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At least 127 records · Page 7Linked to original sources
PubMed1982

Virion DNA of ground squirrel hepatitis virus: structural analysis and molecular cloning.

The structure of the encapsidated DNA genome of ground squirrel hepatitis virus (GSHV) has been examined by restriction endonuclease cleavage, nucleic acid hybridization, and molecular cloning. GSHV virion DNA is a relaxed circular molecule of approximately 3,200 bases in length; most molecules harbor an extensive single-stranded region which is largely confined to one-half of the genome. The full-length viral DNA strand is covalently bound to protein. The single-stranded region can be repaired in vitro by the action of the endogenous virion polymerase, exogenously added DNA polymerase from avian myeloblastosis virus, or both. Restriction enzyme cleavage of viral DNA from different isolates demonstrated that multiple variants of GSHV exist in nature. The genomes of two such strains have been cloned in Escherichia coli, and their physical maps have been determined. Nucleic acid hybridization studies revealed that the strains share sequence homology with the DNA of human hepatitis B virus. Regions homologous to the coding regions for the surface and core antigens of human hepatitis B virus have been localized on the GSHV chromosome. Molecular cloning experiments have also led to the identification of a region of the viral genome which is altered in a procaryotic host.

Animals↗
PubMed1991

Evaluating measurement models in clinical research: covariance structure analysis of latent variable models of self-conception.

Indirect measures of psychological constructs are vital to clinical research. On occasion, however, the meaning of indirect measures of psychological constructs is obfuscated by statistical procedures that do not account for the complex relations between items and latent variables and among latent variables. Covariance structure analysis (CSA) is a statistical procedure for testing hypotheses about the relations among items that indirectly measure a psychological construct and relations among psychological constructs. This article introduces clinical researchers to the strengths and limitations of CSA as a statistical procedure for conceiving and testing structural hypotheses that are not tested adequately with other statistical procedures. The article is organized around two empirical examples that illustrate the use of CSA for evaluating measurement models with correlated error terms, higher-order factors, and measured and latent variables.

Analysis of Variance↗
PubMed1986

Nucleotide sequence of the yeast cell division cycle start genes CDC28, CDC36, CDC37, and CDC39, and a structural analysis of the predicted products.

The nucleotide sequences of the yeast cell division cycle start genes CDC36, CDC37, and CDC39 are presented. An open reading frame corresponding in size and mapped position to the mRNA for each gene was revealed. These sequences, as well as that of the CDC28 gene, were analyzed for the presence of consensus sequences postulated to be transcriptional or translational signals, or to be involved in mRNA processing. In addition, the predicted protein products of the four genes were subjected to a number of structural and statistical analyses including codon usage bias analysis, secondary structure analysis and hydropathicity analysis.

Amino Acid Sequence↗
PubMed1985

Fine structural analysis of the human pro-alpha 1 (I) collagen gene. Promoter structure, AluI repeats, and polymorphic transcripts.

The human pro-alpha 1(I) collagen gene is 18 kilobases long, and its coding information is subdivided in 51 exons. We have recently reported a detailed analysis of this elaborate structure. Here we describe the exact termini of the gene as well as its 5' flanking sequences (promoter region). Sequence comparison between the human and mouse pro-alpha 1(I) gene showed an unusual highly conserved region 5' to the initiation site of transcription, which may be important for pro-alpha 1(I) collagen expression. Sequence comparison between the pro-alpha 1(I) and other collagen genes revealed the presence of a conserved secondary structure possibly implicated in the regulation of collagen mRNA translation and/or collagen secretion. Two short repetitive sequences closely associated with the pro-alpha 1(I) gene have been identified to be different members of the AluI family of repeats. Like the independently co-expressed pro-alpha 2(I) collagen gene, the analysis of the last exon has revealed that the pro-alpha 1(I) gene transcribes two different mRNAs in cultured fibroblasts. The colinearity and exact location of the termini of these transcripts, as well as their monogenic nature, have been conclusively determined.

Base Sequence↗
PubMed1990

Structural analysis of the specific capsular polysaccharide of Rhodococcus equi serotype 1.

The specific capsular polysaccharide produced by Rhodococcus equi serotype 1 was found to be a high molecular weight acidic polymer composed of D-glucose, D-mannose, and D-glucuronic acid. Structural analysis of the polysaccharide employed a combination of chemical and nuclear magnetic resonance techniques, from which it was determined that the polysaccharide possessed a linear repeating tetrasaccharide unit containing a single O-acetyl substituent and and acetal-linked pyruvic acid moiety: [formula: see text] The 1H and 13C nuclear magnetic resonances of O-deacetylated and pyruvic-free serotype 1 polysaccharides were fully assigned by homo- and hetero-nuclear chemical shift correlation methods.

Acetylation↗
PubMed1987

Isolation and structure analysis of bee venom mast cell degranulating peptide.

The venom of Apis mellifera was processed by gel permeation chromatography on Sephadex G-50 and by reversed-phase HPLC. The initial gel permeation step was carried out in the presence of phosphate ions (0.5 M). Ion pair reagents were required to resolve the strongly basic peptides, secapin, mast cell degranulating (MCD-) peptide and apamin, by reversed-phase (RP) HPLC. Using this relatively simple procedure it is possible to isolate these peptides essentially free of melittin (less than 1 in 10(7)) and phospholipase (less than 1 in 10(5] in high yield. The CD spectrum and secondary structure analysis are reported for MCD-peptide and on this basis a solution structure is proposed for this toxin.

Animals↗
PubMed1992

Peptide/protein structure analysis using the chemical shift index method: upfield alpha-CH values reveal dynamic helices and alpha L sites.

The alpha-CH shifts observed by 1H NMR for medium-sized peptides and for an unusual small protein, herein, which has a high density of alpha L conformations within its 43 residue length, reveal that the recently introduced chemical shift index (CSI) analysis places short dynamic helices (alpha R) and alpha L residues in the same category as stable helices. The method appears to be a promising addition to the arsenal of methods for peptide structure analysis and is clearly not limited to rigid protein systems.

Amino Acid Sequence↗
PubMed1992

Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180.

Bullous pemphigoid (BP) is an autoimmune skin disease that is characterized by the presence of subepidermal blisters resulting from a disruption of the adhesive interactions between basal keratinocytes and the cutaneous basement membrane. Autoantibodies from patients suffering from this disorder recognize two epidermal antigens, BP180 and BP230, both of which have been localized to the hemidesmosome, a transmembrane structure of stratified, squamous epithelia that functions in cell-matrix adhesion. In the present study we report the primary structural analysis of BP180 based on the sequence of a series of overlapping cDNA clones encompassing 4,669 bases of the BP180 transcript. A polymerase chain reaction-based protocol was used to confirm the contiguity of the cDNA segments. This cloned portion of the BP180 transcript was found to contain one long open reading frame (ORF) 4.596 bases in length. This ORF encodes a polypeptide of 155,000 Daltons with an isoelectric point of 9.7. The carboxy-terminal half of BP180, a stretch of 916 amino acids, consists of 15 collagen domains of variable length (15 to 242 amino acids) that are separated from one another by short stretches of non-collagen sequences. Located 76 amino acids upstream of the collagenous region is a putative transmembrane domain, a structural feature that distinguishes BP180 from all of the well-characterized members of the collagen family. This membrane-spanning domain is predicted to function as a signal-anchor sequence, directing the C-terminal collagenous segment of this protein to the exterior of the cell. The putative intracellular domain is highly basic with an isoelectric point of 10.37. This molecular analysis predicts that the BP180 antigen is an integral membrane protein of the hemidesmosome that contains a long extracellular collagenous tail. This combination of structural features suggests that BP180 may function as a cell-matrix adhesion molecule, with the collagenous region acting as a potential site of interaction with basement membrane components. Autoantibody-mediated disruption of such an adhesive interaction may play a critical role in the development of sub-epidermal blisters in BP patients.

Amino Acid Sequence↗
PubMed1991

Purification of an endogenous digitalislike factor from human plasma for structural analysis.

In previous reports, we described the isolation and characterization of an endogenous digitalislike factor (EDLF). In this report, we describe a unique combination of bioassay and large-scale purification methodology that made possible the purification of sufficient quantities of this inhibitor of Na+,K(+)-ATPase for structural analysis. Using an initial XAD-2 extraction and preparative high-performance liquid chromatography followed by a batch enzyme affinity extraction and two subsequent semipreparative chromatographic steps, 300 l of human plasma was processed, yielding 31 micrograms (53 nmol) of pure EDLF and representing purification on a dry weight basis in excess of 0.6 billionfold. Four divergent pieces of evidence, including chromatographic, mass spectrometric, immunoreactive, and binding characteristics, suggested that the EDLF purified in the present study was either ouabain or an isomer of ouabain. This material may represent a plasma-borne, naturally occurring, selective, high-affinity ligand for the digitalis binding site that may play a significant role in the modulation of the sodium pump and thereby cellular electrolyte homeostasis in humans.

Antibodies↗
PubMed1975

Structural analysis of compartmental models for the hepatic kinetics of drugs.

The structure of some compartmental models for the analysis of the hepatobiliary kinetics of bromosulphalein (BSP) was studied in order to evaluate their adequacy in the estimation of the processes involved in the hepatic metabolism of drugs, namely uptake, conugation, and biliary excretion. Biological measurements were obtained from 4 cholecystectomized patients with a biliary T-tube. Blood and bile specimens were taken at various intervals after the administration of a single intravenous dose of BSP and analyzed for both direct BSP quantitation and chromatographic separation and estimation of BSP metabolic fractions. The structural analysis was carried out by using a mathematical model that described the kinetics of BSP. By means of computer simulations different measurement situations were analyzed, showing for each experimental condition the available information and the degree of accuracy of each estimated parameter. The obtained results show that the use of compartmental models can provide a useful theoretical framework by which the experimental data can be interpreted for the evaluation of the hepatobiliary metabolism and for a discriminant analysis between different physiopathological conditions.

Bile↗
PubMed1979

Structural analysis of four strains of Paracoccus denitrificans.

Two out of eleven newly isolated strains of Paracoccus denitrificans were investigated by light and electron microscopic methods and compared with two strains of P. denitrificans already kept in culture collections. Samples were taken from different growth phases revealing short rods and nearly spherical cells in the exponential growth phase, and an increasing ratio of nearly spherical cells in the stationary growth phase. Cell division followed the binary fission mode; higher cell aggregates were not observed. Fine structural analysis revealed extracellular surface material stainable with Ruthenium red, a gram-negative cell wall and different storage material inclusions. Structural properties and variations within the four strains under investigation are discussed and compared with those of related bacteria.

Cell Division↗
PubMed1991

Structural analysis of the Neurospora mitochondrial large rRNA intron and construction of a mini-intron that shows protein-dependent splicing.

The gene encoding the Neurospora mitochondrial large rRNA contains a single group I intron of 2.3 kilobases that is not self-splicing in vitro. We showed previously that the splicing of this intron in vivo and in vitro is dependent on the Neurospora cyt-18 protein, mitochondrial tyrosyl-tRNA synthetase. In the present work, we carried out further structural analysis of the intron and constructed mutant derivatives of it in order to identify features that are either required for splicing or prevent it from self-splicing. Previous studies showed that the intron contains a large hairpin structure near the 5' splice site. By mapping RNase III cleavage sites, we identified this hairpin structure as an extended P2 stem. We construct a mini-intron of 388 nucleotides by deleting the 426-amino acid intron open reading frame, most of the 5' intron hairpin, and all of L8. This mini-intron shows the same protein-dependent splicing as the full length intron, but is still not self-splicing. Further deletions, which remove all of P2 or all or part of P4, P6, P7, or P9, inactivate splicing, suggesting that an intact group I intron core structure is required. Strengthening the P1, P10, or P9.0 pairings did not enable the mini-intron to self-splice. Our findings indicate that the inability of the mitochondrial large rRNA intron to self-splice reflects deficiency of a structure or activity required for cleavage at the 5' splice site, either in the intron core itself or in the interaction between the core and the P1 stem.

Base Sequence↗
PubMed1990

Structural analysis of the specific capsular polysaccharide of Rhodococcus equi serotype 2.

The specific capsular polysaccharide produced by Rhodococcus equi serotype 2 is a high-molecular-weight acidic polymer composed of D-glucose, D-mannose, D-glucuronic acid and 3-O-[(S)-1-carboxyethyl]-L-rhamnose in equimolar proportions. Structural analysis, employing a combination of chemical and n.m.r. techniques, established that the polysaccharide is composed of linear repeating tetrasaccharide units. (formula; see text) in which the beta-D-mannose residues carry O-acetyl groups at O-2 and O-3 to the extent of 1.7 mol equivalents. Unequivocal determination of the absolute chirality of the 3-O-[(S)-1-carboxyethyl]-alpha-L-rhamnose residues was achieved by chemical correlation with an authentic synthetic sample. The 1H and 13C-n.m.r. resonances of the native and O-deacetylated serotype 2 polysaccharides were fully assigned by homo- and heteronuclear chemical-shift correlation methods.

Carbohydrate Conformation↗
PubMed1988

Structural analysis of recombinant soluble human interleukin-2 receptor. Primary structure, assignment of disulfide bonds and core IL-2 binding structure.

A purified soluble and functional form of recombinant human interleukin-2 receptor, engineered and expressed in Chinese hamster ovary cells, was structurally characterized. The primary sequence of this 224 amino acid recombinant protein which lacks most of the carboxy-terminal transmembrane and cytoplasmic portions of the intact protein was established by sequence analyses. The disulfide bonds were assigned by comparative peptide mapping of the reduced and non-reduced peptide digests. As in the case of natural interleukin-2 receptor they occur between cysteines 3-147, 46-104, 131-163, and 28/30-59/61. Based on assignment of the disulfide bonds, a structural model of the interleukin-2 receptor for interleukin-2 binding is proposed.

Amino Acid Sequence↗
PubMed1992

Characterization of guanosine diphospho-D-mannose dehydrogenase from Pseudomonas aeruginosa. Structural analysis by limited proteolysis.

Alginate is believed to be a major virulence factor in the pathogenicity of Pseudomonas aeruginosa in the lungs of patients suffering from cystic fibrosis. Guanosine diphospho-D-mannose dehydrogenase (GDPmannose dehydrogenase, EC 1.1.1.132) is a key enzyme in the alginate biosynthetic pathway which catalyzes the oxidation of guanosine diphospho-D-mannose (GDP-D-mannose) to GDP-D-mannuronic acid. In this paper, we report the structural analysis of GMD by limited proteolysis using three different proteases, trypsin, submaxillary Arg-C protease, and chymotrypsin. Treatment of GMD with these proteases indicated that the amino-terminal part of this enzyme may fold into a structural domain with an apparent molecular mass of 25-26 kDa. Multiple proteolytic cleavage sites existed at the carboxyl-terminal end of this domain, indicating that this segment may represent an exposed region of the protein. Initial proteolysis also generated a carboxyl-terminal fragment with an apparent molecular mass of 16-17 kDa which was further digested into smaller fragments by trypsin and chymotrypsin. The proteolytic cleavage sites were localized by partial amino-terminal sequencing of the peptide fragments. Arg-295 was identified as the initial cleavage site for trypsin and Tyr-278 for chymotrypsin. Catalytic activity of GMD was totally abolished by the initial cleavage. However, binding of the substrate, GDP-D-mannose, increased stability toward proteolysis and inhibited the loss of enzyme activity. GMP and GDP (guanosine 5'-mono- and diphosphates) also blocked the initial cleavage, but NAD and mannose showed no effect. These results suggest that binding of the guanosine moiety at the catalytic site of GMD may induce a conformational change that reduces the accessibility of the cleavage sites to proteases. Binding of [14C]GDP-D-mannose to the amino-terminal domain was not affected by the removal of the carboxyl-terminal 16-kDa fragment. Furthermore, photoaffinity labeling of GMD with [32P]arylazido-beta-alanine-NAD followed by proteolysis demonstrated that the radioactive NAD was covalently linked to the amino-terminal domain. These observations imply that the amino-terminal domain (25-26 kDa) contains both the substrate and cofactor binding sites. However, the carboxyl-terminal fragment (16-17 kDa) may possess amino acid residues essential for catalysis. Thus, proteolysis had little effect on substrate binding, but totally eliminated catalysis. These biochemical data are in complete agreement with amino acid sequence analysis for the existence of substrate and cofactor sites of GMD. A linear peptide map of GMD was constructed for future structure/functional studies.

Affinity Labels↗
PubMed1989

[Functional and structural analysis of the Lyb-2 system].

The Lyb-2 system of the mouse is a B cell-specific cell surface molecule encoded by a gene on chromosome 4. We have previously reported that Lyb-2 is involved in an early phase of B cell differentiation, specifically a process mediated by B cell stimulatory factor-1 (BSF-1) or-interleukin 4. It is thus very important to define the functional role and structural features of Lyb-2 molecule for the understanding of the regulatory mechanisms of B cell activation initiated by BSF-1. In our attempt to resolve these issues, we found that Lyb-2 antibody inhibits activation processes mediated by BSF-1 such as induction of Ia antigen on the B cell surface and of IgG1 production in lipopolysaccharide-activated B cells, suggesting that Lyb-2 molecule participates in a signaling event triggered by the binding of BSF-1 to its receptor. In the structural analysis, we found that in contrast to previous reports, Lyb-2 is not a monomer of 45 kilodaltons (kDa) but is composed of two components with molecular weight of 45 kDa and 105 kDa. Reduced and nonreduced two-dimensional electrophoresis analysis further revealed that Lyb-2 molecules are present on the B cell surface in two forms, a disulfide-bonded heterodimer of 45 kDa and 105 kDa chains and a 45 kDa homatrimer. How the structural uniqueness of Lyb-2 is related to its functional expression remains to be determined.

Animals↗
PubMed1989

Observed family interactions among subtypes of eating disorders using structural analysis of social behavior.

Compared observations of family interactions among anorexic, bulimic-anorexic, bulimic, and normal families. A total of 74 family triads participated, including father, mother, and teenage daughter. Each family was videotaped during a 10-min discussion of the daughter's separation from the family. These tapes were coded using Benjamin's structural analysis of social behavior (SASB) model and observational schema. The results showed that the SASB methodology differentiated clinical from normal families and that there were unique patterns among subtypes of eating disorders. Specifically, parents of anorexics communicated a double message of nurturant affection combined with neglect of their daughter's needs to express themselves and their feelings. Anorexic daughters, in turn, were ambivalent about disclosing their feelings versus submitting to their parents. In contrast, bulimics and their parents were hostilely enmeshed and, for them, this appeared to undermine the daughter's separation and self-assertion. These findings are consistent with current theory and research on anorexia and bulimia.

Adolescent↗