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PubMed1989

Characteristics of protective immunity in mice induced by drug-attenuated larvae of Schistosoma mansoni. Antigen localization and antibody responses.

Partial resistance to reinfection developed in mice with immature infections of Schistosoma mansoni treated with the drug Ro11-3128, whereas mice vaccinated with irradiated cercariae and treated in a similar way did not become immune. Persistence of drug-treated parasites in the skin and draining lymph nodes, which prolonged the opportunity for efficient Ag presentation, was necessary for the development of protective immunity. Death and clearance of parasites solely in the skin, was not sufficient to induce protection. The expansion in the number of lymphocytes in the skin-draining nodes after vaccination, was reflected in the contrasting levels of resistance induced by the different drug-treatment regimes. Challenge parasites were eliminated predominantly after they reached the lungs. An investigation of antibody reactivity revealed an immunodominant response against a doublet of Ag of Mr 97 to 99 kDa. Recognition of this complex by antisera from different groups of mice was not related to their immune status. Western blots and inhibition analysis showed that this doublet has epitopes in common with the Sm97/paramyosin protective Ag, originally identified by antisera reactivity from mice immunized with a nonliving vaccine.

Animals↗
PubMed1994

DNA binding specificity of the bovine papillomavirus E1 protein is determined by sequences contained within an 18-base-pair inverted repeat element at the origin of replication.

Bovine papillomavirus type 1 (BPV-1) DNA replicates episomally and requires two virally expressed proteins, E1 and E2, for this process. Both proteins bind to the BPV-1 genome in the region that functions as the origin of replication. The binding sequences for the E2 protein have been characterized previously, but little is known about critical sequence requirements for E1 binding. Using a bacterially expressed E1 fusion protein, we examined binding of the BPV-1 E1 protein to the origin region. E1 strongly protected a 28-bp segment of the origin (nucleotides 7932 to 15) from both DNase I and exonuclease III digestion. Additional exonuclease III protection was observed beyond the core region on both the 5' and 3' sides, suggesting that E1 interacted with more distal sequences as well. Within the 28-bp protected core, there were two overlapping imperfect inverted repeats (IR), one of 27 bp and one of 18 bp. We show that sequences within the smaller, 18-bp IR element were sufficient for specific recognition of DNA by E1 and that additional BPV-1 sequences beyond the 18-bp IR element did not significantly increase origin binding by E1 protein. While the 18-bp IR element contained sequences sufficient for specific binding by E1, E1 did not form a stable complex with just the isolated 18-bp element. Formation of a detectable E1-DNA complex required that the 18-bp IR be flanked by additional DNA sequences. Furthermore, binding of E1 to DNA containing the 18-bp IR increased as a function of overall increasing fragment length. We conclude that E1-DNA interactions outside the boundaries of the 18-bp IR are important for thermodynamic stabilization of the E1-DNA complex. However, since the flanking sequences need not be derived from BPV-1, these distal E1-DNA interactions are not sequence specific. Comparison of the 18-bp IR from BPV-1 with the corresponding region from other papillomaviruses revealed a symmetric conserved consensus sequence, T-RY--TTAA--RY-A, that may reflect the specific nucleotides critical for E1-DNA recognition.

Base Sequence↗
PubMed1998

Interictal paroxysmal discharges in the electroencephalograms of epileptic dogs.

Paroxysmal discharges (PD) consisting of spikes, multiple spike complexes, sharp waves, multiple sharp-wave complexes, and spike-and-slow-wave complexes can be recorded in the interictal electroencephalograms of sedated epileptic dogs. With appropriate methods, focal PD can be detected and the hemisphere of origin and rostrocaudal location of their sources identified. Electrode montages that permit ready recognition of so-called instrumental phase reversal are especially helpful. PD that are presumably generalized can be recorded from dogs with signalments suggesting inherited/"idiopathic" epilepsy. These PD are described as "presumably" generalized because of the lack of recordings from ventral and ventrolateral cerebral regions. Further study is necessary, attempting to correlate types of PD with types of epilepsy.

Animals↗
PubMed2002

Crystal structure of a human mitochondrial deoxyribonucleotidase.

5' nucleotidases are ubiquitous enzymes that dephosphorylate nucleoside monophosphates and participate in the regulation of nucleotide pools. The mitochondrial 5'-(3') deoxyribonucleotidase (dNT-2) specifically dephosphorylates dUMP and dTMP, thereby protecting mitochondrial DNA replication from excess dTTP. We have solved the structure of dNT-2, the first of a mammalian 5' nucleotidase. The structure reveals a relationship to the HAD family, members of which use an aspartyl nucleophile as their common catalytic strategy, with a phosphoserine phosphatase as the most similar neighbor. A structure-based sequence alignment of dNT-2 with other 5' nucleotidases also suggests a common origin for these enzymes. Here we study the structures of dNT-2 in complex with bound phosphate and beryllium trifluoride plus thymidine as model for a phosphoenzyme-product complex. Based on these structures, determinants for substrate specificity recognition and the catalytic action of dNT-2 are outlined.

5'-Nucleotidase↗
PubMed2001

Investigation of disaccharide recognition by molecularly imprinted polymers.

The selectivity of carbohydrate-imprinted polymers for several disaccharides, namely cellobiose, maltose, lactose and gentiobiose, is investigated. An ternary ligand-Cu(II)-carbohydrate complex was formed in alkaline solution and captured afterwards in the polymer. The accessibility of the polymer matrix for disaccharides was investigated by HPLC analysis, refractometry and 1H NMR spectroscopy applying excess of the original template during rebinding experiments under saturation conditions in unbuffered, aqueous solution at neutral pH and 20 degrees C. The selective discrimination of the alpha- and beta-glycosidic linkage of cellobiose and maltose is demonstrated. It is further shown, that the disaccharide-imprinted polymers slightly distinguish between the 1,4-beta- and the 1,6-beta-glycosidic linkage of cellobiose and gentiobiose, while cellobiose and lactose are not selectively recognized. Due to the weak apparent binding constant of the functional Cu(II) monomers with the targeted disaccharides at physiological pH, the recognition process is dominated by the shape of the created imprinted cavity under the applied conditions.

Carbohydrate Sequence↗
PubMed2006

The Mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary C-type lectin surfactant protein A.

Tuberculosis is still a major health problem, and understanding the mechanism by which Mycobacterium tuberculosis (Mtb) invades and colonizes its host target cells remains an important issue for the control of infection. The innate immune system C-type lectins (C-TLs), including the human pulmonary surfactant protein A (PSP-A), have been recently identified as determinant players in the early recognition of the invading pathogen and in mounting the host defense response. Although the antigenic lipoglycan mannosylated lipoarabinomannan is currently considered to be the major C-TL target on the mycobacterial surface, the recognition by some C-TLs of the only mycobacterial species composing the "Mtb complex" indicates that mannosylated lipoarabinomannan cannot account alone for this specificity. Thus, we searched for the mycobacterial molecules targeted by human PSP-A, focusing our attention on the Mtb surface glycoproteins. We developed an original functional proteomic approach based on a lectin blot assay using crude human bronchoalveolar lavage fluid as a source of physiological PSP-A. Combined with selective cell-surface protein extraction and mass spectrometry peptide mapping, this strategy allowed us to identify the Apa (alanine- and proline-rich antigenic) glycoprotein as new potential target for PSP-A. This result was supported by direct binding of PSP-A to purified Apa. Moreover, EDTA addition or deglycosylation of purified Apa samples completely abolished the interaction, demonstrating that the interaction is calcium- and mannose-dependent, as expected. Finally, we provide convincing evidence that Apa, formerly considered as mainly secreted, is associated with the cell wall for a sufficiently long time to aid in the attachment of PSP-A. Because, to date, Apa seems to be restricted to the Mtb complex strains, we propose that it may account for the selective recognition of those strains by PSP-A and other immune system C-TLs containing homologous functional domains.

Adhesins, Bacterial↗
PubMed2006

Mechanism of DNA recognition at a viral replication origin.

Recognition of the DNA origin by the Epstein-Barr nuclear antigen 1 (EBNA1) protein is the primary event in latentphase genome replication of the Epstein-Barr virus, a model for replication initiation in eukaryotes. We carried out an extensive thermodynamic and kinetic characterization of the binding mechanism of the DNA binding domain of EBNA1, EBNA1452-641, to a DNA fragment containing a single specific origin site. The interaction displays a binding energy of 12.7 kcal mol-1, with 11.9 kcal mol-1 coming from the enthalpic change with a minimal entropic contribution. Formation of the EBNA1452-641.DNA complex is accompanied by a heat capacity change of -1.22 kcal mol-1 K-1, a very large value considering the surface area buried, which we assign to an unusually apolar protein-DNA interface. Kinetic dissociation experiments, including fluorescence anisotropy and a continuous native electrophoretic mobility shift assay, confirmed that two EBNA1.DNA complex conformers are in slow equilibrium; one dissociates slowly (t1/2 approximately 41 min) through an undissociated intermediate species and the other corresponds to a fast twostep dissociation route (t1/2 approximately 0.8 min). In line with this, at least two parallel association events from two populations of protein conformers are observed, with on-rates of 0.25-1.6x10(8) m-1 s-1, which occur differentially either in excess protein or DNA molecules. Both parallel complexes undergo subsequent firstorder rearrangements of approximately 2.0 s-1 to yield two consolidated complexes. These parallel association and dissociation routes likely allow additional flexible regulatory events for site recognition depending on site availability according to nucleus environmental conditions, which may lock a final recognition event, dissociate and re-bind, or slide along the DNA.

Animals↗
PubMedDate not supplied

Determination of the structure of the RNA complex of a double-stranded RNA-binding domain from Drosophila Staufen protein.

We have determined using NMR the structure of the complex between the third double-stranded RNA-binding domain (dsRBD3) of Drosophila Staufen protein and a RNA stem-loop with optimal binding properties in vitro. This work was designed to understand how dsRBD proteins bind RNA and to investigate the role of Staufen dsRBDs in the localization of maternal RNAs during early embryonic development. The structure determination was challenging, because of weak, nonsequence specific binding and residual conformational flexibility at the RNA-protein interface. In order to overcome the problems originated by the weak interaction, we used both new and more traditional approaches to obtain distance and orientation information for the protein and RNA components of the complex. The resulting structure allowed the verification of aspects of RNA recognition by dsRBDs matching the information obtained by a related crystallographic study. We were also able to generate new observations that are likely to be relevant to dsRBD-RNA binding and to the physiological role of Staufen protein.

Animals↗
PubMed1993

The evolutionary origin of the major histocompatibility complex: polymorphism of class II alpha chain genes in the cartilaginous fish.

T cells recognize antigen (Ag) in the form of peptides bound to the major histocompatibility complex (MHC) molecule. One of the important issues in evolutionary immunology is to identify the stage in phylogeny when this mode of Ag recognition emerged. At present, there is a considerable controversy as to whether the cartilaginous fish have the bona fide MHC. In our previous study, we showed that the nurse shark, a member of the cartilaginous fish, has (a) gene(s) capable of encoding MHC class II alpha chains. In the present study, we examined the polymorphism of nurse shark MHC class II alpha chain genes designated Gici-DAA and Gici-DBA using the polymerase chain reaction. The Gici-DAA and Gici-DBA genes had six and five alleles, respectively, and individual alleles usually differed by multiple nucleotides. In addition, most of the nucleotide substitutions were located at the putative Ag-binding sites, where non-synonymous substitutions occurred more frequently than synonymous substitutions. The fact that the Gici-DAA and Gici-DBA genes display a polymorphism pattern essentially similar to that of mammalian MHC genes playing a major role in Ag presentation suggests that the cartilaginous fish have the bona fide MHC. Thus, the MHC-peptide-based T cell recognition system appears to have arisen at or before the emergence of the cartilaginous fish.

Alleles↗
PubMed2006

Comparability of the Rey-Osterrieth Complex Figure, the Taylor Complex Figure, and the Modified Taylor Complex Figure in a normal sample of Japanese speakers.

The comparability of recall performance was examined on the Rey-Osterrieth Complex Figure, the Taylor Complex Figure, and the Modified Taylor Complex Figure as measures of visuospatial abilities and visual memory in 66 healthy Japanese university students. A between-subjects design was used in which each of the participants received the three figures. The interrater reliability of each was excellent. While the three figures yielded equivalent copy scores, the Taylor Complex Figure produced significantly higher scores than the other two on both short (3-min.) and delayed (30-min.) recall tests. There were no significant differences between recall scores of the Rey-Osterrieth and the Modified Taylor figures. These results are consistent with the findings of the 2002 study of English-speakers by Hubley and Tremblay, and support that the Modified Taylor Figure is superior to the original one for the companion figure to the Rey-Osterrieth Complex Figure.

Adolescent↗
PubMed2005

Convergence on a distinctive assembly mechanism by unrelated families of activating immune receptors.

Activating receptors in cells of hematopoetic origin include members of two unrelated protein families, the immunoglobulin (Ig) and C type lectins, which differ even in the orientation of the transmembrane (TM) domains. We examined assembly of four receptors with diverse function: the NK receptors KIR2DS and NKG2C/CD94, the Fc receptor for IgA, and the GPVI collagen receptor. For each of the four different receptors studied here, assembly results in the formation of a three-helix interface in the membrane involving two acidic TM residues from the signaling dimer and a basic TM residue from the ligand recognition module, an arrangement remarkably similar to the T cell receptor (TCR)-CD3 complex. The fact that the TM domains of Ig family and C type lectins adopt opposite orientations proves that these receptor families independently evolved toward the same structural arrangement of the interacting TM helices. This assembly mechanism is thus widely utilized by receptors in cells of hematopoetic origin.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Efficient fluorescent sensors of oligopeptides by dithiobis(2-benzoylamide)-bridged bis(beta-cyclodextrin)s: structure in solution, binding behavior, and thermodynamic origin.

Two 6,6'-bis(beta-cyclodextrin)s linked by 2,2'-dithiobis[2-(benzoylamino)ethyleneamino] and 2,2'-dithiobis[2-(benzoylamino)diethylenetriamino] bridges (1 and 2) have been synthesized as cooperative multipoint recognition receptor models for non-aromatic oligopeptides. Their structures in solution and inclusion complexation mechanism are comprehensively investigated by means of circular dichroism, 2D NMR spectra and temperature-dependent fluorescence titrations. The results show that the cooperative 'host-linker-guest' binding mode and the extensive desolvation effect jointly contribute to the guest-induced fluorescence enhancement of bis(beta-cyclodextrin)s. Further examinations on the binding behavior of hosts 1-2 with a series of di- and tri-peptides demonstrate that bis(beta-cyclodextrin) 1 can recognize not only the size/shape of oligopeptides but also the dipeptide sequence, giving an exciting residue selectivity up to 37.5 for Gly-Gly-Gly/Glu-Glu pair and a high sequence selectivity up to 5.0 for Gly-Leu/Leu-Gly pair. These fairly good selectivities are discussed from the viewpoint of cooperative binding, multiple recognition and induced-fit interactions between host and guest.

Fluorescent Dyes↗
PubMed1995

A chloroplast homologue of the signal recognition particle subunit SRP54 is involved in the posttranslational integration of a protein into thylakoid membranes.

The mechanisms involved in the integration of proteins into the thylakoid membrane are largely unknown. However, many of the steps of this process for the light-harvesting chlorophyll a/b protein (LHCP) have been described and reconstituted in vitro. LHCP is synthesized as a precursor in the cytosol and posttranslationally imported into chloroplasts. Upon translocation across the envelope membranes, the N-terminal transit peptide is cleaved, and the apoprotein is assembled into a soluble "transit complex" and then integrated into the thylakoid membrane via three transmembrane helices. Here we show that 54CP, a chloroplast homologue of the 54-kDa subunit of the mammalian signal recognition particle (SRP54), is essential for transit complex formation, is present in the complex, and is required for LHCP integration into the thylakoid membrane. Our data indicate that 54CP functions posttranslationally as a molecular chaperone and potentially pilots LHCP to the thylakoids. These results demonstrate that one of several pathways for protein routing to the thylakoids is homologous to the SRP pathway and point to a common evolutionary origin for the protein transport systems of the endoplasmic reticulum and the thylakoid membrane.

Animals↗
PubMedDate not supplied

Factors influencing women's decisions about timing of motherhood.

OBJECTIVE: To examine the factors that influence women's decisions about the timing of motherhood from a life span perspective. DESIGN: Qualitative. SETTING: Large Western Canadian city with a high rate of infants born to women aged 35 years and older. PARTICIPANTS: 45 Canadian women aged 20 to 48 years. RESULTS: Independence, a stable relationship, and declining fertility influenced women's decisions about the timing of motherhood. Women integrated child developmental transitions into a projected life plan as they considered the timing of motherhood. Partner readiness and family of origin influences played a lesser role. Delayed childbearing has become more socially acceptable, with subsequent negative connotations associated with younger motherhood. Parental benefits have limited influence on the timing of motherhood. CONCLUSIONS: Recognition by nurses of the various and complex factors that influence women's decisions about the timing of motherhood may flag the importance of pregnancy-related counseling for woman across the fertility life span. Policy decision makers must be cognizant of the need for additional high-risk obstetric and neonatal health services when societal norms encourage women to delay childbearing in favor of completing education and establishing a career.

Adult↗
PubMed2004

The nuclease domain of adeno-associated virus rep coordinates replication initiation using two distinct DNA recognition interfaces.

Integration into a particular location in human chromosomes is a unique property of the adeno-associated virus (AAV). This reaction requires the viral Rep protein and AAV origin sequences. To understand how Rep recognizes DNA, we have determined the structures of the Rep endonuclease domain separately complexed with two DNA substrates: the Rep binding site within the viral inverted terminal repeat and one of the terminal hairpin arms. At the Rep binding site, five Rep monomers bind five tetranucleotide direct repeats; each repeat is recognized by two Rep monomers from opposing faces of the DNA. Stem-loop binding involves a protein interface on the opposite side of the molecule from the active site where ssDNA is cleaved. Rep therefore has three distinct binding sites within its endonuclease domain for its different DNA substrates. Use of these different interfaces generates the structural asymmetry necessary to regulate later events in viral replication and integration.

Binding Sites↗
PubMed1992

Reconstitution of pyruvate dehydrogenase multienzyme complexes based on chimeric core structures from Azotobacter vinelandii and Escherichia coli.

Two unique restriction sites were introduced by site-directed mutagenesis at identical positions in the DNA encoding the dihydrolipoyltransacetylase (E2p) components of the pyruvate dehydrogenase complex from Azotobacter vinelandii and from Escherichia coli. In this manner each DNA chain could be cut into three parts, coding for the lipoyl domain, which consists of three lipoyl subdomains, the binding domain and the core-forming catalytic domain, respectively. Chimeric E2p components were constructed by exchanging the three domains between E2p from A. vinelandii and E. coli on gene level. The six chimeric E2p proteins were expressed and purified from E. coli TG2. All chimeras were catalytically active, 24-subunit E2p proteins. Interactions of the peripheral components E1p and E3 with the wild-type enzymes from A. vinelandii and E. coli and with the chimeric proteins were studied by gel-filtration experiments, analytical ultracentrifugation and reconstitution of the overall activity of the complex. A. vinelandii E3 interacts only with those chimeras that contain the A. vinelandii binding domain, whereas E. coli E3 interacts with all chimeras. Exchange of the lipoyl or catalytic domain did not influence the binding properties of E3. Recognition of E1p depends on the origin of both the binding domain and the catalytic domain. E. coli E1p interacts strongly with those chimeras in which both the binding domain and the catalytic domain were derived from E. coli E2p and weakly with chimeras that contained either the binding domain or the catalytic domain from E. coli E2p. No binding of E. coli E1p was observed when both domains were of A. vinelandii origin. A. vinelandii E1p recognizes E2p from A. vinelandii and E. coli, but strong interaction required that the binding and catalytic domain were of the same origin. Exchange of lipoyl domains had no effect on the binding properties of the E1p component. These observations confirm previous conclusions, based on site-directed mutagenesis of A. vinelandii E2p [Schulze, E., Westphal, A. H., Boumans, H., and de Kok, A. (1991) Eur. J. Biochem. 202, 841-848], that the binding site for E1p consists of amino acid residues derived from both the binding and the catalytic domain and extend these conclusions to E. coli E2p. Dissociation of the 24 subunit E2p core was only detected when the chimeric E2p proteins contained the catalytic domain from A. vinelandii E2p. Dissociation depends on the binding of peripheral components to the E1p-binding sites, pointing to differences in the inter-trimer contacts between the E2p proteins from both species.(ABSTRACT TRUNCATED AT 400 WORDS)

Azotobacter vinelandii↗
PubMed1995

Testing the mental model theory of propositional reasoning.

Johnson-Laird, Byrne, and Schaeken (1992) present a theory of propositional reasoning by mental models. The present study provides a comprehensive test of the predictions of that theory for the difficulty of simple disjunctive and conditional inferences. The results are largely consistent with the complex pattern of predictions. They are discussed in the light of recently proposed modifications of the original theory.

Adult↗
PubMed1991

G-protein beta gamma forms: identity of beta and diversity of gamma subunits.

Signal-transducing G-proteins are heterotrimers composed of GTP-binding alpha subunits in association with a tightly bound complex of beta and gamma subunits. While the alpha subunits are recognized as a family of diverse structures, beta and gamma subunits have also been found as heterogeneous isoforms. To investigate the diversity and tissue specificity of the beta gamma complexes, we have examined homogeneous oligomeric G-proteins from a variety of sources. The beta and gamma subunits isolated from the major-abundance G-proteins from bovine brain, bovine retina, rabbit liver, human placenta, and human platelets were purified and subjected to biochemical and immunological analysis. Protease mapping and immune recognition revealed an identical profile for each of the two distinctly migrating beta isoforms (beta 36 and beta 35) regardless of tissue or G-protein origin. Digestion with V8 protease revealed four distinct, clearly resolved terminal fragments for beta 36 and two for beta 35. Trypsin and chymotrypsin digestion yielded numerous bands, but again each form had a unique profile with no tissue specificity. Tryptic digestion was found to be conformationally specific with the most resistant structure being the native beta gamma complex. With increasing trypsin, the complex was digested but in a pattern distinct from that for denatured beta. In contrast to the two highly homologous beta structures, examination of this set of proteins revealed at least six distinct gamma peptides. Two unique gamma peptides were found in bovine retinal Gt and three gamma peptides in samples of bovine brain derived Go/Gi. Human placental and platelet Gi samples each contained a unique gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗