and all of these methods may significantly benefit from more accurate secondary structure predictions. Although there are many different secondary structure prediction methods available in the literature, their cross-validated prediction accuracy is generally <80%. In order to increase the prediction accuracy, we developed a novel hybrid algorithm called Consensus Data Mining (CDM) that combines our two previous successful methods: (1) Fragment Database Mining (FDM), which exploits the Protein Data Bank structures, and (2) GOR V, which is based on information theory, Bayesian statistics, and multiple sequence alignments (MSA). In CDM, the target sequence is dissected into smaller fragments that are compared with fragments obtained from related sequences in the PDB. For fragments with a sequence identity above a certain sequence identity threshold, the FDM method is applied for the prediction. The remainder of the fragments are predicted by GOR V. The results of the CDM are provided as a function of the upper sequence identities of aligned fragments and the sequence identity threshold. We observe that the value 50% is the optimum sequence identity threshold, and that the accuracy of the CDM method measured by Q(3) ranges from 67.5% to 93.2%, depending on the availability of known structural fragments with sufficiently high sequence identity. As the Protein Data Bank grows, it is anticipated that this consensus method will improve because it will rely more upon the structural fragments.

Algorithms↗

PubMed1974

Beta-galactosidase from termination and deletion mutant strains.

beta-Galactosidase fragments were isolated from strains of Escherichia coli with mutations in the lacZ gene. The polypeptide obtained from a termination mutant (lacZNG125) appeared to be the intact gene product, containing the first half of the beta-galactosidase amino acid sequence. From an internal deletion mutant strain (lacZU163), an aggregate was obtained of several partially degraded polypeptides. Each of these was smaller than predicted from genetic data for the fragment. Introduction of the lacZU163 mutation into a protein degradation-deficient strain (Deg(-)) resulted in the protection of the amino-terminal region of the protein. Some of the BrCN peptides from the U163 polypeptides were separated and identified. From such experiments it was shown that in both Deg(-) and Deg(+) strains the COOH-terminal region is rapidly degraded. This indicates that the complete gene product of lacZU163 has not been detected. The use of genetically defined enzyme fragments in studying structure-function relationships and in determination of primary structure is discussed.

Amino Acid Sequence↗