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At least 127 records · Page 7Linked to original sources
PubMed1991

Structural analysis of two-dimensional arrays of cholera toxin B-subunit.

Two-dimensional arrays of cholera toxin B-subunit (CTB) have been obtained by specific interaction with lipid films, as described by Ludwig et al. (1986). The relationship between two types of array, of either rectangular or hexagonal geometry, was analyzed using crystallographic methods of electron image analysis. Our results showed that the type of array obtained was highly dependent on the negative stain used and that both arrays presented related lattice parameters, indicating that they originated from a common unstained structure. Image analysis of hexagonal arrays at 17 A resolution revealed variable CTB projected structures, ranging from annularly symmetric particles to highly asymmetric particles, very distinct from the pentameric structure resolved from rectangular crystals. The present data suggest that hexagonal arrays result from an imperfect staining of CTB rectangular crystals. The staining distortion is such that the stain layer does not match faithfully the pentameric protein distribution whereas the regular organization of the specimen is maintained.

Cholera Toxin↗
PubMed1992

Secondary structural analysis of two recombinant murine proteins, interleukins 1 alpha and 1 beta: is infrared spectroscopy sufficient to assign structure?

The secondary structure for two murine recombinant proteins, interleukins 1 alpha and 1 beta (rmIL-1 alpha and -1 beta), has been analyzed by Fourier transform infrared (IR) spectroscopy and then compared to results obtained by X-ray diffraction, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. The IR results obtained here for rmIL-1 alpha and -1 beta suggested that their secondary structures consisted predominantly of beta-sheets or strands. However, the analysis also revealed a significant absorption band near 1656 cm-1, which is typically assigned to alpha-helical or random structures. When these same murine polypeptides were analyzed by CD, no evidence of alpha-helical structures was observed. Further, published X-ray diffraction and NMR studies characterizing the human forms of IL-1 alpha and -1 beta indicate the absence of alpha-helices and that the human proteins are composed mainly of beta-strands (i.e., greater than 55%), with approximately 24% of the amino acids involved in large loops connecting the strands. The murine IL-1 proteins, when compared to their respective human counterparts, each show greater than 80% sequence homology. Given this fact, the CD analyses, and the result that this IR band amounted to 21% of the overall integrated area, the absorption peak at 1656 cm-1 was attributed to the presence of large loops rather than to alpha-helical or random structures. Such a structural assignment appears reasonable and is totally consistent with the established existence of large loops in the human forms as well as in other proteins found to fold similarly (viz., human bFGF).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

[Improved reproducibility of computer-assisted structural analysis of the optic papilla].

Longitudinal monitoring of the optic disc structure by means of automated nervehead analysis (Optic Nerve Head Analyzer, ONHA) can facilitate the diagnosis of ongoing glaucoma. Until recently, however, longitudinal disc monitoring was complicated by considerable fluctuations of the results measured with the Optic Nerve Head Analyzer. Since March/1990 there is a new software version of the equipment (ONHA-plus). The determination of disc parameters using ONHA-plus proved to be significantly more reliable than the determination using the former ONHA: the mean fluctuation of the neuroretinal rim area measured on 18 normal eyes was 0.07 mm2 (ONHA-plus) compared to 0.094 mm2 (ONHA); the variation coefficient dropped from 7.9% (ONHA) to 4.6% (ONHA-plus). A further improvement of the reproducibility can be obtained by double examinations, because the mean examination time using ONHA-plus is about half as long as the examination time using ONHA. Moreover, ONHA-plus enables the recognition and elimination of unreliable results caused by poor image quality. Due to this increased reliability the ONHA-plus system is a considerably improved equipment for the clinical monitoring of glaucoma patients.

Glaucoma↗
PubMed1989

Protein and carbohydrate structural analysis of a recombinant soluble CD4 receptor by mass spectrometry.

The primary structure of a soluble form of the CD4 receptor (sCD4) expressed in Chinese hamster ovary cells has been confirmed by mass spectrometric peptide mapping and and tandem mass spectrometry. These studies corroborated 95% of the 369-amino acid-long sequence and established the fidelity of translation of the NH2 and COOH terminal including the absence of "ragged ends." The arrangement of the three disulfide bonds in recombinant sCD4 was also established by mass spectrometry and comparative high performance liquid chromatography mapping and shown to be identical to that expected from previous studies of intrachain disulfide bonding in T4 antigens derived from sheep and mouse. No other arrangements of disulfides were detected. Carbohydrate mapping by mass spectrometry was used to establish that both potential Asn-linked glycosylation sites in sCD4 (Asn271 and Asn300) have oligosaccharides attached. Structural characterization by mass spectrometry and methylation analysis of the heterogeneous family of oligosaccharides at each of the specific attachment sites indicates that the major components of both families of oligosaccharides have the following biantennary structures: (Formula, see text) where m + n = 0-2, and x = 0,1. Minor carbohydrate components having three N-acetylneuraminic acid (NeuAc) groups and an additional hexose-hexosamine unit were detected by high performance anion-exchange chromatography.

Amino Acid Sequence↗
PubMed1992

Structural analysis of lipooligosaccharide produced by Neisseria gonorrhoeae, strain MS11mk (variant A): a precursor for a gonococcal lipooligosaccharide associated with virulence.

We studied the structure of the lipooligosaccharide (LOS) that is produced by a variant A of strain MS11mk. This variant produces a single LOS that is recognized by monoclonal antibody (MAb) 2-1-L8. In a recent study of the pathogenesis of Neisseria gonorrhoeae in male volunteers, variant A gave rise to other phase variants that produce higher molecular weight LOSs, and these LOS were associated with virulence. Definition of the structure of the variant A LOS is important to understand the biosynthesis of LOS and its expression in vivo. The dephosphorylated oligosaccharide (OS) structure derived from the variant A LOS was analyzed by two-dimensional NMR and methylation analysis. The OS structure was found to be a truncated form of the LOS produced by strain F62 [Yamasaki et al. (1991) Biochemistry 30, 10566-10575]; the variant A OS is a hexamer, a beta-lactosyl residue linked to a tetrasaccharide: Gal beta 1-->4Glc beta 1-->4[GlcNAc alpha 1-->2Hep alpha 1-->3]Hep alpha 1-->KDO. We determined that the variant A LOS is a precursor for the synthesis of higher MW LOS. We also studied expression of the MAb 2-1-L8-defined epitope present on the variant A LOS. Our data indicate that the MAb-defined epitope is not a linear beta-lactosyl residue but its specificity is directed toward the phosphorylated GlcNAc-Hep-Hep residue. Since this MAb binds to gonococci, at least part of the phosphorylated diheptose area is exposed on the gonococcal surface.

Antibodies, Monoclonal↗
PubMed1991

Rat urate oxidase: cloning and structural analysis of the gene and 5'-flanking region.

The structural gene (UOX) encoding rat urate oxidase (UOX) spans at least 23 kb and is composed of eight exons and seven introns. All of the exon-intron splice junction sequences conformed to the GT/AG consensus established for eukaryotic genes. The transcription start point (tsp) was determined using S1-type nuclease protection riboprobe, and assigned to an adenine 54 nucleotides (nt) upstream of the ATG start codon. A 456-bp 5'-terminal fragment, starting at the ATG codon, carries a putative TATA (ATAAAA) sequence at -32, and two putative 'CAAT box' sequences at -62 and -71 bp upstream from the tsp. No sequence resembling 'GC' box hexanucleotides (GGGCGG or CCGCCC) was found. The structural features of the 5'-flanking region of the UOX gene are distinct from the 5'-flanking sequences of peroxisomal beta-oxidation system genes which contain one or more 'GC' box elements but lack TATA- and CAAT-like features [Osumi et al., J. Biol. Chem. 262 (1987) 8138-8143; Ishii et al., J. Biol. Chem. 262 (1987) 8144-8150]. The 5'-flanking region of the UOX gene reveals a sequence, TTAGTAATT at nt -276 from the tsp, which appears to be complementary to the underlined part of the liver-specific LF-B1/HNF-1 consensus sequence, GTTAATNATTAAC (where N = A, C, T, G or no nt).

Amino Acid Sequence↗
PubMed1991

Structural analysis of cataract incision construction.

This report examines the structural components of incision construction for cataract surgery so the causes of corneal instability and astigmatism can be understood. Changing the shape of the external incision, from limbus-parallel to linear to curved away from the limbus, results in a more stable external incision. The internal entry incision is more directly associated with corneal instability, however, and its construction and closure are crucial. Corneal stability improves as the closure changes from radial to horizontal to corneal valve autoclosure. Hyphema rates also improve with this progression because blood is directed into the anterior chamber with posterior incision closure, but out of the eye with anterior corneal valve closure.

Astigmatism↗
PubMed1992

Structural analysis of ternary complexes of vaccinia RNA polymerase.

The structure of the elongation complex of vaccinia RNA polymerase halted at discrete template positions was examined by DNase I footprinting. The leading edge of the footprint bore a constant relationship to the catalytic template position, being 22-24 nucleotides (nt) in advance on the nontemplate strand and 17 nt on the template strand. DNase hypersensitivity of the nontemplate strand at the leading edge suggested that the DNA might be distorted as it entered the polymerase molecule. The region of DNA unwinding at the transcription bubble extended at least 12 nt 5' from the catalytic center, as indicated by the reactivity of adenosine residues to diethylpyrocarbonate. Cu-phenanthroline-hypersensitive sites located 13 nt 5' and 4 nt 3' of the growing point appeared to demarcate the margins of the bubble. Strand asymmetry of chemical modification within the bubble was consistent with an RNA-DNA hybrid of no more than 10 base pairs.

Base Sequence↗
PubMed1989

In situ structural analysis of microsomal UDP-glucuronyltransferases by radiation inactivation.

The structure of the UDP-glucuronyltransferases in microsomes from guinea pig and rat liver was examined in situ by radiation inactivation analysis. The p-nitrophenol conjugating activity of guinea pig microsomes increased at lower doses of radiation; at higher doses (greater than or equal to 36 megarads), activity showed a first order decline yielding a target size of 71 +/- 9 kDa. Treating microsomes with Triton X-100 eliminated the activation seen at lower doses of radiation and yielded a simple exponential decrease in activity which gave a larger target size (95 +/- 18 kDa). A monoexponential decrease in activity was seen in sonicated microsomes, at greater than or equal to 36 megarads. The same response was obtained when the reaction was assayed in the reverse direction. The estrone conjugating activity of guinea pig microsomes was similarly activated at lower doses of radiation and declined at higher doses (greater than or equal to 36 megarads), with a target size of 57 +/- 11 kDa. Allosteric activation of the enzyme by UDP-N-acetylglucosamine was eliminated by lower doses of radiation. Thus, activation of the enzyme by radiation, detergent, sonication, and UDP-N-acetylglucosamine appear to be interdependent. These activations are postulated to be due to the existence of the enzyme in an oligomeric form which can be dissociated into monomers with higher activity. The same biphasic activation-inactivation curves were obtained for p-nitrophenol conjugation in rat liver microsomes. The target sizes were 54 +/- 8 kDa (p-nitrophenol in the forward direction) and 66 +/- 10 kDa (p-nitrophenol in the reverse direction). Thus, the enzyme appears to be smaller in rat liver as compared with guinea pig liver. Lithocholate glucuronidating activity in rat liver microsomes (at greater than 36 megarads) gave a target size of 74 +/- 1 kDa.

Animals↗
PubMed2026

Characterisation of Carbapenem-Resistant Raoultella planticola and Structural Analysis of NDM Composite Plasmids.

OBJECTIVE: This study aimed to investigate the molecular characteristics, resistant plasmid structures and phylogeny of a carbapenem-resistant Raoultella planticola (CRRP) strain from a patient with pneumonia to inform antimicrobial resistance control strategies. METHODS: We performed strain identification using MALDI-TOF MS, the BD Phoenix 100 system and whole-genome sequencing (WGS). We assessed antimicrobial susceptibility and resistance gene transfer using PCR, conjugation and stability assays, plasmid structure using a bioinformatics tool and phylogeny using a core-genome phylogenetic tree. RESULTS: WGS confirmed the isolate as R. planticola (average nucleotide identity (ANI) > 98.9% with reference type strains), co-harbouring blaKPC-2 and blaNDM-1. It was resistant to 19 antimicrobial agents and susceptible to only polymyxin, amikacin and chloramphenicol. Resistance genes were present on two conjugative plasmids: pzwx_KPC (IncFIA) and pzwx_NDM (a novel repFIB/repHI5B hybrid assembled via non-homologous end joining). Both plasmids demonstrated efficient transfer and stable inheritance over 12 passages. pzwx_KPC was highly homologous to plasmids from Klebsiella pneumoniae. Phylogenetic analysis revealed the closest relationship with German R. planticola strains. CONCLUSION: CRRP carries highly transmissible and stable resistance plasmids. Strengthened monitoring in immunocompromised patients and improved environmental disinfection are recommended. The risk of misidentification by automated systems underscores the importance of WGS for accurate pathogen identification.

Carbapenem resistance↗
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↗
PubMed1991

Crystal structure analysis of the B-DNA dodecamer CGTGAATTCACG.

The crystal structure of the DNA dodecamer C-G-T-G-A-A-T-T-C-A-C-G has been determined at a resolution of 2.5 A, with a final R factor of 15.8% for 1475 nonzero reflections measured at 0 degrees C. The structure is isomorphous with that of the Drew dodecamer, with the space group P2(1)2(1)2(1) and cell dimensions of a = 24.94 A, b = 40.78 A, and c = 66.13 A. The asymmetric unit contains all 12 base pairs of the B-DNA double helix and 36 water molecules. The structure of C-G-T-G-A-A-T-T-C-A-C-G is very similar to that of C-G-C-G-A-A-T-T-C-G-C-G, with no major alterations in helix parameters. Water peaks in the refined structure appear to represent a selection of peaks that were observed in the Drew dodecamer. The minor-groove spine of hydration at 2.5 A is fragmentary, but as Narendra et al. (1991) [Biochemistry (following paper in this issue)] have observed, lowering the temperature leads to a more complete representation of the spine.

Base Sequence↗
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↗
PubMed1991

Three-dimensional structural analysis of the group B polysaccharide of Neisseria meningitidis 6275 by two-dimensional NMR: the polysaccharide is suggested to exist in helical conformations in solution.

The solution conformations of the group B polysaccharide of Neisseria meningitidis were analyzed by DQF-COSY and pure absorption 2D NOE NMR with three mixing times. The pyranose ring of the sialic acid residue was found to be in the 2C5 conformation. The DQF-COSY analysis indicated that the orientations of H6 and H7 and of H7 and H8 are both gauche. In order to overcome the difficulties in analyzing the NOE data due to the two sets of proton overlaps, molecular modeling of alpha-2,8-linked sialic acid oligomers was carried out to investigate possible conformers, and theoretical NOE calculations were performed by using CORMA (complete relaxation matrix analysis). Our analysis suggests that the polysaccharide adopts helical structures for which the phi (defined by O6-C2-O8-C8) and psi (C2-O8-C8-C7) angles are in the following ranges: phi -60 to 0 degrees, psi 115-175 degrees or phi 90-120 degrees, psi 55-175 degrees. The weak affinity of anti-B antibodies for smaller alpha-2,8-linked oligosaccharides may be due to the fact that such oligomers are more flexible and may not form an ordered structure as the poly(sialic acid) does.

Antigens, Bacterial↗
PubMed1985

Human T-cell lymphotropic virus type III: immunologic characterization and primary structure analysis of the major internal protein, p24.

The major internal structural protein of human T-cell lymphotropic virus type III (HTLV-III), a virus etiologically implicated in acquired immunodeficiency syndrome (AIDS), was purified to homogeneity. This 24,000-molecular-weight protein (p24) was shown to lack immunologic cross-reacting antigenic determinants shared by other known retroviruses, including HTLV-I and HTLV-II, with the exception of equine infectious anemia virus (EIAV). A broadly reactive competition immunoassay was developed in which antiserum to EIAV was used to precipitate 125I-labeled HTLV-III p24. Although the major structural proteins of HTLV-III and EIAV competed in this assay, other type B, C, and D retroviral proteins lacked detectable reactivity. Thus, HTLV-III is more related to EIAV than to any other retroviruses. That the HTLV-III isolate is very distinct from HTLV-I and HTLV-II was further confirmed by the amino acid compositions of the major internal antigens of all three isolates. Moreover, comparison of the amino-terminal amino acid sequence of HTLV-III p24 with analogous sequences for HTLV-I and HTLV-II p24 showed that these proteins do not share significant sequence homology. In an attempt to evaluate immune response in individuals exposed to HTLV-III, sera from AIDS and lymphadenopathy syndrome patients as well as from clinically normal blood donor controls were tested for antibodies to HTLV-III p24. The results showed that sera from 93% of lymphadenopathy syndrome patients and 73% of AIDS patients exhibited high-titered antibodies to HTLV-III p24. In contrast, none of the normal control sera showed detectable reactivity to HTLV-III p24.

Acquired Immunodeficiency Syndrome↗