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PubMed1989

Women and autonomy: using structural analysis of social behavior to find autonomy within connections.

In their work to construct psychological theories about women's development, Carol Gilligan and Jean Baker Miller both highlight the centrality of interpersonal connections in women's lives. As they describe how women's senses of self and morality are organized around relationships, Gilligan and Miller tend to contrast affiliation with autonomy. The message that readers often take from this view is that autonomy has no meaning for women--is somehow beneath them, beyond them, or unnatural to them. Although Miller and Gilligan dichotomize affiliation and autonomy, they also provide numerous examples in which women's feelings of worth, ability, and self-consideration enhance relatedness. We argue that autonomy can be understood as a sense of freedom and personal integrity that encompasses these same characteristics, and we use the Structural Analysis of Social Behavior to clarify how autonomy makes critical contributions to interpersonal connections.

Female↗
PubMed1987

Use of N-glycanase to release asparagine-linked oligosaccharides for structural analysis.

An enzymatic procedure for releasing asparagine-linked oligosaccharides from glycoproteins by treatment with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase) has been investigated. Ribonuclease B, transferrin, fetuin, and alpha 1-acid glycoprotein were treated with N-glycanase and the released oligosaccharides were radiolabeled with NaB3H4. Lectin staining of the N-glycanase-treated proteins indicated that the deglycosylation reactions had proceeded to completion. The labeled carbohydrate chains were analyzed by HPLC on Micro-Pak AX-5 and AX-10 columns. The proportion of high-mannose and bi-, tri-, and tetraantennary complex chains obtained from each glycoprotein was in agreement with literature values. These results demonstrate that N-glycanase provides a simple method to release all common classes of asparagine-linked oligosaccharides from a glycoprotein in a form that can be radiolabeled directly for structural analysis.

Borohydrides↗
PubMed1988

Physiological and structural analysis of light-harvesting mutants of Rhodobacter sphaeroides.

Two mutants of Rhodobacter sphaeroides defective in formation of light-harvesting spectral complexes were examined in detail. Mutant RS103 lacked the B875 spectral complex despite the fact that substantial levels of the B875-alpha polypeptide (and presumably the beta polypeptide) were present. The B800-850 spectral complex was derepressed in RS103, even at high light intensities, and the growth rate was near normal at high light intensity but decreased relative to the wild type as the light intensity used for growth decreased. Mutant RS104 lacked colored carotenoids and the B800-850 spectral complex, as well as the cognate apoproteins. This strain grew normally at high light intensity and, as with RS103, the growth rate decreased as the light intensity used for growth decreased. At very low light intensities, however, RS104 would grow, whereas RS103 would not. Structural analysis of these mutants as well as others revealed that the morphology of the intracytoplasmic membrane invaginations is associated with the presence or absence of the B800-850 complex as well as of carotenoids. A low-molecular-weight intracytoplasmic membrane polypeptide, which may play a role in B800-850 complex formation, is described, as is a 62,000-dalton polypeptide whose abundance is directly related to light intensity as well as the absence of either of the light-harvesting spectral complexes. These data, obtained from studies of mutant strains and the wild type, are discussed in light of photosynthetic membrane formation and the abundance of spectral complexes per unit area of membrane. Finally, a method for the bulk preparation of the B875 complex from wild-type strain 2.4.1 is reported.

Bacterial Proteins↗
PubMed1978

The covalent structure of bovine liver rhodanese. NH2-terminal sequence and partial structural analysis of tryptic peptides from the citraconylated protein.

Nineteen tryptic peptides produced by cleavage at 18 of the 20 arginyl residues in citraconylated S-carboxymethylcysteinyl-rhodanese have been isolated by a combination of gel filtration and high voltage paper electrophoresis. These Tc fragments account for all of the 293 residues in the parent polypeptide and their partial or complete sequences have been determined by automated and manual Edman degradation. In some cases, sequence analyses were completed by degradation of peptides derived by secondary cleavages of the decitraconylated Tc fragments with trypsin, chymotrypsin, or the protease from Staphylococcus aureus. Automated Edman degradation of intact S-carboxymethylcysteinyl-rhodanese was performed for 60 cycles; the information thus obtained permitted the alignment of seven of the Tc fragments and gave the sequence of the first 79 residues in the polypeptide chain. The Tc peptide at the COOH terminus of rhodanese was placed by virtue of the fact that it contained no arginine. Structural analysis of the Tc peptides provided the sequences surrounding all five of the methionyl residues in the enzyme. One of the methionines was found in a 19-residue Tc fragment which also contained the cysteinyl residue essential for catalysis.

Amino Acid Sequence↗
PubMed1987

Benzamide-DNA interactions: deductions from binding, enzyme kinetics and from X-ray structural analysis of a 9-ethyladenine-benzamide adduct.

The interaction of benzamide with the isolated components of calf thymus poly(ADP-ribose) polymerase and with liver nuclei has been investigated. A benzamide-agarose affinity gel matrix was prepared by coupling o-aminobenzoic acid with Affi-Gel 10, followed by amidation. The benzamide-agarose matrix bound the DNA that is coenzymic with poly(ADP-ribose) polymerase; the matrix, however, did not bind the purified poly(ADP-ribose) polymerase protein. A highly radioactive derivative of benzamide, the 125I-labelled adduct of o-aminobenzamide and the Bolton-Hunter reagent, was prepared and its binding to liver nuclear DNA, calf thymus DNA and specific coenzymic DNA of poly(ADP-ribose) polymerase was compared. The binding of labelled benzamide to coenzymic DNA was several-fold higher than its binding to unfractionated calf thymus DNA. A DNA-related enzyme inhibitory site of benzamide was demonstrated in a reconstructed poly(ADP-ribose) polymerase system, made up from purified enzyme protein and varying concentrations of a synthetic octadeoxynucleotide that serves as coenzyme. As a model for benzamide binding to DNA, a crystalline complex of 9-ethyladenine and benzamide was prepared and its X-ray crystallographic structure was determined; this indicated a specific hydrogen bond between an amide hydrogen atom and N-3 of adenine. The benzamide also formed a hydrogen bond to another benzamide molecule. The aromatic ring of benzamide does not intercalate between ethyladenine molecules, but lies nearly perpendicular to the planes of stacking ethyladenine molecules in a manner reminiscent of the binding of ethidium bromide to polynucleotides. Thus we have identified DNA as a site of binding of benzamide; this binding is critically dependent on the nature of the DNA and is high for coenzymic DNA that is isolated with the purified enzyme as a tightly associated species. A possible model for such binding has been suggested from the structural analysis of a benzamide-ethyladenine complex.

Adenine↗
PubMed1991

Structural analysis and expression of human desmoglein: a cadherin-like component of the desmosome.

Desmosomes are adhesive cell junctions found in great abundance in tissues that experience mechanical stress. The transmembrane desmosomal glycoproteins have been proposed to play a role in cell adhesion; desmoglein I (DGI) is a major member of this class of desmosomal molecules. However, evidence supporting a role for DGI in cell adhesion or in the plaque is lacking. In order to begin to understand DGI function we have identified human cDNA clones encoding the entire mature polypeptide of 1000 amino acids. Our data suggest that like the bovine DGI molecule human DGI is highly related to the calcium-dependent class of cell adhesion molecules known as cadherins. Four related extracellular domains located in the amino-terminal domain of the molecule contain putative calcium binding sites originally identified in the cadherins. The highest degree of similarity between human N-cadherin and human DGI, and likewise between bovine DGI and human DGI, is greatest in the most amino-terminal extracellular domain. This suggests a conserved functional role for the extracellular domains, perhaps in calcium-mediated cell adhesion. The cytoplasmic portion of the molecule contains a cadherin-like region and, like bovine DGI, a carboxy-terminal tail that is not present in the cadherins, comprising three additional domains. One of these contains a novel repeating motif of 29 +/- 1 residues, first identified in bovine DGI. Each of the highly homologous repeating units is likely to consist of two beta-strands and two turns with special characteristics. Five amino acids that are identical in bovine and human DGI lie in the second of the two predicted beta-strands, and intriguingly contain putative target sites for protein kinase C. On the basis of structural analysis, a model predicting the disposition of human DGI domains in the desmosome is proposed. Northern analysis suggests that unlike bovine epidermis, which expresses a single mRNA of reported size approximately 7.6 kb, human foreskin and cultured keratinocytes display a complex pattern with bands of approximately 7.2, 4.0 and 3.0 kb. Each of these cross-hybridizing mRNAs is coordinately expressed in normal human keratinocytes in response to long-term culture and increased calcium.

Amino Acid Sequence↗
PubMed1992

Decomposition of multivariate phenotypic means in multigroup genetic covariance structure analysis.

Observed differences in phenotypic means between groups such as parents and their offspring or male and female twins can be decomposed into genetic and environmental components. The decomposition is based on the assumption that the difference in phenotypic means is due to a difference in the location of the normal genetic and environmental distributions underlying the phenotypic individual differences. Differences between the groups in variance can be accommodated insofar as they are due to differences in unique variance or can be modeled using a scale parameter. The decomposition may be carried out in the standard analysis of genetic covariance structure using, for instance, LISREL. Illustrations are given using simulated data and twin data relating to blood pressure. Other possible applications are mentioned.

Blood Pressure↗
PubMed1992

Structural analysis of the carbohydrate chains isolated from mistletoe (Viscum album) lectin I.

Two glycopeptide fractions prepared from mistletoe (Viscum album) lectin I by Pronase digestion were fractioned by affinity chromatography on a concanavalin A-Sepharose column. With 400-MHz 1H NMR spectroscopy, in conjunction with sugar analysis, the following oligosaccharide structures could be determined: two oligomannose-type glycans in the ratio 4:1, one containing six mannose and the other containing five mannose units, both with two 2-acetamido-2-deoxyglucose units. In addition, a mannotriosyl-->N,N'-diacetylchitobiose glycan containing a xylosyl group and an alpha-fucosyl group (1-->3)-linked to the 2-acetamido-2-deoxyglycosyl-1 residue, a common core element of many plant glycoproteins, was also observed.

Carbohydrate Sequence↗
PubMed1979

Proposed replicative role of the NS polypeptide of vesicular stomatitis virus: structural analysis of an electrophoretic variant.

The structural lesion in the temperature-sensitive mutant E1 of the New Jersey serotype of vesicular stomatitis virus has been assigned to the NS protein. Although the packaged wild-type and mutant NS proteins were similarly phosphorylated, the mutant NS protein migrated faster than the wild-type NS protein in polyacrylamide slab gels electrophoresed in the presence of sodium dodecyl sulfate. The resolution appears to be the result of conformational rather than size differences since the two proteins comigrated in polyacrylamide gels which contained 4 M urea in addition to sodium dodecyl sulfate. Peptide maps, obtained by limited proteolysis of 32P-labeled wild-type and mutant NS proteins with Staphylococcus aureus V8 protease and papain, revealed striking differences which suggested that the mutant alteration could involve an aspartic or glutamic acid residue. Since NS proteins obtained from naturally occurring revertants of E1 were indistinguishable from the wild-type protein in all of these analyses, the structural alteration in the mutant NS protein correlates with the functional lesion. Because E1 is defective in the RNA replication pathway at the restrictive temperature, a replicative role is proposed for the NS protein.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

A structural analysis of the genotoxic and carcinogenic potentials of cyclosporin A.

The structural determinants identified by CASE, a knowledge-based structure--activity relational expert system, as contributing to toxicological effects have been distilled from the informational content of over 2000 molecules that have been adequately tested. This methodology has been applied to an analysis of the potential structural determinants of the mutagenicity, clastogenicity and carcinogenicity of cyclosporin A. The analysis predicts that cyclosporin A is devoid of mutagenicity, clastogenicity and DNA-modifying activity. There is however, a structural basis for its carcinogenicity in rodents, i.e. it appears to be a 'non-genotoxic' carcinogen. As a group, 'non-genotoxic' carcinogens are considered by some to pose a far lesser risk to humans than 'genotoxic' ones. The analysis is consistent with the interpretation that the carcinogenicity and immunosuppressive properties of cyclosporin A derive from different mechanisms and are, therefore, separable.

Animals↗
PubMed1988

Structural analysis of polymers of sickle cell hemoglobin. I. Sickle hemoglobin fibers.

The structure of fibers of deoxyhemoglobin S has been under investigation for several years and a number of different models have been proposed for the arrangement of molecules within the particles. We have used reconstruction and modeling techniques in our analysis of these structures. Several new approaches have been employed in this analysis in order to provide improved estimates of the co-ordinates, pairing, and polarity of the hemoglobin S molecules. Fibers have a variable pitch and, in order to minimize distortions in the reconstructed density maps associated with these variations in pitch, we have developed an iterative procedure to measure the instantaneous pitch and have modified the reconstruction algorithm to incorporate the measured values. This procedure improves the accuracy with which the hemoglobin S molecules can be located in the density maps. Furthermore, the determination of the instantaneous pitch allows us to measure directly the rotation of the individual hemoglobin molecules. These measurements are in excellent agreement with the values predicted using a random angular walk model (as originally proposed for F-actin) to describe the variable pitch. The reconstructions confirm that the fiber consists of 14 strands of hemoglobin S arranged in a hexagonally shaped cross-section. We have determined the pairing of the molecules to form double strands directly from the density maps by identifying the molecules that have intermolecular distances that conform to those of double strands in the Wishner-Love crystal. The seven double strands identified in this manner are consistent with the strand pairings proposed by Dykes et al. (1979) rather than the alternate pairings proposed by Rosen & Magdoff-Fairchild (1985). In addition, we have for the first time determined the polarity of the double strands directly from the reconstruction data. This was achieved using a procedure that amounts to essentially "dissecting" individual double strands from the reconstructed density maps so that their density distribution could be examined independently of the neighboring double strands. Knowledge of the relative polarities of the double strands is essential for determining the intermolecular interactions that stabilize the fiber.

Crystallography↗
PubMed1986

Larval salivary gland secretion proteins in Drosophila structural analysis of the Sgs-5 gene.

The structure of the Drosophila melanogaster salivary gland secretion gene Sgs-5 has been determined by DNA sequence analysis of cloned genomic DNA. This developmentally and tissue-specific gene is a member of the third instar intermolt gene set and is under control of the insect molting hormone ecdysterone. RNA protection experiments show that the RNA coding region of Sgs-5 contains 769 nucleotides and is divided into three exons by two small introns. The protein-coding region appears to begin after a short untranslated RNA leader (33 nucleotides) and to result in a protein of 163 amino acids. The first 18 amino acids give the amino-terminal end the highly hydrophobic nature characteristic of a signal peptide.

Amino Acid Sequence↗
PubMed1991

Escherichia coli aspartate carbamoyltransferase: the probing of crystal structure analysis via site-specific mutagenesis.

Crystal structures are known for aspartate carbamoyltransferase (ATCase) in the T and R states, with and without the allosteric activator adenosine triphosphate (ATP) or inhibitor cytidine triphosphate (CTP). Visual inspection of X-ray crystal structures does not provide all of the information necessary for the determination of structure--function relationships in protein molecules. This problem is compounded because the crystalline states of the molecule may introduce effects due to crystal packing, restricted flexibility and less than optimum enzymatic conditions. Therefore, alternative techniques are required to test mechanisms conjectured from three-dimensional crystal structures of proteins. The technique of site-specific mutagenesis allows the researcher to test structure--function models based on three-dimensional structures and to obtain further insight into characteristics of the enzyme. Site-specific mutagenesis has been used to probe residues believed to be critical in the structure and function of ATCase. Selection of residues to be mutated has depended extensively on three-dimensional crystal structures of the enzyme. To date, 48 site-specific mutations at 37 different amino acid sites have been published. Although a total of 118 mutants at 58 different sites has been communicated to our laboratory, only published mutants will be considered in this review. In this paper, we compile for the first time, review, and analyze the site-specific mutants of ATCase. Site-specific mutagenesis of proteins has become a powerful technique in modern-day molecular biology, especially in studying a molecule as large as aspartate carbamoyltransferase. In this review, the role of site-specific mutagenesis of ATCase is discussed and improvements in the analysis are suggested.

Amino Acid Sequence↗
PubMed1987

Fluorescence method for the structural analysis of oligomannose-type sugar chains by partial acetolysis.

Fluorescence labeling was used in the analysis of partial acetolysis products of oligomannose-type sugar chains with five to nine mannose residues. The principle of the method was the pyridylamination of fragments obtained by the partial acetolysis of pyridylamino sugar chains and the identification of the fragments with an HPLC apparatus equipped with a fluorescence spectrophotometer. The method was tested by analysis of eight oligomannose-type sugar chains with known chemical structures and was found to be effective for analysis of branching structures with samples of 0.5 nmol.

Carbohydrate Sequence↗
PubMed1986

The complete sequence and structural analysis of human apolipoprotein B-100: relationship between apoB-100 and apoB-48 forms.

We have isolated and sequenced overlapping cDNA clones covering the entire sequence of human apolipoprotein B-100 (apoB-100). DNA sequence analysis and determination of the mRNA transcription initiation site by S1 nuclease mapping showed that the apoB mRNA consists of 14,112 nucleotides including the 5' and 3' untranslated regions which are 128 and 301 nucleotides respectively. The DNA-derived protein sequence shows that apoB-100 is 513,000 daltons and contains 4560 amino acids including a 24-amino-acid-long signal peptide. The mol. wt of apoB-100 implies that there is one apoB molecule per LDL particle. Computer analysis of the predicted secondary structure of the protein showed that some of the potential alpha helical and beta sheet structures are amphipathic, whereas others have non-amphipathic neutral to apolar character. These latter regions may contribute to the formation of the lipid-binding domains of apoB-100. The protein contains 25 cysteines and 20 potential N-glycosylation sites. The majority of cysteines are distributed in the amino terminal portion of the protein. Four of the potential glycosylation sites are in predicted beta turn structures and may represent true glycosylation positions. ApoB lacks the tandem repeats which are characteristic of other apolipoproteins. The mean hydrophobicity the mean value of H1 and helical hydrophobic moment the mean value of microH profiles of apoB showed the presence of several potential helical regions with strong polar character and high hydrophobic moment. The region with the highest hydrophobic moment, between amino acid residues 3352 and 3369, contains five closely spaced, positively charged residues, and has sequence homology to the LDL receptor binding site of apoE. This region is flanked by three neighbouring regions with positively charged amino acids and high hydrophobic moment that are located between residues 3174 and 3681. One or more of these closely spaced apoB sequences may be involved in the formation of the LDL receptor-binding domain of apoB-100. Blotting analysis of intestinal RNA and hybridization of the blots with carboxy apoB cDNA probes produced a single 15-kb hybridization band whereas hybridization with amino terminal probes produced two hybridization bands of 15 and 8 kb. Our data indicate that both forms of apoB mRNA contain common sequences which extend from the amino terminal of apoB-100 to the vicinity of nucleotide residue 6300. These two messages may have resulted from differential splicing of the same primary apoB mRNA transcript.

Adult↗
PubMed1989

Age-related changes in the cholinergic components within the central nervous system of CW1 female mice. I. Structural analysis.

Histomorphometric analysis of age-related structural changes in the brain was performed in CW1 female mice, 3, 9, 24 and 32 months of age. Cholinergic regions, such as the hippocampus, NBM and the medial habenula (MH) were investigated in more detail focusing on morphological parameters. The thickness of the frontoparietal cortex (FPC), and the surface area of the dorsal hippocampus and the MH were found to decrease significantly from 9 to 24 months of age. Except for the unique appearance of pseudo-cysts within the FPC, the structural changes culminated by 24 months. Cells' degeneration, in the CA3 hippocampal subfield, was noted already by 9 months of age whereas in other regions the cells' surface area decreased only between 9 and 24 months. Lipofuscin accumulation was most pronounced in the large neurons of the cortex, hippocampus and NBM at 24 months of age.

Aging↗
PubMed1989

[Antipeptide antibodies to the porcine lactate dehydrogenase isoenzyme M4 active center fragment as a probe for the structural analysis of active centers of human lactate dehydrogenase isoforms].

Antipeptide antibodies (AB) to the fragment of the active center of porcine lactate dehydrogenase M4 isoform were used for the analysis of antigenic properties and structural comparison of active centers of human lactate dehydrogenase isoforms. Selective precipitation of the M-subunit-containing isoforms using an immunoadsorbent based on antipeptide AB as well as selective inhibition of the enzymic activity of the M4 isoform by antipeptide AB testify to the specific binding of isoforms to antipeptide AB. The experimental results confirm the literary data on conformational changes in the structure of the active centers of corresponding human lactate dehydrogenase isoforms. The specific interaction of antipeptide AB with human lactate dehydrogenase isoforms suggests that the site of the amino acid sequence (residues 180-214) in both human and porcine M4 isoenzymes is immunochemically identical. The data obtained suggest that antipeptide AB are convenient probes for detecting differences (including minor ones) in the primary and spatial structure of enzymes.

Animals↗
PubMed1990

Negative ion fast atom bombardment-tandem mass spectrometry for structural analysis of isoprenoid diphosphates.

Applicability of negative ion fast atom bombardment (FAB)-tandem mass spectrometry (MS/MS) was examined in trace mixture analyses and structural assignments of some isoprenoid diphosphates. Negative ion FAB-MS spectra using a glycerol matrix of these isoprenoid diphosphates showed predominantly molecular ions (M-H)- together with fragment ions at m/z 177 (H3P2O7)-, 176 (H2P2O7)-, 159 (HP2O6)-, and 79 (PO3)- which were characteristic of the diphosphate ester moiety. The molecular ions did not overlap with peaks arising from any impurities even when crude sample such as butanol extracts from enzymatic reaction mixtures were directly analyzed without any purification. Moreover, collisionally activated dissociation spectra of the molecular ion showed many structurally significant fragment ions which enabled us to elucidate the structures of such irregular alkyl chain moieties as those having a homoisoprenoid skeleton or substituted structures. These studies indicate that negative ion FAB-MS/MS is a simple and useful technique for trace mixture analysis and structure elucidation of isoprenoid diphosphates.

Esters↗