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PubMed1991

Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy.

A protease-resistant form of the protein PrP (PrP-res) accumulates in tissues of mammals infected with scrapie, Creutzfeldt-Jakob disease, and related transmissible neurodegenerative diseases. This abnormal form of PrP can aggregate into insoluble amyloid-like fibrils and plaques and has been identified as the major component of brain fractions enriched for scrapie infectivity. Using a recently developed technique in Fourier transform infrared spectroscopy which allows protein conformational analysis in aqueous media, we have studied the secondary structure of the proteinase K resistant core of PrP-res (PrP-res 27-30) as it exists in highly infectious fibril preparations. Second-derivative analysis of the infrared spectra has enabled us to quantitate the relative amounts of different secondary structures in the PrP-res aggregates. The analysis indicated that PrP-res 27-30 is predominantly composed of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn (31%) and alpha-helix (17%) were identified, indicating that amyloid-like fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within PrP-res 27-30.

Acetylglucosamine↗
PubMed1992

An antiplatelet aggregation principle and X-ray structural analysis of cis-khellactone diester from Peucedanum japonicum.

Three structurally related khellactone coumarins, 1-3, were isolated from the aerial parts of Peucedanum japonicum (Umbelliferae). Compound 2 was identified as a new coumarin (cis-3'-isovaleryl-4'-senecioylkhellactone) by spectral and chemical analysis. Conformation of the dihydropyrano ring of cis-3',4'-disenecioylkhellactone [3] was elucidated by X-ray crystallographic analysis. These three natural khellactone esters were subjected to the antiplatelet aggregation bioassay where cis-3',4'-diisovalerylkhellactone [1] showed significant activity (at 50 micrograms/ml).

Animals↗
PubMed2026

Molecular Cloning, Recombinant Expression, and In Silico Structural Analysis of Cu/Zn-Superoxide Dismutase from Trachyspermum ammi.

Superoxide dismutase (SOD) is an essential antioxidant metalloenzyme that is critical for the cellular defense against oxidative damage, as it scavenges superoxide radicals and maintains the redox status. Cytosolic Cu/Zn-SOD is particularly important in the regulation of oxidative stress among different isoforms in higher plants. While Cu/Zn-SODs from several plant species have been characterized, molecular information is limited for Trachyspermum ammi, a medicinally important member of a family Apiaceae with antioxidant potential.In the present study, an integrated molecular and in silico approach has been taken to clone and analyze a Cu/Zn type SOD gene from T. ammi to get insight into its structural and evolutionary characteristics. PCR amplification yielded an open reading frame of 456 bp encoding a protein of 152 amino acids. Sequence analysis showed that plant Cu/Zn-SODs, especially those from Daucus carota, were highly similar to one another (about 90-95%).Multiple sequence alignment confirmed the presence of conserved catalytic motifs and metal-binding histidine residues, both of which are crucial for enzymatic function. Physicochemical analysis predicted the protein to be stable, hydrophilic and compatible with cytosolic localization. The analysis of secondary structure indicated a predominance of β-strands, consistent with the conserved β-barrel architecture of plant Cu/Zn-SODs.The three-dimensional structure was built by homology modeling using a closely related plant Cu/Zn-SOD template with high sequence identity. Structural validation demonstrated an acceptable stereochemical quality with 86.3% residues in the favored region of Ramachandran plot, satisfactory ERRAT and Verify3D scores, and a low RMSD value of 0.104 Å on structural superimposition. Phylogenetic analysis placed the enzyme in the Apiaceae lineage, suggesting evolutionary conservation among related plant species. In conclusion, this study presents the first molecular and structural characterization of Cu/Zn-SOD from T. ammi and confirms the existence of a conserved structural framework typical of plant Cu/Zn-SODs. These results provide a basis for further studies concerning recombinant expression, enzymatic validation and potential relevance in antioxidant and plant stress biology.

Cloning, Molecular↗
PubMed1992

Three-dimensional structure of acylphosphatase. Refinement and structure analysis.

We report here the complete determination of the solution structure of acylphosphatase, a small enzyme that catalyses the hydrolysis of organic acylphosphates, as determined by distance geometry methods based on nuclear magnetic resonance information. A non-standard strategy for the distance geometry calculations was used and is described here some detail. The five best structures were then refined by restrained energy minimization and molecular dynamics in order to explore the conformational space consistent with the experimental data. We address the question of whether the solution structure of acylphosphatase follows the general principles of protein structure, i.e. those learned from analysing crystal structures. Static and dynamic features are discussed in detail. An uncommon beta-alpha-beta motif, so far found only in procarboxypeptidase B and in an RNA-binding protein, is present in acylphosphatase.

Acid Anhydride Hydrolases↗
PubMed1992

Design and synthesis of novel 6,7-imidazotetrahydroquinoline inhibitors of thymidylate synthase using iterative protein crystal structure analysis.

Antifolate inhibitors of thymidylate synthase (TS) have primarily been based on the structure of folic acid. This paper describes the identification and development of novel 6,7-imidazotetrahydroquinoline TS inhibitors by iterative ligand design, synthesis, and crystallographic analysis of protein-inhibitor complexes. Beginning with a high-resolution crystal structure of E. coli TS (TS, EC 2.1.1.45), an imidazotetrahydroquinoline inhibitor was designed de novo to occupy the folate binding pocket. Structural modifications of the initial compound 1h (Ki approximately 5 microM human/E. coli TS) were then made on the basis of feedback from additional cocrystal structures and activity data. An amino group in the 2-position of the imidazole was found to increase the potency of the series by 1-2 orders of magnitude. Other substitutions on the imidazole ring (1-CH3, 2-CH3, 2-NHCH3, 2-SCH3) generally led to weaker inhibition. Additional improvements in activity were obtained by modification of the substituents on the tetrahydroquinoline nitrogen, bringing the Ki of three of the compounds below 15 nM against the human TS enzyme. The compounds were tested for cytotoxicity and were shown to inhibit the growth of three tumor cell lines in vitro.

Adenocarcinoma↗
PubMed1977

A genetic fine structure analysis of the suppressor 3 locus in Saccharomyces.

A meiotic fine structure map of a yeast tyrosine-inserting ochre suppressor, SUP3-omicron, was constructed. This was accomplished by examining ten intragenic suppressor-inactive revertants for their relationship to each other and to the original SUP3-omicron mutation. The second-site revertants map on both sides of the SUP3-omicron mutation. The meiotic map length based on the summation of short intervals is 45+/10(5) asci.

Chromosome Mapping↗
PubMed1977

Structural analysis of the surface polysaccharide of Staphylococcus aureus M.

The chemical structure of the surface polysaccharide from Staphylococcus aureus M was investigated by a combination of methanolytic, hydrolytic, and chromatographic techniques. The repeating unit that was most consistent with the data was a hexasaccharide composed of N-acetyl-D-aminogalacturonic acid, N-acetyl-D-fucosamine, and taurine in molar ratios of 4:2:1. A disaccharide was isolated and characterized, by combined gas-liquid chromatography-mass spectrometry, as N-acetyl-D-aminogalacturonyl-(1 leads to 3)-N-acetyl-D-fucosamine. Taurine is linked to a carboxyl group of N-acetyl-D-aminogalacturonic acid via an amide bond.

Amino Acids↗
PubMed1975

Quantitative structural analysis of pulmonary vessels in isolated ventricular septal defect in infancy.

Structural changes in the pulmonary circulation were studied in the lungs of 5 infants dying with ventricular septal defect. Applying precise quantitative morphological techniques to the pulmonary vessels, it was possible to correlate pathological change with clinical and haemodynamic findings, and to identify two patterns of response. Three of the infants (group I) ppresnted in cardiac failure with a large pulmonary blood flow, dilated and tortuous pulmonary arteries, and fewer intra-acinar vessels than normal. Medial hypertrophy was moderate and affected chiefly the larger arteries, i.e. those with a diameter greater than 200 mum. The other 2 infants (group 2) had a high pulmonary vascular resistance with an intermittent right-to-left shunt. The pulmonary arteries were of normal size and the reduction in the number of the arteries was less striking. Medial hypertrophy was greater than in the first group and affected all sizes of artery including those less than 200 mum in diameter. In both groups, muscle extended further along the axial pathway. Muscular hypertrophy was found also in the vein wall in most cases and, as with the arteries, was more severe in those with a higher pulmonary vascular resistance. The findings illustrate the variation in pulmonary vascular response in infants with a ventricular septal defect. It is suggested that in patients with a ventricular septal defect, arterial muscularity usually regresses after birth and a left-to-right shunt develops; secondary hypertrophy of the media then develops in reaponse to the shunt. Our findings also suggest, however, that in some infants arterial muscle fails to regress postnatally so that pulmonary blood flow is never high and a right-to-left shunt develops soon after birth.

Blood Pressure↗
PubMed1975

A structural analysis of the myogenic control systems of the human Fallopian tube.

The structural basis for myogenic and neurogenic control of motility of human Fallopian tubes was studied. In the ampulla distinct muscle layers were absent, and bundles of cells oriented in different axes were in contact with one another by means of randomly located simple apposition contacts; no nexuses were present. The organization was consistent with a spread of electrical activity in either direction throughout all muscle bundles. Nerves were not found in close proximity to muscle and were mostly peripheral, surrounded by connective tissue sheaths and by Schwann cells within the sheath. They could play little role in control of ampullar motility. In the isthmus muscle bundles were grouped into innermost longitudinal layer, circular layer, and outermost layer of mixed orientation. Cell-to-cell connections were mainly by simple apposition contacts; no nexuses were found. There were such connections between cells of bundles oriented in different axes. The organization of muscles suggested that electrical activity could spread in either direction and throughout all layers. Nerves like those in the ampulla were located peripherally, and smaller bundles without connective tissue sheaths were in circular muscle. Few close contacts between nerve and muscle were observed.

Action Potentials↗
PubMed2026

Genome-wide SNP-based genomic diversity and population structure analysis in alpaca populations from Europe and Peru.

This study aimed to analyze the genetic diversity and population structure of alpacas in Germany, Switzerland, and Austria (German-speaking regions, GSR) and to compare with that of the country of origin of the species (Peru). A total of 179 animals from GSR and 151 from Peru were genotyped with a species-specific 76k SNP array. The observed and expected heterozygosity was 0.305 and 0.311 for GSR and 0.310 and 0.312 for Peru. The mean FROH values were 0.029 for GSR and 0.023 for Peru. In general, results show that breeders in both analyzed regions efficiently maintain genetic diversity. Principal component analysis identified the GSR and Peru populations as separate from each other, but the relative proximity of both clusters indicates the shared genetic heritage. FST and XPEHH methods identified genomic regions under selection for traits such as coat color and adaptation. Genome-wide association studies comparing black and brown with white or gray alpacas identified associated genome regions containing the ASIP and KIT genes, respectively. The association of a recently identified keratin locus on chromosome 16 with differences in fleece type in alpacas was confirmed, while the putative causality of a TRPV3 variant was rejected.

Animals↗
PubMed1992

Structural analysis of the N-linked oligosaccharides from human urinary trypsin inhibitor.

The N-linked oligosaccharides from human urinary trypsin inhibitor were purified and their structures were investigated by compositional analysis, the two-dimensional sugar map method and 500 MHz 1H-NMR. The results revealed that they were composed of disialosyl, monosialosyl and asialosyl oligosaccharides, which have the common biantennary core structure; Gal1-4GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)M an1-4GlcNAc1-4GlcNAc.

Carbohydrate Conformation↗
PubMed1979

Similarity of the structure of ferritin and iron . dextran (imferon) determined by extended X-ray absorption fine structure analysis.

Ferritin, a natural complex of iron oxide encased in protein, and iron . dextran, a synthetic complex of iron oxide coated with dextran, have the similar properties of maintaining high concentrations of iron in solution at physiological pH and releasing iron relatively slowly in vivo. Extended x-ray absorption fine structure (EX-AFS) analysis was performed on each complex and compared to see if the structures of the iron cores were similar. The results obtained from the extended x-ray absorption fine structure technique show that the near-neighbor environment around the average iron atom in ferritin and iron . dextran is identical, within experimental uncertainty, for the first three shells. The similarity of the iron cores in both complexes may explain the similarity of iron release in vivo. Ferritin has a protein coat which is composed of 24 subunits arranged in a hollow sphere with six channels through which the iron may move during deposition and release. However, little is known about the requirements of the protein structure in ferritin for the maintenance of high concentrations of iron in a soluble, nontoxic form or about the role of the protein in the release of iron from ferritin. The results suggest that iron . dextran will be a useful model compound in studies of the relation of the iron core and protein in ferritin to function.

Dextrans↗
PubMed1992

Structural analysis of 2',3'-dideoxyinosine, 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine and 2',3'-dideoxycytidine by 500-MHz 1H-NMR spectroscopy and ab-initio molecular orbital calculations.

Solution structure of anti-AIDS drug, 2',3'-dideoxyinosine (ddI) has been assessed by NMR spectroscopy and pseudorotational analysis in conjunction with its analogues: 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyguanosine (ddG) and 2',3'-dideoxycytidine (ddC). The absence of 3'-hydroxyl groups in these compounds has prompted us to establish the relationship between proton-proton and corresponding endocyclic torsion angles in the 2',3'-dideoxyribofuranose moiety on the basis of five available crystal structures of 2',3'-dideoxynucleosides. A subsequent pseudorotational analysis on ddI (1), ddA (2), ddG (3) and ddC (4) shows that the twist C2'exo-C3'-endo forms of sugar are overwhelmingly preferred (75-80%) over the C2'-endo envelope forms. The phase angles (P) for North and South conformers with the corresponding puckering amplitude (psi m) for ddI (1), ddA (2) and ddG (3) are as follows: PN = 0.1 degrees, PS = 161 degrees and psi m = 34.1 degrees for ddI (1); PN = 1.4 degrees, PS = 160 degrees and psi m = 34.2 degrees for ddA (2) and PN = 2.4 degrees, PS = 163 degrees and psi m = 33.6 degrees for ddG (3). The predominant North conformer of ddC (4) is intermediate between twist C2'-exo-C3'-endo and C3'-endo envelope (P = 10.9 degrees) with a psi m of 34.7 degrees. Note that these preponderant North-sugar structures (approx. 75-80%) found in the solution studies of ddI (1), ddA (2), dG (3) and ddC (4) are not reflected in the X-ray crystal structures of 2',3'-dideoxyadenosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures denosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures are found to be in the South-type geometry (ddA crystalizes in C3'-exo envelope form, while ddC adopts the form intermediate between the C3'-exo envelope and C3'-endo-C4'-exo twist form). This means that X-ray structures of ddA (2) and ddC (4) only represent the minor conformer of the overall pseudorotamer population in solution. An assumption that the structure of the pentofuranose sugar (i.e. P and psi m) participating in conformational equilibrium described by the two-state model remains unchanged at different temperatures has been experimentally validated by assessing five unknown pseudorotational parameters with eight unique observables (3J1'2', 3J1'2", 3J2'3', 3J2'3", 3J2"3', 3J2"3", 3J3'4' and 3J3"4') for 2',3'-dideoxynucleosides.(ABSTRACT TRUNCATED AT 400 WORDS)

Didanosine↗
PubMed1991

A detailed functional and structural analysis of a major thyroid hormone inhibitory element in the human thyrotropin beta-subunit gene.

The first exon of the human thyrotropin-beta (hTSH beta) gene has been demonstrated in our laboratory to contain a major thyroid hormone inhibitory element. In order to characterize fully this element, we have performed a detailed functional and structural scanning mutational analysis of this element. Various -1192 to +37 (base pairs) bp fragments of the hTSH beta gene containing consecutive five deoxythymidine substitution mutations of the first exon were inserted into a luciferase reporter plasmid and transiently transfected into human embryonal cells (293) and stably transfected into rat pituitary cells (GH3). Two domains (domain 1 and 2) were identified by scanning mutations that were essential for function of the thyroid hormone inhibitory element: +3 to +13 bp and +28 to +37 bp. Biotinylated DNA fragments containing -12 to +43 bp of the hTSH beta gene and the identical scanning mutations demonstrate that in vitro synthesized c-erbA-beta binding is disrupted as much as 95% by mutations from -3 to +17 bp and to a lesser extent (20-30%) by mutations from +23 to +27 bp and from +33 to +43 bp. Domain 1 displayed a higher affinity for c-erbA-beta than domain 2 in avidin-biotin complex DNA-binding and gel-mobility assays. Using increasing amounts of in vitro synthesized c-erbA-beta, we were unable to demonstrate more than one protein-DNA complex in gel-mobility assays. However, using the avidin-biotin complex DNA-binding assay and the cross-linking reagent, 1,6-bismaleimidohexane, we were able to demonstrate thyroid hormone receptor dimer formation on domain 1 but not to any significant extent on domain 2. In conclusion, functional and DNA-binding studies suggest that the thyroid hormone receptor binds to two distinct regions in the first exon of the hTSH beta gene. The upstream site (domain 1) binds c-erbA-beta with higher affinity and is capable of binding c-erbA-beta as a dimer under some conditions, while the downstream site (domain 2) appears to bind a single molecule of c-erbA-beta with lower affinity. These results suggest that thyroid hormone receptor, binding to at least two sites in the first exon, act in conjunction to mediate T3 inhibition of hTSH beta expression.

Animals↗
PubMed1991

Structural analysis of unstable intermediate and stable forms of recombinant fowlpox virus.

The stability and structure of the products of recombination in a fowlpox virus (FPV) system using the thymidine kinase (TK) gene as the insertion site were examined. A 4.6 kb chimeric DNA fragment from the pUV1 expression vector, containing the bacterial lacZ gene and the vaccinia virus P7.5 promoter, was ligated into the XbaI site of the FPV TK gene. The resulting vector, pFTKlacZb, was transfected into chicken embryo fibroblast cultures infected with FPV at an m.o.i. of 0.1. Recombinants were screened for the expression of beta-galactosidase. Five recombinants were isolated and plaque-purified to 80 to 90% for expression of beta-glucosidase. Serial cell culture passage of the recombinants led to the gradual reappearance of the non-recombinant parental phenotype. Southern hybridization analysis of EcoRI fragments from all five recombinants indicated that a single cross-over homologous recombination had occurred between either the 5' or the 3' end fragments of the TK gene, generating unstable intermediate recombinants incorporating the entire pFTKlacZb vector. Secondary intermolecular or intramolecular recombination of intergenic repetitive sequences within the intermediate recombinants appears to have resulted in frequent regeneration of the parental genotype and an infrequent generation of more stable recombinants. A method was developed to select stable recombinants by passage of the intermediate recombinants in chicken embryo fibroblast cultures treated with 5-bromo-2'-deoxyuridine.

Animals↗
PubMed1976

Partial structural analysis of the basic chromosomal protein of rat spermatozoa.

An investigation of the primary structure of the basic chromosomal protein of rat spermatozoa by automated Edman degradation and by carboxypeptidase digestion has provided a general structural outline of the entire molecule. The exact or approximate location of virtually all residues other than arginine or cysteine is reported. Of particular interest, because of the occurrence of phosphorylated derivatives of this protein, is the location of three of the four serine residues.

Amino Acid Sequence↗
PubMed1992

Structure analysis of purified histone H5 and of H5 in nuclei by limited proteolysis.

The structure of purified histone H5 in 1 M-NaClO4 and of H5 in nuclei was analysed by digestion with either one of three endoproteinases, papain, subtilisin or elastase, which preferentially cleave unstructured protein regions (and additionally with trypsin). Digestion with papain and subtilisin produced 'limiting' resistant peptides (p1 and s1) that contain the central region between residues 18-20 and residue 114. Digestion of purified H5 with elastase generated resistant peptides e1 and e2, and that of H5 in nuclei, peptide e2. Peptides e1 and e2 contain the region from residues 22 to 114 and 109 respectively. These results show that a central region of H5 encompassing the sequence between residues 18-22 and residue 114 is folded into a compact structure. A central structured 'core' domain ranging from residues 22 to 100 is defined by the limit trypsin peptide t3, which is identical to the previously described fragment GH5 [Aviles et al. (1978) Eur. J. Biochem. 88, 363-371]. Generation of peptides e2 and t3, as well as of resistant peptides of lower abundance, shows that the sites near Lys-100 and Lys-109 exhibit some proteolytic sensitivity, which may result either from an exposed location or from a locally less compact conformation. Significantly, all these structural features of H5 are manifested in the purified form as well as in nuclei. A role of the structured region from residues 101 to 114 for the interaction with linker DNA and the determination of its path is discussed.

Amino Acid Sequence↗
PubMed1992

Structural analysis of the operator binding domain of Tn10-encoded Tet repressor: a time-resolved fluorescence and anisotropy study.

An engineered Tn10-encoded Tet repressor, bearing a single Trp residue at position 43, in the putative alpha-helix-turn-alpha-helix motif of the operator binding domain, was studied by time-resolved fluorescence and anisotropy. Fluorescence intensity decay data suggested the existence of two classes of Trp-43, defined by different lifetimes. Analysis of anisotropy data were consistent with a model in which each class was defined by a different lifetime, rotational correlation time, and fluorescence emission maximum. The long-lifetime class had a red-shifted spectrum, similar to that of tryptophan zwitterion in water, and a short rotational correlation time. In contrast, the spectrum of the short-lifetime class was blue-shifted 10 nm compared to that of the long-lifetime class. Its correlation time was similar to that of the protein, which showed that Trp in this class was entirely constrained. Trp in this latter class could not be quenched by iodide, whereas most of the long-lifetime class was easily accessible. Presence of disruptive agents, such as 1 M GuCl or 3 M KCl, did not alter markedly the lifetimes but increased the weight of the short-lifetime component. In the same time, the rotational correlation time of this component was dramatically reduced. Taken together, our data suggest that the long-lifetime class could correspond to the tryptophan residues exposed to solvent whereas the short-lifetime class would correspond to the tryptophan residues embedded inside the hydrophobic core holding the helix-turn-helix motif. Destabilization of hydrophobic interactions would lead to an increase in the weight of the latter class for entropic reasons. Analysis of the fluorescence parameters of Trp-43 could provide structural information on the operator binding domain of Tet repressor.

DNA Transposable Elements↗