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PubMed1997

Structural analysis of disaccharides synthesized by beta-D-glucosidase of Bifidobacterium breve clb and their assimilation by Bifidobacteria.

Circulation of a solution of 1 M D-fucose and 1 M D-glucose through a reaction system consisting of serial columns of immobilized recombinant beta-D-glucosidase of Bifidobacterium breve clb and activated charcoal gave two oligosaccharides. Structural analysis identified these oligosaccharides as D-fucosylglucose (6-O-beta-D-Fucopyranosyl-D-glucose) and gentiobiose (6-O-beta-D-Glucopyranosyl-D-glucose). The D-fucosylglucose obtained was well assimilated by many Bifidobacteria but not by the other intestinal bacteria tested.

Animals↗

PubMed1999

Computer-assisted differential diagnosis of malignant mesothelioma based on syntactic structure analysis.

BACKGROUND: Malignant mesothelioma, a mesoderm-derived tumor, is related to asbestos exposure and remains a diagnostic challenge because none of the genetic or immunohistochemical markers have yet been proven to be specific. To assist in the identification of mesothelioma and to differentiate it from other common lesions at the same location, we have tested the performance of syntactic structure analysis (SSA) in an automated classification procedure. MATERIALS AND METHODS: Light-microscopic images of tissue sections of malignant mesothelioma, hyperplastic mesothelium, and adenocarcinoma were analyzed using parameters selected from the Voronoi diagram, Gabriel's graph, and the minimum spanning tree which were classified with a K-nearest-neighbor algorithm. RESULTS: Results showed that mesotheliomas were diagnosed correctly in 74% of the cases; 76% of the adenocarcinomas were correctly graded, and 88% of the mesotheliomas were correctly typed. The performance of the parameters was dependent on the obtained classification (i.e., tumor-tumor versus tumor-benign). CONCLUSIONS: Our results suggest that SSA is valuable in the differential classification of mesothelioma and that it supplements a visually appraised diagnosis. The recognition scores may be increased by a combination of SSA with, for example, cellular or nuclear parameters, measured at higher magnifications to form a solid base for fully automated expert systems.

Adenocarcinoma↗

PubMed1996

Structural analysis of unsaturated hexasaccharides isolated from shark cartilage chondroitin sulfate D that are substrates for the exolytic action of chondroitin ABC lyase.

The enzymatic action of highly purified chondroitin ABC lyase from Proteus vulgaris is dependent on the size of the substrate, and the enzyme does not cleave tetrasaccharides, irrespective of their sulfation profiles [Sugahara, K., Shigeno, K., Masuda, M., Fujii, N., Kurosaka, A. & Takeda, K. (1994) Carbohydr. Res. 255, 145-163]. To characterize the enzyme action in more detail, we isolated nine sulfated hexasaccharides from commercial shark cartilage chondroitin sulfate D, after partial digestion with highly purified chondroitin ABC lyase, by means of gel chromatography and HPLC on an amine-bound silica column. Structural analysis by 500-MHz H-NMR spectroscopy, and enzymatic digestion in conjunction with HPLC, demonstrated that these hexasaccharides, with the common core saccharide structure delta 4 HexA (alpha 1-3)GalNAc(beta 1-4)GlcA(beta 1-3)GalNAc(beta 1-4) GlcA(beta 1-3)GalNAc(where delta 4 HexA and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid and glucuronic acid, respectively) bear three or four sulfate groups in different combinations. In the hexasaccharides, the D, disaccharide unit GlcA2-SO3 (beta 1-3) GalNAc4SO(3-) which is characteristic of chondroitin sulfate D, was arranged on the reducing side of the A disaccharide unit GlcA(beta 1-3)GalNAc4SO(3)-, and thus formed an A-D tetrasaccharide sequence GlcA(beta 1-3)GalNAc4SO(3)-(beta 1-4)GlcA2SO(3)-(beta 1-3) GalNAc6SO(3)-. Analysis of the degradation products of these hexasaccharides with highly purified chondroitin ABC lyase indicated that the enzyme preferentially acted on the unsaturated hexasaccharides in an exolytic fashion and removed an unsaturated disaccharide unit from the non-reducing termini, irrespective of the sulfation profiles of the hexasaccharides.

Animals↗

PubMedDate not supplied

[Structural analysis of alphoid DNA of primates. I. Heterogeneity of nucleotide sequence of alphoid repeats in human DNA].

The nucleotide sequence of two cloned fragments of human alphoid DNA was established. These fragments were earlier characterized in our laboratory as molecular markers of the 3rd (pHS05) and 11th (pHS53) chromosomes. Fragment pHS53 (2546 bp) contains alphoid repeats tandemly arranged and organized into three highly homologous pentamers. The heterogeneity of monomeric sequences within individual pentamers reaches 24-33%. Structural analysis of EcoRI subfragment pHS05 showed that this alphoid tetramer consists of two dimers 340 bp long. These dimers differ up to 16% from each other and from the so-called consensus sequence of the EcoRI-340 bp-restriction fragments family reported earlier by Wu and Manuelidis. The primary structure of four cloned fragments of EcoRI-340 bp-family was established. The data show that human alphoid DNA is highly heterogeneous. This conclusion is opposite to the view suggesting that alphoid DNA is a highly homogeneous class of reiterated sequences of the human genome.

Animals↗

PubMed1984

18,19-Dihydroxydeoxycorticosterone, a new metabolite produced from 18-hydroxydeoxycorticosterone by cytochrome P-450(11) beta. Chemical synthesis and structural analysis by 1H NMR.

A new metabolite was produced from 18-hydroxydeoxycorticosterone by the cytochrome P-450(11) beta linked hydroxylase system purified from bovine adrenocortical mitochondria. It was identified as 18,19-dihydroxydeoxycorticosterone by chemical synthesis on the basis of high-performance liquid chromatography, gas chromatography-mass spectrometry, and proton nuclear magnetic resonance (1H NMR) spectroscopy, and detailed structural analysis of it was performed by 1H NMR spectroscopy. The methylene protons at the C-19 position of the steroid were nonequivalent and coupled with each other, having a coupling constant of 10.6 Hz. These protons had different coupling constants, 6.7 and 3.4 Hz, for the hydroxy proton at the C-19 position. Due to these couplings, the signals of the methylene protons were observed around 3.9 ppm as two double doublets. The methylene protons at the C-21 position were also nonequivalent, having a coupling constant of 11.1 Hz. Coupling constants between these methylene protons and the hydroxy proton at the C-21 position were 8.2 and 4.2 Hz, respectively. These results indicate that both hydroxymethyl groups at the C-19 and C-21 positions do not freely rotate in chloroform solution. The signals of hydroxy protons at the C-19 and C-21 positions were found at 1.25 and 1.87 ppm, respectively, by means of decoupling of the corresponding methylene protons. The hydroxy proton at the C-18 position was found to scarcely couple with any proton. This fact suggests that this hydroxy group is linked to the C-20 position, making a hemiketal bridge between the C-18 and the C-20.

18-Hydroxydesoxycorticosterone↗

PubMed2002

Bias and heteroscedastic memory error in self-reported health behavior: an investigation using covariance structure analysis.

BACKGROUND: Frequent use of self-reports for investigating recent and past behavior in medical research requires statistical techniques capable of analyzing complex sources of bias associated with this methodology. In particular, although decreasing accuracy of recalling more distant past events is commonplace, the bias due to differential in memory errors resulting from it has rarely been modeled statistically. METHODS: Covariance structure analysis was used to estimate the recall error of self-reported number of sexual partners for past periods of varying duration and its implication for the bias. RESULTS: Results indicated increasing levels of inaccuracy for reports about more distant past. Considerable positive bias was found for a small fraction of respondents who reported ten or more partners in the last year, last two years and last five years. This is consistent with the effect of heteroscedastic random error where the majority of partners had been acquired in the more distant past and therefore were recalled less accurately than the partners acquired more recently to the time of interviewing. CONCLUSIONS: Memory errors of this type depend on the salience of the events recalled and are likely to be present in many areas of health research based on self-reported behavior.

Adult↗

PubMed1994

Overproduction of Thermus aquaticus DNA polymerase and its structural analysis by ion-spray mass spectrometry.

We cloned the pol gene from the Thermus aquaticus YT-1 strain into a plasmid vector and constructed a high-level expression system of the gene in Escherichia coli. Six codons in the translational start region were changed to simple AT-type codons or codons which are most frequently used in E. coli by the genetic engineering techniques with retention of the amino acid sequence of the native enzyme. The modified pol genes were expressed under the lac promoter of pUC-type plasmid and 266,418 units of activity was obtained in a sonicated and heated crude extract from 2 g of E. coli cells bearing one of the recombinant plasmids, pTAQ9. Highly purified protein was subjected to structural analysis using a protein sequencer and an ion-spray mass spectrometer combined with reversed-phase HPLC (LC-MS). The primary structure of the DNA polymerase was identical with the amino acid sequence deduced from the nucleotide sequence of the pol gene as far as examined (about 95% of the sequence); though, several regions where small peptides of less than 5 residues were produced by lysyl endopeptidase digestion could not be sequenced.

Amino Acid Sequence↗

PubMed1975

Primary structural analysis of sulfhydryl protease inhibitors from pineapple stem.

Pineapple stem acetone powder provides a rich source of the sulfhydryl protease bromelain and of a family of compositionally similar but chromatographically distinct polypeptide inihibtors of this enzyme. The isoinhibitors have molecular weights of 5600, and they contain five disulfide bonds and about 50 amino acids each (Perlstein, S. H., AND Kezdy, F.J. (1973) J. Supramol. Struct. 1, 249-254). Primary structural analysis of one of the seven inhibitor fractions (VII) revealed extensive microheterogeneity. Each of the inhibitor molecules in Fraction VII was shown to be composed of two peptide chains joined by disulfide bonds. These chains, designated A and B on the basis of size, comprise 41 and 10-11 residues, respectively, and the amino acid sequence of one of each are given below: (see article for formular). On the basis of ionization properties and yields of the A and B chains, it would appear that one of the major inhibitor species in Fraction VII is the covalently linked complex of the two chains shown, namely [A-1, B-2]. The second major inhibitor component of Fraction VII is identical in structure with [A-1, B-2i1 except that residues 1 and 8 in the A chain are pyroglutamate and threonine, respectively, and in the B chain glutamine 11 is replaced by arginine. The third inhibitor in Fraction VII is a minor constituent identical with the second, except that residue 1 in the A chain is glutamate rather than pyroglutamate. This microheterogeneity in the inhibitors of Fraction VII is further increased by the fact that B chains may lack threonine 1, in which case they are decapeptides beginning with alanine. On the basis of the striking homology of the cysteine residues with those of other protease inhibitors, it is proposed that the bromelain inhibitors are generated enzymatically from single chain precursors by excision of a "bridge" paptide which links the NH-2 termal A chain to the COOH-terminal B chain.

Amino Acids↗

PubMed1995

Basic cerebellar events relating sensorimotor adaptation--structural analysis on extracellular records of Purkinje cells.

The heterosynaptic facilitation or suppression of synaptic efficacy between parallel fibers and the dendritic spines of Purkinje cells (PCs) in the cerebellar cortex has been for decades the basic idea of sensorimotor adaptation. Great efforts in order to get direct evidence failed, or were not accepted as direct proofs. A new facility was introduced with the structural analysis of intradendritic records of the PC. These records reveal a generally double (rarely single, triple or quadruple) rhythmic pattern of small spikes, which are proposed to be prespikes of dendritic origin. Moreover, they may take their origin at functionally separated dendritic compartments resulting in a nonlinear, phase-sensitive integrative process, performed by the compound spike generating mechanism of the cerebellar PC.

Action Potentials↗

PubMed1981

Structural analysis of human hemoglobin variants with field desorption mass spectrometry.

Tryptic hydrolyzates of normal and abnormal human globin chains were analyzed with mass spectrometry, using field desorption ionization technique. All the peptides, including core peptides, were detected as protonated molecular ions in field desorption mass spectra. Since the technique makes it possible to determine the mass number of each peptide, it is very useful for structural analysis of human hemoglobin variants, even those with electrophoretically and chromatographically silent mutations.

Adult↗

PubMed1993

Structural analysis of the Actinobacillus pleuropneumoniae-RTX-toxin I (ApxI) operon.

Actinobacillus pleuropneumoniae-RTX-toxin I (ApxI), an important virulence factor, is secreted by serotypes 1, 5, 9, 10, and 11 of A. pleuropneumoniae. However, sequences homologous to the secretion genes apxIBD of the ApxI operon are present in all 12 serotypes except serotype 3. The purpose of this study was to determine and compare the structures of the ApxI operons of the 12 A. pleuropneumoniae serotypes. We focused on the nucleotide sequence comparison of the ApxI-coding genes, the structures of the ApxI operons, and the transcription of the ApxI operons. We determined the nucleotide sequences of the toxin-encoding apxICA genes of serotype 9 and found that the gene for the structural toxin, apxIA, was almost identical to the apxIA gene of serotype 1. The toxin-encoding genes of the other serotypes are also similar for the main part; nevertheless, two variants were identified, one in serotypes 1, 9, and 11 and one in serotypes 5 and 10. The two apxIA variants differ mainly within the distal 110 nucleotides. Structural analysis demonstrated that intact ApxI operons, consisting of the four contiguous genes apxICABD, are present in serotypes 1, 5, 9, 10, and 11. ApxI operons with a major deletion in the apxICA genes are present in serotypes 2, 4, 6, 7, 8, and 12. Serotype 3 does not contain ApxI operon sequences. We found that all ApxI operons are transcriptionally active despite the partial deletion of the operon in some serotypes. The implications of these data for the expression and secretion of ApxI and the other Apx-toxins, ApxII and ApxIII, as well as for the development of a subunit vaccine against A. pleuropneumoniae will be discussed.

Actinobacillus pleuropneumoniae↗

PubMed2003

Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein MobA.

Molybdopterin guanine dinucleotide (MGD) is the form of the molybdenum cofactor that is required for the activity of most bacterial molybdoenzymes. MGD is synthesized from molybdopterin (MPT) and GTP in a reaction catalyzed by the MobA protein. Here we report that wild type MobA can be copurified along with bound MPT and MGD, demonstrating a tight binding of both its substrate and product. To study structure-function relationships, we have constructed a number of site-specific mutations of the most highly conserved amino acid residues of the MobA protein family. Variant MobA proteins were characterized for their ability to support the synthesis of active molybdenum enzymes, to bind MPT and MGD, to interact with the molybdenum cofactor biosynthesis proteins MobB and MoeA. They were also characterized by x-ray structural analysis. Our results suggest an essential role for glycine 15 of MobA, either for GTP binding and/or catalysis, and an involvement of glycine 82 in the stabilization of the product-bound form of the enzyme. Surprisingly, the individual and double substitution of asparagines 180 and 182 to aspartate did not affect MPT binding, catalysis, and product stabilization.

Amino Acid Sequence↗

PubMed1996

Structural analysis of the glycosyl-phosphatidylinositol membrane anchor of the merozoite surface proteins-1 and -2 of Plasmodium falciparum.

Plasmodium falciparum accumulates the two merozoite surface proteins-1 and -2 during schizogony. Both proteins are proposed to be anchored in membranes by glycosyl-phosphatidylinositol membrane anchors. In this report the identity of these GPI-anchors is confirmed by labelling with tritiated precursors and additionally by specific enzymatic and chemical treatments. Detailed structural analysis of the core-glycans showed that the GPI-anchors of both proteins possess an extra alpha 1-2 linked mannose at the conserved trimannosyl-core-glycan. MSP-1 and MSP-2 labelled with tritiated myristic acid possess primarily radioactive myristic acid at inositol rings in both GPI-anchors. Additionally the hydrophobic fragments released from [3H]myristic acid labelled GPI-anchors were identified as diacyl-glycerols, carrying preferentially [3H]palmitic acid in an ester-linkage.

Animals↗

PubMed1990

Structured analysis: methodology for developing a model for defining nursing information system requirements.

Including nurses in decisions to purchase or design information systems is an increasingly common phenomenon. Without previous informatics experience, nurses may need help defining specific requirements for information systems that will assist in managing and administering nursing care. The Model for Defining Nursing Information System Requirements was developed to serve as a guiding framework for deriving nursing information system (NIS) requirements. Structured analysis techniques were used to develop the model. The model was tested by surveying 75 registered nurses who had made decisions about NISs. Subjects supported the model's completeness and usefulness for defining NIS requirements.

Decision Trees↗

PubMed1999

Peptide Mapping and Primary Structure Analysis of hEGFby Mass Spectrometry.

Liquid chromatography-mass spectrometry(LC-MS) was adopted to analyze peptide mapping of the hEGF digested by TPCK-trypsin and V8 to determine the molecular weight of each peptide fragment. Most of the peptide fragments were sequenced by tandem MS(MS-MS) to obtain the amino acid sequence. In addition, the number of disulfide bonds was confirmed. This method will facilitate the determination and structural analysis for nature, synthesis and recombinant proteins and peptides. The accuracy and resolution are both better than that of the routine method.

Journal Article↗

PubMed2003

Overexpression, purification, and structural analysis of the hydrophobic E5 protein from human papillomavirus type 16.

The E5 proteins of human papillomavirus (HPV) are highly hydrophobic transmembrane proteins that display weak transforming activity. The HPV E5 proteins are localized largely to intracellular membranes, such as the Golgi apparatus and endoplasmic reticulum, but also appear in the plasma membrane. Infection with HPV16 is the cause of over 90% of human cervical cancers. HPV E5 is known to interact with growth factor receptors and gap junction proteins and is believed to play a role during the initiation of neoplasia. The structure of HPV E5 and the mechanism of its interactions with growth factor receptors remain largely unknown. In the present studies, the E5 protein of HPV16 was cloned into the pBAD/TOPO vector fused to an N-terminal thioredoxin leader and a C-terminal His-tag, and expressed in Escherichia coli. The identity of the protein was confirmed by immunoblotting using antibodies against a V5-epitope tag engineered into the protein. Due to formation of high molecular mass superaggregates of the protein, two chromatography steps were employed for its purification: (1) gel filtration chromatography to separate the superaggregated protein from other soluble proteins and (2) Ni-chelate affinity chromatography in the presence of detergent. The superaggregates of the E5-fusion protein were broken down to monomers and various oligomers by sonication in the presence of 0.2% SDS. The purified E5-fusion protein was then reconstituted into lipid vesicles and initial structural analysis of the protein was performed using circular dichroism spectroscopy.

Chromatography, Affinity↗

PubMed1995

Structure analysis of breast lesions using neighborhood graphs.

The College of American Pathologists divides proliferative lesions of the breast into three categories, but the diagnostic criteria for classification are not easy to apply. Therefore, many morphologic, immunohistochemical and morphometric features were studied in order to clarify this subdivision; however, the reported results are not very satisfactory. We also attempted to identify and differentiate the architectural features useful for the diagnosis of proliferative breast lesions. The morphologic characteristics of proliferative lesions of the breast were considered amenable to a graph theoretical approach. From a set of images of mammary ducts acquired from a hematoxylin-eosin-stained section, a hierarchy of graphs was built. The main graphs representing the structure are: (1) neighborhood graph, (2) planar graph, derived from (1) and (3) a dual graph. A simplified prototype of a system for structure analysis of the lesions was implemented and tested. Forty duct images classified by two expert pathologists were acquired and subjected to the system. The data obtained were analyzed, and some significant graph features validating our approach to the structure representation of the lesions were discovered. This approach can also be applied to other, similar classification problems and may help in understanding the biologic significance of proliferative lesions of the breast.

Breast Neoplasms↗

PubMed2003

Structuring parameters for assessment of bone quality using a morphological filter and star volume analysis: structuring property in the cancellous bone of the human femoral head.

This study examines structuring parameters for the assessment of bone quality based on the relationship of bone strength to the trabecular structure or bone mineral density (BMD). Thirty-nine human cancellous bone blocks sampled from resected Ward's triangle area were investigated. The trabecular bone pattern of each bone block was binarized into its corresponding trabecular skeletal pattern using computed radiography with a morphological filter. Every binarized trabecular skeletal pattern was quantified to find the trabecular skeletal pixel percentage (SkP = volume parameter of trabecular skeletal signal component) and skeletal star volume (Vt = connection parameter of trabecular skeletal structure). After BMD and elasticity for each bone block was measured by dual X-ray absorptiometry and the breaking test, the correlations to SkP and Vt were determined. In addition, an attempt was made to detect changes of the trabecular structure in the Ward's triangle area of the femoral head after patients had received treatment for osteoporosis. This evaluation used the structuring parameters SkP and Vt. The results showed that the larger the BMD value, the greater the elasticity, although some samples with similar BMD values had considerably different elasticities. With increased Vt values the trabecular skeletal pattern that had horizontal skeletal elements orientated perpendicular to the loading direction showed higher elasticity. No correlation between SkP and either elasticity or BMD was observed in either the subset (sequential images of morphological filter) or sumset images (combined with subset images). On the other hand, the correlation coefficient between Vt and elasticity was similar to that between BMD and elasticity for subset image ( n = 6). For sumset images (3-6 and 4-7), the correlation coefficient of Vt was equal or higher than that of BMD and the connectivity of the trabecular skeletal structure was more closely linked to bone strength as compared to BMD. Therefore, it was concluded that because no high correlation could be detected between Vt and BMD, BMD and the connectivity of trabeculae are independently related to bone strength. In addition, this method was used to evaluate patients after treatment for osteoporosis, and noticeable changes were seen in the bone trabecular skeletal structure in the Ward's triangle area of the femoral head. In reference to these clinical images, variations in SkP and Vt were quantitatively assessed. These results suggest that the SkP and Vt obtained using computed radiography, morphological filter, and star volume analysis are useful structuring parameters for the assessment of bone quality.

Aged↗