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Equilibria in 5-S ribosomal RNA secondary structure. Bulges and interior loops in 5-S RNA secondary structure may serve as articulations for a flexible molecule.

The basic assumption in this paper is that the secondary structure of a 5-S ribosomal RNA cannot be represented by a single model. We propose that the molecule can adopt, at least within the ribosome, a series of slightly different structures of nearly equal stability. The different structures arise from the existence of ambiguous base-pairing opportunities in bulged helices and the adjacent interior loops. In eubacterial 5-S RNAs there is one such an area, in eukaryotic 5-S RNAs two such areas that can give rise to structural switches. We explain how a change in secondary structure in these areas may influence the relative orientation of the surrounding helices, in other words how bulges and interior loops may serve as articulations and give rise to a flexible tertiary structure.

Animals↗

Primary and secondary structural patterns in eukaryotic cytochrome P-450 families correspond to structures of the helix-rich domain of Pseudomonas putida cytochrome P-450cam. Indications for a similar overall topology.

An extensive sequence analysis of the eukaryotic cytochrome P-450 (P-450) protein families was conducted with a view to identifying conserved regions that might be related to secondary structural features in the Pseudomonas putida camphor hydroxylase (P-450cam). All sequences available on-line were collected, classified and aligned within families. Distinctively different sequences were chosen from each of seven eukaryotic families, and an unbiased multi-alignment was constructed. Profile patterns of the most conserved regions were generated and screened against the sequence of P-450cam, the structure of which has been elucidated by X-ray crystallography. While some of these profiles did not map on the P-450cam sequence, the structurally most important helices were clearly identified and the correlations were found to be statistically significant. Our analysis suggests that the helix-rich domain with the cysteine pocket and the oxygen-binding site is conserved in all P-450 forms. Helices I and L from P-450cam can be easily identified in all eukaryotic P-450 forms. Other helices which seem to exist in all P-450 forms include helices C, D, G and J. K. In the helix-poor domain of P-450cam, only structures b3/b4 seem to have been conserved. The obvious sequence conservation throughout the helix-rich domain of the P-450cam protein might be expected for a molecular class whose overall topology is preserved. Additional support for the conservation of structure between eukaryotic cytochromes P-450 and P-450cam comes from secondary structure prediction of the eukaryotic sequences.

Amino Acid Sequence↗

Heat-induced synthesis of sigma32 in Escherichia coli: structural and functional dissection of rpoH mRNA secondary structure.

The heat shock response in Escherichia coli depends primarily on the increased synthesis and stabilization of otherwise scarce and unstable sigma32 (rpoH gene product), which is required for the transcription of heat shock genes. The heat-induced synthesis of sigma32 occurs at the level of translation, and genetic evidence has suggested the involvement of a secondary structure at the 5' portion (nucleotides -19 to +247) of rpoH mRNA in regulation. We now present evidence for the mRNA secondary structure model by means of structure probing of RNA with chemical and enzymatic probes. A similar analysis of several mutant RNAs with a mutation predicted to alter a base pairing or with two compensatory mutations revealed altered secondary structures consistent with the expression and heat inducibility of the corresponding fusion constructs observed in vivo. These findings led us to assess the possible roles of each of the stem-loop structures by analyzing an additional set of deletions and base substitutions. The results indicated not only the primary importance of base pairings between the translation initiation region of ca. 20 nucleotides (the AUG initiation codon plus the "downstream box") and the internal region of rpoH mRNA but also the requirement of appropriate stability of mRNA secondary structures for characteristic thermoregulation, i.e., repression at a low temperature and induction upon a temperature upshift.

Base Sequence↗

Secondary structure model of the Mason-Pfizer monkey virus 5' leader sequence: identification of a structural motif common to a variety of retroviruses.

A stable secondary structure model is presented for the region 3' of the primer-binding site to 130 bases into the gag sequence of the prototype type D retrovirus Mason-Pfizer monkey virus. Using biochemical probing of RNA from this region in association with free energy minimization, we have identified a stem-loop structure in the region, which from other studies has been shown to be important for genomic RNA encapsidation. The structure involves a highly stable stem of five G-C pairs terminating in a heptaloop. Comparison of the Mason-Pfizer monkey virus structure with one predicted for squirrel monkey retrovirus demonstrates an identical stem and a common ACC motif in the loop. Free energy studies of the secondary structure of the 5' regions of eight other retroviruses predict stem loops which have similar GAYC motifs. We believe this may represent a common structural and sequence motif which among other functions may be involved in genomic RNA packaging in these viruses.

Animals↗

The development of the Toronto Structured Interview for Alexithymia: item selection, factor structure, reliability and concurrent validity.

BACKGROUND: Assessments of personality constructs increasingly use self-report and structured interview instruments, which allow for a multimethod measurement approach and decrease specific measurement method bias. The aim of this study was to develop a valid and reliable structured interview for assessing the alexithymia construct. METHODS: Sixty interview questions were written initially, each with a set of scoring criteria and prompts and probes to elicit information assisting in the scoring of the respondents' answers. RESULTS: After pilot testing, the number of questions was reduced to 43, which were administered to 136 community participants and 97 psychiatric outpatients. A series of item and scale analyses further reduced the item pool to 24 items. Principal component analysis and confirmatory factor analysis of these 24 items revealed preliminary evidence of a hierarchical, four-factor structure, with four lower factors nested within two higher-order latent factors. This structural configuration resulted in the Toronto Structured Interview for Alexithymia (TSIA) with two domain scales and four facet scales. The TSIA and its six scales demonstrated acceptable levels of interrater, internal, and retest reliability. The TSIA and its scales correlated modestly but significantly with the 20-item Toronto Alexithymia Scale and its three factor scales, providing some support for the concurrent validity of this interview. CONCLUSION: The TSIA appears to be a promising structured interview for assessing alexithymia.

Adult↗

Process and structure: resource management and the development of sub-unit organisational structure.

Resource Management (RM) requires hospital units to manage their work in new ways, and the new management processes affect, and are affected by, organisation structure. This paper is concerned with these effects, reporting on the basis of a three-year evaluation of the national RM experiment that was commissioned by the DH. After briefly indicating some of the major characteristics of the RM process, the two main types of unit structures existing in the pilot sites at the beginning of the experiment, unit disciplinary structure and clinical directorates, are analysed. At the end of the experiment, while clinical directorates had become more popular, another variant, clinical grouping, had replaced the unit disciplinary structure. Both types of structure represent a movement towards sub-unit organisation, bringing the work and interests of the service providers and unit managers closer together. Their properties are likewise analysed and their implications, particularly in terms of training and organisational development (OD), are then considered. The paper concludes by considering the causes for these structural changes, which, in the immediate time-scale, appear to owe as much to the NHS Review as to RM.

Decision Making, Organizational↗

The apical structure in Perophora annectens (tunicate) spermatozoa: fine structure, differentiation and possible role in fertilization.

The apical structure in Perophora annectens spermatozoa is approximately 4 micron in length and it is helically coiled. Its major component is a striated structure, which may be analogous to a perforatorium. The plasmalemma enclosing the anterior quarter of the apical structure is covered by extracellular materials, the anterior ornaments. During spermiogenesis, the apical structure is first recognized as a small blister of the plasmalemma at the apex of the young spermatid. It develops into a conical protrusion and then into a finger-like process (approximately 1 micron in length). This process is transformed into an elongated process (approximately 4 micron in length) with electron-dense material in its core. Finally, the elongated process is helically coiled to form an apical structure in which electron-dense material forms dense striations. Vesicles (50-70 nm in diameter), presumably derived from the Golgi apparatus, have been recognized in the blisters of younger spermatids, and can be followed through to the finger-like process. In the finger-like process these vesicles are transformed into smaller vesicles (20-30 nm in diameter), which probably fuse with the anterior plasmalemma of the finger-like process. This suggests that chorion lysin(s) is associated with the anterior membrane enclosing the apical structure in these spermatozoa.

Animals↗

Structural properties of visual images constructed from poorly or well-structured verbal descriptions.

Previous research has found a linear relation between distance and scanning times for spatial configurations when the spatial configurations were learned perceptually and when they were constructed from well-structured verbal descriptions. The current research replicated the time-distance relation when the images were constructed from repetitions of well-structured descriptions but not when the images were generated from three repetitions of a description that presented information in a random order. Six exposures to the randomly ordered information yielded the expected time-distance relation in image scanning. We posited that additional exposure to the poorly structured information allowed the image to develop the structural coherence and resolution needed to support consistent scanning. Thus, the structure of descriptions can affect the intrinsic structure of images of described objects and hence the mental operations performed subsequently on these images. Another experiment indicated that image coherence and resolution improves even after the verbal description is accurately recalled.

Adult↗

Sequence, structure, and expression of a wasp venom protein with a negatively charged signal peptide and a novel repeating internal structure.

An expression cDNA library prepared from mRNA from the venom gland of a parasitic wasp, Chelonus sp. near curvimaculatus, was screened with polyclonal antibodies against a 33-kDa venom protein from this wasp. Immunoreactive clones were sequenced, yielding a complete inferred sequence for a protein with an NH2 terminus identical with that of the 33-kDa protein. The structure of the cDNA showed an apparent encoded signal peptide, which was unusual in possessing 2 glutamic acid residues juxtapositioned next to, or replacing, the conventional basic residues. The bulk of the mature protein sequence which follows the NH2-terminal, 5000-kDa hydrophobic domain is composed of a dozen tandem repeats of a highly charged, approximately 14-residue sequence, except for the truncated eighth repeat which terminates in the only proline in this large domain. The primary structure is not closely related to any sequence in the GenBank data bank. Secondary structure analysis identified a turn occurring at or near each of 12 invariantly conserved serine residues. Further, the codon used for this serine was invariant, whereas other serines in the protein (including a nearly invariant serine 2 residues away) used various codons. Results of epitope mapping experiments supported a proposed tertiary structure in which the NH2-terminal 5-kDa forms a hydrophobic core, overlain with the charged repeats. Northern analysis of poly(A) RNA from the venom gland of young adult female wasps showed expression of a single 1-kilobase transcript, for which there is no corresponding message in normal or parasitized host larvae. The remarkable structure of this protein and structural data on other wasp venom proteins suggest an evolutionary pattern in which some proteins critical for venom function evolve by internal tandem duplication, and which are secreted after biosynthesis by a different mechanism from that used for proteins with classical signal peptides containing basic residues.

Amino Acid Sequence↗

Structural studies on the glycosylphosphatidylinositol membrane anchor of Trypanosoma cruzi 1G7-antigen. The structure of the glycan core.

The 1G7-antigen is expressed by the infective metacyclic trypomastigote stage of the protozoan parasite Trypanosoma cruzi. The 1G7-antigen is a 90-kDa glycoprotein, present at about 40,000 copies/cell, which is anchored in the plasma membrane via a glycosylphosphatidylinositol (GPI) membrane anchor. The glycan of the GPI anchor has been isolated from immunopurified 1G7-antigen and its structure determined using a combination of methylation linkage analysis and exoglycosidase sequencing. The structure of the glycan is Man alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2. The glucosamine residue is in glycosidic linkage to a phosphatidylinositol moiety. The penultimate nonreducing alpha-Man residue is substituted with phosphate, which is most likely part of an ethanolamine phosphate bridge linking the GPI anchor to the 1G7-antigen polypeptide. The glycan sequence was obtained from 1.1 nmol of glycoprotein isolated from a detergent lysate of whole cells. The procedures reported here represent a high sensitivity protocol for determining GPI glycan structures from small quantities of biological material. The structure of the 1G7-antigen GPI anchor is consistent with the conserved core structure of all GPI anchors analyzed to date and is similar to that of the T. cruzi lipopeptidophosphoglycan. The biosynthesis of GPI anchors and lipopeptidophosphoglycan in T. cruzi is discussed in the light of this structural homology.

Animals↗

Structures of the asparagine-linked oligosaccharide chains of human von Willebrand factor. Occurrence of blood group A, B, and H(O) structures.

The asparagine-linked oligosaccharide chains of human von Willebrand factor (vWF) purified from pooled plasma were quantitatively liberated from the polypeptide moiety by hydrazinolysis. After N-acetylation, these were fractionated by paper electrophoresis and sequential chromatography on lectin-affinity columns of concanavalin A, Phaseolus vulgaris erythrophytohemagglutinin, Datura stramonium agglutinin, Ricinus communis agglutinin 120, and Ulex europaeus agglutinin I and on a Bio-Gel P-4 column. Their structures were investigated by sequential exoglycosidase digestion in conjunction with methylation analysis. The glycoprotein was shown to be unique in its great diversity of oligosaccharide structures. Another noteworthy finding which had not been reported previously was the occurrence of asparagine-linked oligosaccharide chains with blood group A, B, and H(O) structures. In the present study, this glycoprotein was shown to contain mono- (0.4% of the total oligosaccharides), bi-(78.2%), tri- (12.3%), and tetraantennary (2.3%) complex type oligosaccharides in addition to a series of high mannose type oligosaccharides, Man6-9GlcNAc2 (0.8%). Biantennary complex type oligosaccharide chains were those with (8.2%) and without (70.0%) a bisecting GlcNAc residue and approximately 13.2%, 2.2%, and 0.4% of these contained blood group H(O), A, and B structures, respectively. The tri- and tetraantennary complex type chains were those with and without N-acetyllactosamine repeats, and about 13.0% of the triantennary chains without the N-acetyllactosamine repeat contained the blood group H(O) structure. Occurrence of these asparagine-linked oligosaccharides with blood group A and B structures suggest that the repeated use of factor VIII/vWF pooled concentrate for the treatment of hemophiliacs could result in the production of antibodies against vWF with a different blood group from that of the patient, and this development may be pathogenic.

ABO Blood-Group System↗

Structural studies of immunomodulators. Part 2: Crystal structure and conformation of azimexon (BM 12.531) an immunostimulant and an anti-tumor drug.

Azimexon [2-cyanaziridinyl-2-carbamoyl-aziridinyl-1-propane] is a derivative of 2-cyanaziridine. Azimexon is an immunostimulant which shows therapeutic effects in tumor models and experimental infections in mice in vitro, enhances T lymphocyte transformation in vitro and increases phagocytosis of latex particles by mouse peritoneal cells. In cancer patients it increases blood active T rosettes, increases T4/T8 ratio and is used in the treatment of melanomas. The crystal structure analysis of azimexon has been undertaken to study the conformation of the molecule in the solid state as a first step in the investigation of a possible structure-function relationship of immunomodulators. Crystals of azimexon are triclinic, space group P1, with a = 6.342(2), b = 6.804(1), c = 13.106(2) A, alpha = 75.17(1), beta = 89.17(2), gamma = 83.26(2) degrees, V = 542.8 A3, Z = 2, D0 = 1.18 g cm-3 and Dc = 1.189 g cm-3. The structure was solved using CAD-4 data by multisolution techniques and refined to a final R value of 0.057. There are two independent molecules in the asymmetric unit with very different conformations relating the two aziridine rings in the molecule. The mean lengths of C-N and C-C in the aziridine rings are 1.461 and 1.494 A and the configuration of the ring nitrogen is pyramidal. The mean C-C-N and C-N-C bond angles in the ring are 59.2 and 60.6 degrees respectively. The two molecules, which differ significantly in the relative orientation of their aziridine rings, are linked by hydrogen bonding involving the amino group of one molecule as the donor and the cyano nitrogen and carbamoyl oxygen of the other molecule as the acceptor. There are a number of C-H...O and C-H...N interactions in the structure. The mode of action of azimexon is unknown. It has been suggested that azimexon may alkylate DNA. The knowledge of the three-dimensional structure should facilitate distance geometry calculations that would determine which modes of crosslinking would be possible. The end-to-end distances between the two ends of the molecule is 9.0 A which is of the same order as the intrastrand distances in the DNA double helix. The present structural work of azimexon strongly indicates that azimexon crosslinks with DNA using its two reactive groups at its ends. Further study is necessary in order to determine which modes of crosslinking would be possible.

Adjuvants, Immunologic↗

Secondary structure analysis of apolipoprotein II mRNA using enzymatic probes and reverse transcriptase. Evaluation of primer extension for high resolution structure mapping of mRNA.

Primer extension has been employed to locate sites of cleavage made in apolipoprotein II (apo-II) mRNA by structure-specific nucleases. This approach permits structural analysis of specific mRNAs within a complex population. Electrophoretic analysis of cDNAs synthesized from T1 RNase-treated and mock-treated apo-II mRNA revealed that most cleavage sites can be mapped with single nucleotide accuracy. However, some T1 RNase-dependent cDNAs demonstrated mobilities corresponding to one nucleotide longer than the mRNA template, suggesting that reverse transcriptase can add a single nucleotide to full-length cDNAs in a template-independent reaction. This approach has been used to map double-stranded and single-stranded accessible domains of the 3' noncoding region of apo-II mRNA with cobra venom, T1, and S1 ribonucleases. Cleavage profiles of apo-II mRNA renatured under a variety of buffer and temperature conditions were identical and in no case was overlap observed between sites of cleavage by double strand- and single strand-specific enzymes. These results suggest that apo-II mRNA possesses a predominant, stable secondary structure. A computer-generated structure model, consistent with these nuclease cleavage data, is presented. In addition to the analysis of mRNA higher order structure in mixed RNA populations, this approach also appears suitable for the analysis of protein-mRNA interactions. Termination sites of incomplete cDNAs produced when untreated or mock-treated RNA is used as a template for primer extension were also mapped. This analysis revealed an over-representation of termination at the dinucleotides CA and CU, suggesting that termination of some incomplete apo-II cDNAs is related to primary and not secondary structure. Such sequence dependence could reflect in vivo degradation by an endogenous cytidine-specific nuclease.

Animals↗

[A theoretical analysis of structural restructuring during formation of secondary RNA structures].

An improved method for predicting the RNA secondary structure is proposed. The process of self-organization of structure is considered as a Markov chain. The kinetic of secondary structure is analysed by the Monte-Carlo method. The topological compatibility of helices is discussed. On the base of analysis it follows that the dynamical process of secondary structure formation is so that it is impossible to define a static set of complementary pairs. The method was used for predicting the mRNA secondary structure of a series of recombinant plasmids, containing the cro gene. The observed variation in expression can be explained by secondary structure.

Computer Simulation↗

Secondary structure and limited three-dimensional structure of bovine amelogenin.

Secondary structural features of bovine amelogenin, a hydrophobic protein of developing enamel implicated in ename mineralization, are derived using 2D NMR spectroscopy in solution and molecular mechanics-dynamics studies. A beta-turn: beta-sheet model with some "unordered" segments was previously proposed from circular dichroism, Fourier-transform infrared and Raman spectroscopy augmented by Chou-Fasman predictive algorithm. The proposed structure contains a repetitive beta-turn segment, "beta-spiral" between Gln112 and Leu138 residues containing a (Pro, Leu, Gln) rich segment. The beta-spiral structure offers a probable site for interaction of Ca++ ions. Assignment of proton resonances using 2D COSY spectroscopy is presently in progress. Preliminary 2D NOESY spectra have revealed the presence of Tyr residues (TRAP segment) on the surface of amelogenin molecule and clusters of cross peaks reminiscent of beta-turns and sheets which are consistent with the primary structure and proposed secondary structures of amelogenin. The channel-like beta-spiral structure embedded in amelogenin provides a novel mechanism for trapping of Ca++ ions and their passage for a hydrophobic protein sparse in Ser(P) and charged amino acid residues.

Amelogenin↗

Studies on the structure of avian myeloblastosis virus (AMV) RNA. III. Electron microscopic definition of secondary structure.

The secondary structure of avian myeloblastosis virus (AMV) RNA was characterized by electron microscopy under moderately denaturing spreading conditions. Under denaturation by aqueous 44% formamide or 77% formamide in the presence of salts, partly stretched RNA molecules with measurable double-stranded regions were observed. This approach allowed the localization from 5 to 11 regions of preserved secondary structure on AMV RNA molecules. Topographic analysis revealed a nonrandom occurrence of stable secondary structures in several prevalent regions. These regions with higher secondary structure stability revealed certain similarity to hairpin structures localized by electron microscopy on Rous sarcoma virus RNA or to highly structured regions found on this RNA by T1 ribonuclease oligonucleotide analysis.

Avian Leukosis Virus↗

[Dynamic structures and nonlinear spectral transformations based on structure interaction reactions].

A notion of dynamic excitation structure (DES) is introduced. Such structure is a mobile picture which adequately reflects external nonstationary excitation spectra. The stimulated structure transitions are possibly caused by DES generation if the substrate structure has space-time polimorphism. The stimulated associative transitions can be a basis of structure-holographic information processing in biological systems. Possibility of associative transformations of nonstationary processes is discussed and elements of associative structure logic are given. The model of semantic organization of nonequilibrium cooperative systems is discussed. This model may be useful for explaining the gnosiological activity of biological objects.

Kinetics↗

[Determination of the secondary structure of proteins from their circular dichroism spectra. I. Protein reference spectra for alpha-, beta- and irregular structures].

It is shown that to obtain the protein-derived basic CD spectra for alpha-helical, beta-structural and irregular regions it is necessary to use a common criterion for isolation of secondary structures for all the reference proteins. Using the "rigid" criterion proposed by Finkelstein, Ptitsyn, Kozytsyn and the "mild" one (as proposed by Levitt and Greer) isolation of the alpha- and beta-structural regions for 5 reference proteins (myoglobin, lysozyme, ribonuclease A, papain, lactate dehydrogenase) has been carried out. Using the f alpha, f beta and f irregular thus obtained and the experimental CD spectra of these proteins, the basic CD spectra for alpha-helical, beta-structural and irregular regions in the proteins have been calculated. It is shown that the use of criterions common for all the proteins leads to good agreement between the calculated basic CD spectra for alpha-helical, beta-structural and irregular forms and the CD spectra of polylysine in the corresponding conformations. The secondary structure of the proteins studied has been analysed using the new protein-derived basic spectra and fairly good quantitative agreement with the X-ray data was achieved.

Animals↗