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PubMed1990

[Structural analysis of melanocytic pigment nevi using epiluminescence microscopy. Review and personal experiences].

Epiluminescent microscopy is now used frequently for the differential diagnosis of pigmented skin lesions. In order to improve the distinction between benign and malignant melanocytic tumours it seemed advisable to develop a standardized pathway of epiluminescent microscopical analysis and to determine the frequency of structures recognizable with the microscope. A total of 600 melanocytic lesions were examined by epiluminescent microscopy, photographed and classified histologically after excision, revealing 426 naevocellular naevi and 174 melanoma. The experience achieved during the course of the investigation was used as the basis of a procedure for stepwise analysis of keratin layer, pigment structures and blood vessels. The most important findings are described and referred to malignancy. The photographs were analysed for the frequency of occurrence of various pigment structures in different types of lesions. The results differ in several aspects from previous findings.

Diagnosis, Differential↗
PubMed1995

In vivo structural analysis of spliced leader RNAs in Trypanosoma brucei and Leptomonas collosoma: a flexible structure that is independent of cap4 methylations.

The formation of the mRNA 5' end in trypanosomatid protozoa is carried out by trans-splicing, which transfers a spliced leader (SL) sequence and its hypermethylated cap (cap4) from the SL RNA to the pre-mRNA. Previous in vitro studies with synthetic uncapped RNAs have shown that the SL sequence of Leptomonas collosoma can assume two alternate conformations, Form 1 and Form 2, with Form 1 being the dominant one. To gain information about the structure of the SL RNA in vivo, in its protein-rich environment, we have used permeable Trypanosoma brucei and L. collosoma cells for chemical modification experiments. We introduce the use in vivo of the water-soluble reagents CMCT and kethoxal. In contrast to the in vitro results, the Form 2 secondary structure predominates. However, there are chemically accessible regions that suggest conformational flexibility in SL RNPs and a chemically inaccessible region suggestive of protection by protein or involvement in tertiary interactions. Using complementary 2'-O-methyl RNA oligonucleotides, we show that T. brucei SL RNA can be induced to switch conformation in vivo. SL RNA stripped of proteins and probed in vitro does not display the same Form 2 bias, indicating that SL RNA structure is determined, at least in part, by its RNP context. Finally, the methyl groups of the cap4 do not seem to affect the secondary structure of T. brucei SL RNA, as shown by chemical modification of undermethylated SL RNA probed in vivo.

Aldehydes↗
PubMed1983

The carbohydrate of human thrombin: structural analysis of glycoprotein oligosaccharides by mass spectrometry.

The carbohydrate structure of human thrombin has been determined by direct probe mass spectrometry of the oligosaccharides released by trifluoroacetolysis from the asialo glycoprotein. The free oligosaccharides were studied as permethylated and N-trifluoroacetylated oligosaccharide alditols. The structure was confirmed by sequential exoglycosidase digestion of intact thrombin and sugar and methylation analysis of the oligosaccharides by gas-liquid chromatography-mass spectrometry. The results indicate the following structure: (formula; see text) with Fuc present on only about 50% of the oligosaccharides.

Carbohydrate Sequence↗
PubMed1992

Immunochemical and structural analysis of the O polysaccharides of Salmonella zuerich [1,9,27,(46)].

Salmonella zuerich [1,9,27,(46)] has been shown to exhibit two levels of structural heterogeneity. The bacterium carries two distinct O-polysaccharide molecules with and without O:factor 1. Both sets of molecules (1+ and 1-) carry the two O:factors 27 and 46 on the same O chain, but they are expressed unevenly; in contrast to factor 27, O:factor 46 is always weakly expressed. Part of this weak expression was thought to be due to strong inhibition of factor 46 by factor 1. In this study, serological analysis gave more detailed information on the sizes of the different O:factor epitopes. Structural analysis of S. zuerich O polysaccharides showed that they are constructed of the expected chemical sequences characteristic of factors 1, 9, 27, and 46. No modification in the sugar sequence could account for the weak expression of O:factor 46. Factors 27 and 46 are present on oligosaccharides carrying either an alpha-Man (factor 27) or a beta-Man (factor 46) residue. In S. zuerich, the alpha-Man configuration is predominant, corroborating the expression of strong factor 27 and weak factor 46 on the bacteria. Questions raised by the existence of such heterogeneous O polysaccharides on the specificity of the O chain polymerase, as well as the place of S. zuerich in Salmonella evolution, are dicussed in this paper.

Carbohydrate Sequence↗
PubMed1995

Mechanism of post-segregational killing: secondary structure analysis of the entire Hok mRNA from plasmid R1 suggests a fold-back structure that prevents translation and antisense RNA binding.

The hok/sok system of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells. The Hok mRNA is very stable and can be translated into Hok killer protein. Translation of the Hok mRNA is inhibited by the small unstable Sok antisense RNA. Translation of hok is coupled to an overlapping reading frame termed mok. Translation of mok is tightly regulated by Sok RNA, and Sok RNA thus regulates hok translation indirectly through mok. The rapid decay of Sok RNA explains the onset of Hok synthesis in newborn plasmid-free segregants. However, a second control level is superimposed on this simple induction scheme, since the full-length Hok mRNA was found to be translationally inactive whereas a 3'-end truncated version of it was active. We have therefore previously suggested, that the 3'-terminal region of the full-length Hok mRNA encodes an element which prevents its translation. This element was termed fbi (fold-back inhibition). Here we describe the in vitro secondary structure of the entire Hok mRNA. Our results suggest a closed structure in which the 3'-end of the full-length Hok mRNA folds back onto the translational initiation region of mok. This structure explains why full-length Hok mRNA is translationally silent. The proposed structure was further supported by results obtained using mutations in the 3'-end fbi element. These "structure closing" mutations affected the structure much further upstream in the mok translational initiation region and concomitantly prevented antisense RNA binding to the same region of the mRNA. These results lend further support to the induction model that explains onset of Hok mRNA translation in plasmid-free segregants. The most important regulatory element in this model is the FBI structure formed between the 3'-end and the mok translational initiation region. This structure renders Hok mRNA translationally inactive and prevents antisense RNA binding, thus allowing the accumulation of a pool of mRNA which, by slow 3'-end processing, is activated in plasmid-free segregants, eventually leading to the elimination of these cells.

Bacterial Proteins↗
PubMed1991

Structural analysis of Bacillus licheniformis 86 surfactant.

A tentative structure and composition of a surfactant, BL-86, produced by Bacillus licheniformis 86 is described. The surfactant is a mixture of lipopeptides with the major components ranging in size from 979 to 1091 Da and varying in increments of 14 Da. The variation in molecular weight represents changes in the number of methylene groups in the lipid and/or peptide portion of the surfactant. There are 7 amino acids per molecule. The peptide portion is composed of the following amino acids: glutamic acid or glutamine (glx), aspartic acid or asparagine (asx), valine, leucine, and isoleucine at a ratio of 1.0:1.0:1.4:3.0:0.6, respectively. The leucine is present as both the D and L isomers at a ratio of about 2:1, respectively. Forty percent of the molecules contain L-valine instead of L-isoleucine. The glx and asx are present as a combination of L-glutamic acid and L-asparagine and/or L-glutamine and L-aspartic acid. The N-terminus of the peptide is blocked, most likely by a peptide bond to the lipid portion. An ester carbonyl structure is present, which could be a part of a lactone ring connecting the beta position of the lipid to one of the carbonyl groups in the peptide. The lipid portion is composed of, on average, 8-9 methylene groups, and contains a mixture of linear and branched tails. Results of DCI-MS and FAB-MS analyses, as well as surface tension measurements, of purified BL-86 HPLC fractions support the proposed composition.

Amino Acid Sequence↗
PubMed1989

Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution.

The structure of human lactoferrin has been refined crystallographically at 2.8 A (1 A = 0.1 nm) resolution using restrained least squares methods. The starting model was derived from a 3.2 A map phased by multiple isomorphous replacement with solvent flattening. Rebuilding during refinement made extensive use of these experimental phases, in combination with phases calculated from the partial model. The present model, which includes 681 of the 691 amino acid residues, two Fe3+, and two CO3(2-), gives an R factor of 0.206 for 17,266 observed reflections between 10 and 2.8 A resolution, with a root-mean-square deviation from standard bond lengths of 0.03 A. As a result of the refinement, two single-residue insertions and one 13-residue deletion have been made in the amino acid sequence, and details of the secondary structure and tertiary interactions have been clarified. The two lobes of the molecule, representing the N-terminal and C-terminal halves, have very similar folding, with a root-mean-square deviation, after superposition, of 1.32 A for 285 out of 330 C alpha atoms; the only major differences being in surface loops. Each lobe is subdivided into two dissimilar alpha/beta domains, one based on a six-stranded mixed beta-sheet, the other on a five-stranded mixed beta-sheet, with the iron site in the interdomain cleft. The two iron sites appear identical at the present resolution. Each iron atom is coordinated to four protein ligands, 2 Tyr, 1 Asp, 1 His, and the specific Co3(2-), which appears to bind to iron in a bidentate mode. The anion occupies a pocket between the iron and two positively charged groups on the protein, an arginine side-chain and the N terminus of helix 5, and may serve to neutralize this positive charge prior to iron binding. A large internal cavity, beyond the Arg side-chain, may account for the binding of larger anions as substitutes for CO3(2-). Residues on the other side of the iron site, near the interdomain crossover strands could provide secondary anion binding sites, and may explain the greater acid-stability of iron binding by lactoferrin, compared with serum transferrin. Interdomain and interlobe interactions, the roles of charged side-chains, heavy-atom binding sites, and the construction of the metal site in relation to the binding of different metals are also discussed.

Amino Acid Sequence↗
PubMed1977

Structural analysis of the ridge count data of Australian Europeans using multivariate analysis.

Multivariate analyses are used to study the structural patterns of ridge counts of fingers. There is a significant difference between the ridge counts of two hands, chiefly due to the first and fourth fingers. There is asymmetry in the covariance matrix of the ridge counts. The correlations between fingers are significant and the decrease in correlation with increasing distance between fingers is not significant.

Australia↗
PubMed1990

Engineering a heavy atom derivative for the X-ray structure analysis of cyclodextrin glycosyltransferase.

Based on a preliminary structural model of cyclodextrin glycosyltransferase from Bacillus circulans (EC 2.4.1.19), Ser428 and Ser475 of the enzyme were mutated to cysteines in order to produce suitable heavy atom derivatives. Mutant Ser475----Cys could not be expressed as protein. Mutant Ser428----Cys was expressed in Escherichia coli and purified. It crystallized isomorphously and gave rise to a mercury derivative that improved the electron density map. The structural results show that the new mercury-binding site is in a pocket at the protein surface.

Bacillus↗
PubMed1992

The S-layer of Caulobacter crescentus: three-dimensional image reconstruction and structure analysis by electron microscopy.

The regular surface protein structure (S-layer) of Caulobacter crescentus was analyzed by electron microscopy and three-dimensional image reconstruction to a resolution of 2 nm. Projections showed that the S-layer is an array of ring structures, each composed of six subunits that are arranged on a lattice with p6 symmetry. Three-dimensional reconstructions showed that the ring subunits were approximately rod-shaped structures and were perpendicular to the plane of the array, with a linker arm emanating from approximately the middle of the rod, accounting for the connections between the rings. The calculated subunit mass was ca. 100 kDa, very close to the size of RsaA (the protein known to be at least the predominant species in the S-layer) predicted from the DNA sequence of the rsaA gene. The core region of the rings creates an open pore 2.5 to 3.5 nm in diameter. The size of the gaps between the neighboring unit cells is in the same range, suggesting a uniform porosity predicted to exclude molecules larger than ca. 17 kDa. Attempts to remove membrane material from S-layer preparations with detergents revealed that the structure spontaneously rearranged into a mirror-image double layer. Negative-stain and thin-section electron microscopy examination of colonies of C. crescentus strains with a mutation in a surface molecule involved in the attachment of the S-layer showed that shed RsaA protein organized into large sheets. The sheets in turn organized into stacks that tended to accumulate near the upper surface of the colony. Image reconstruction indicated that these sheets were also precise mirror-image double layers, and thickness measurements obtained from thin sections were consistent with this finding. The sheets were absent when these mutant strains were grown without calcium, supporting other data that calcium is involved in attachment of the S-layer to a surface molecule and perhaps in subunit-subunit interactions. We propose that when the membrane is removed from S-layer fragments by detergents or the attachment-related surface molecule is absent, the attachment sites of the S-layer align precisely to form a double layer via a calcium interaction.

Bacterial Outer Membrane Proteins↗
PubMed1976

Shapes of carcinogenic benz[alpha]anthracenes: an x-ray crystal structure analysis of 12-methylbenz[alpha]anthracene.

The crystal structure of the moderately active carcinogen 12-methylbenz[alpha]anthracene (12-MBA) has been determined by application of direct methods to X-ray single-crystal diffraction data. Least-squares refinement to a residual R = 0.09 over 929 independent reflections enabled carbon positions to be established with apparent e.s.d.s. of atomic coordinates about 0.008 A. Deviation from planarity is exemplified by the 15.5 degrees inclination of the benz ring (A) to the anthracene nucleus and by the 0.89 A distance of the methyl carbon out of the best plane through the whole benzanthracene nucleus. Comparison with the structure of the highly carcinogenic 7,12-dimethylbenz[alpha]anthracene (7,12-DMBA), and with the recently solved structures of the weak carcinogen 1-MBA and the extremely weak carcinogen 1,12-DMBA, shows a close similarity in the anthracene parts; in 1-MBA, and 1,12-DMBA, the phenanthrenic K-region bond is close to 1.34 A and the M-region bond about 1.38 A. In 12-MBA, overcrowding in the 'bay' region causes the central anthracene ring C and the benz ring A each to be bent about 10 degrees in opposite directions from the phenanthrenic B ring, much as in 1-MBA and 7,12-DMBA, but less than in 1,12-DMBA; the 12-methyl carbon lies about the same distance (0.55 A) above the anthracene plane in 12-MBA as in 1,12-MBA and 7,12-DMBA.

Benz(a)Anthracenes↗
PubMed1988

[X-ray crystallographic studies of diclofenac-sodium--structural analysis of diclofenac-sodium tetrahydrate].

The crystal structure of the hydrate of the antiphlogistic drug diclofenac sodium was determined to explain the unusual dissolution behaviour in ethyl acetate. The compound crystallizes in the monoclinic space group P2(1)/m with a = 9.464(2), b = 39.405(7), c = 9.972(3) A und beta = 90.74(2) degrees. The unit cell contains two symmetry independent formula units (C14H10Cl2NO2)-Na+.3,94 H2O. There are molecule layers and sodium-water layers which show order-disorder phenomena.

Crystallization↗
PubMed1990

The structural analysis of a levan produced by Streptococcus salivarius SS2.

The structure of a water-soluble levan produced by Streptococcus salivarius SS2 has been determined by means of various chemical and instrumental methods. Methylation and periodate oxidation studies demonstrate that the levan is comprised of D-fructofuranosyl backbone residues linked beta-(2----6) (about 70%) with beta-(2----1) branches (about 30%). 13C-N.m.r. spectral analysis of the polymer is consistent with the structure determined by chemical means.

Carbohydrate Sequence↗
PubMed1995

Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co-expressing recombinant furin.

Thorough analysis of multimer composition and molecular structure of recombinant von Willebrand factor (r-vWF) produced by recombinant CHO cells demonstrated r-vWF to be more intact and less proteolytically degraded than plasma-derived vWF (pd-vWF) [B. Fischer et al. (1994) FEBS Lett. 351, 345-348]. In contrast to pd-vWF, r-vWF preparations consisted of pro-vWF (vWF containing covalently attached propeptide) as well as mature vWF subunits forming homo- and hetero-multimers. In order to ensure complete propeptide processing, a r-vWF-producing CHO cell clone was transfected with the cDNA of the human propeptide processing enzyme Furin. A r-vWF/r-Furin co-expressing cell clone was cultivated at industrial scale in high cell density perfusion fermenters. r-vWF obtained from these cells was fully processed. Analysis of r-vWF by multimer analysis revealed a multimer pattern equal in number of high molecular weight multimer to pd-vWF, but absence of satellite bands. Two-dimensional electrophoretic analysis of both the primary dimer and the complete multimer pattern of r-vWF showed that the recombinant coagulation factor was composed exclusively of intact and mature subunits. Since the triplet structure typical to pd-vWF is known to reflect proteolytic degradation, r-vWF thus exhibits an integrity far superior compared to pd-vWF.

Animals↗
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↗