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At least 199 records · Page 11Linked to original sources
PubMed1986

Human liver sulphamate sulphohydrolase. Determinations of native protein and subunit Mr values and influence of substrate agylcone structure on catalytic properties.

Human sulphamate sulphohydrolase was purified at least 20,000-fold to homogeneity from liver with a three-step four-column procedure, which consisted of a concanavalin A-Sepharose/Blue A agarose coupled step, and Bio-Gel HT step and then a CM-Sepharose step. The procedure was also used to purify enzyme from kidney and placenta. The subunit Mr of liver, kidney and placenta sulphamate sulphohydrolase was assessed to be 56,000 by using SDS/polacrylamide-gel electrophoresis. The native protein Mr of enzyme from all three tissue sources was assessed by gel-permeation chromatography to be approx. 120,000 on Sephacryl S-300 and 100,000 on Fractogel TSK. It is probable that the native enzyme results from dimerization of subunits. Kinetic parameters (km and kcat.) of human liver sulphamate sulphohydrolase were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin and heparan sulphate. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are turned over up to 372000 times faster than the monosaccharide substrate 2-sulphaminoglucosamine. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue C-6 carboxy and C-2 sulphate ester groups and a post-penultimate 2-sulphaminoglucosamine residue. The C-4 hydroxy group of the 2-sulphaminoglucosamine under enzymic attack is involved in binding of substrate to enzyme. The presence of C-6 sulphate ester on the non-reducing end 2-sulphaminoglucosamine stimulates sulphamate bond hydrolysis and substrate affinity if the adjacent monosaccharide residue is idose or 2-sulphoidose, but strongly inhibits hydrolysis if the adjacent monosaccharide residue is iduronic acid. Sulphamate sulphohydrolase is an exoenzyme, since activity toward internal sulphamate bonds was not detected. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone C-2 sulphate ester and aglycone C-6 carboxy groups and C-6 sulphate ester groups on the 2-sulphaminoglucosamine residue under attack considerably affect the pH response. Structurally complex substrates had two pH optima. Incubation temperature and buffer ionic strength markedly influenced pH optima and enzyme activity. Cu2+ and SO4(2-)ions are potent inhibitors of enzyme activity.

Catalysis↗
PubMed1991

[New possibilities for studying complex macromolecular structures of the cell by encasing cells in polyacrylamide microspheres].

A method is proposed for incorporation of mammalian cells into spheres permeable for protein molecules, the dimensions of these spheres not overcoming those of the cells. The method permits fractionation of the cells, preserving them as discrete units resistant to mechanical and hydrodynamic destruction in the course of routine laboratory manipulations, as well as using flow cytometry for their analysis. With this approach, the flow cytometric measurements were made of DNA being shielded in the chromatin complex which prevents from binding fluorescent dyes with low-molecular weights.

Acrylic Resins↗
PubMed1987

A study of intercellular bridges during spermatogenesis in the rat.

A morphological evaluation of intercellular bridges was undertaken during rat spermatogenesis. The dimensions and relationships of the bridges were shown to vary during different phases of spermatogenesis. Cellular divisions of spermatogonia and spermatocytes resulted in the partitioning of pre-existing bridges by complex structures termed bridge partitioning complexes, which are described in detail, as is the process whereby new bridges are formed. The structure of premeiotic bridges was generally consistent; however, during spermiogenesis, the structure of bridges and bridge contents were modified at specific phases of their development. The plasma membrane density associated with the cytoplasmic aspect of early step 1 spermatids separated into multiple dense bands that encircled the peripheral aspect of late step 1 spermatid bridges. By step 2 of spermiogenesis, these dense bands became associated with several cisternae of endoplasmic reticulum, which later coalesced into a single saccule that completely encircled the bridge structure by step 4. At steps 10-13 of spermiogenesis, the single saccule of endoplasmic reticulum vesiculated into many smaller cisternae. Also, filament-bounded densities (measuring 10-12 nm in diameter) appeared within the bridge channel. At step 17 of spermiogenesis, the filament-bounded densities were no longer apparent, but an anastomosing network of endoplasmic reticulum, often in the configuration of a sphere, occupied the entire central region of the bridge. In step 19 spermatids, the smooth endoplasmic reticulum within the bridge channel and the multiple cisternae lining the bridge density were gradually displaced. The subsurface density of bridges gradually lost its prominence. Some cytoplasmic lobes were connected by extremely narrow (approximately 22 nm) cytoplasmic channels. Similar-appearing channels were seen on the surface zone of cytoplasmic lobes or residual bodies, this observation suggesting that channels were sites of severence of bridges. Just prior to the separation or disengagement of the spermatid from the cytoplasmic lobe, selected bridges appeared to open to form large masses. After spermiation, residual bodies were not found joined by bridges; but from the size of some of the residual bodies, it was suspected that they were formed by coalescence of more than one cytoplasmic lobe. Freeze-fracture demonstrated few intramembranous particles on either the P or E face of the plasma membrane forming the bridge; this finding suggested bridge structures restricted free lateral movement of membrane constituents across the bridge.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Signal transduction by the T cell antigen receptor.

The T cell antigen receptor (TCR) must recognize antigen, and translate this recognition event into intracellular signal transduction events. Two signal transduction events are regulated by the TCR: the activation of a protein tyrosine kinase (PTK) and phospholipase C (PLC). Recent studies suggest that the TCR-activated PTK regulates PLC activation by the phosphorylation of tyrosine residues of PLC gamma 1. The complex structure of the TCR is now being related to its signal transduction function. Studies with chimeric receptors reveal that the antigen binding Ti heterodimer communicates with the subunits involved with signal transduction, the CD3 chains and zeta dimers, through the carboxy-terminal regions of the Ti chains that surround and include the transmembrane domains. Other chimeras have helped demonstrate that the zeta chain family of dimers function to couple the TCR to intracellular signal transduction mechanisms. The signal transduction function of the TCR can be regulated in a number of ways and by other T cell surface molecules. The plasma membrane tyrosine phosphatase CD45, plays a critical role to specifically regulate TCR-mediated activation of PTK's and PLC. Thus, an understanding of the complex structure of the TCR and the intricacies of its signal transduction function is rapidly emerging.

Animals↗
PubMed1985

Determination of equilibrium constants for a sequential model of dioxygen binding by hemoglobin-inositol hexaphosphate complexes: the structural pathway from deoxy- to oxy-hemoglobin.

The affinity of human hemoglobin (Hb4) for dioxygen was determined in 0.050 M bistris, 0.005 M inositol hexaphosphate (IHP) at pH 7.0 and 20.0 degrees C. Binding of dioxygen by Hb4 was determined by detailed spectroscopic analysis of the absorption spectrum in the region from 460 to 620 nm. The absorption spectrum of samples at intermediate values of fractional saturation (F) could not be resolved into components of Hb4 and (HbO2)4 without generating a residual spectrum, the amplitude of which was greatest at F from 0.4 to 0.5 and least at values of F of 0 and 1. An equation of state for dioxygen binding by the Hb4-IHP complex was formulated and tested by its ability to predict (i) the equilibrium binding curve and (ii) the variation in amplitude of the residual spectrum with F. The equilibrium binding data was fitted to the following equation of state: (Formula: see text) where K1 is the equilibrium constant for binding of dioxygen to an alpha chain of the Hb4-IHP complex, K2 is the constant for the second alpha chain, K3 is the equilibrium constant for the large-scale conformational change, K4 is the equilibrium constant for binding of oxygen by both beta chains, and (L) is the ligand concentration. The best-fitting values were as follows: K1, 0.03497 mm Hg-1; K2, 0.01368 mm Hg-1; K3, 2.44; K4, 0.0008867 mm Hg-2. The residual spectra were attributed to differential loading of dioxygen by the alpha and beta chains. Equations of state for F of each chain are presented, and the amplitude of the residual spectra was shown to be accurately predicted by the differences in F of each chain when subjected to the constraint that the best-fitting values of K1-K4 be used in predicting saturation of each chain with dioxygen.

Binding Sites↗
PubMed1991

Analysis of splicing patterns of human spumaretrovirus by polymerase chain reaction reveals complex RNA structures.

Mapping of transcripts of the human foamy virus genome was carried out in permissive human embryonic fibroblast cells by Northern blot hybridization and S1 nuclease analysis. Since several splice sites that are localized within a relatively narrow genomic region were detected, the polymerase chain reaction (PCR) was employed, and cloning and sequencing of the splice site junctions of the corresponding viral cDNAs were subsequently performed. All spumavirus transcripts have a common but relatively short leader RNA. Genomic, singly spliced env mRNAs and several singly and multiply spliced subgenomic transcripts were identified. The multiply spliced viral mRNAs consist of various exons located in the central or 3' part of the viral genome. At least four novel gene products, termed Bet, Bes, Beo, and Bel3, are predicted to exist. The poly(A) addition site that defines the boundary of the R and U5 region in the 3' long terminal repeat was determined. The pattern of spumavirus splicing is more complex than that of oncoviruses and more similar to that of lentiviruses. One of the characteristic features of spumavirus transcription is the existence of singly spliced bel1 and bel2 mRNAs that alternatively are multiply spliced, thereby generating a complexity comparable to, but different from, that of lentiviruses and from that of other known retroviruses. The complex spumavirus transcriptional pattern of human spumavirus and the coding potential of the 10 exons identified are discussed.

Amino Acid Sequence↗
PubMed1991

[Synthesis of partial structure of complex types of N-glycoproteins].

Comparable syntheses of beta-D-GlcpNAc-(1----2)-[beta-D-GlcpNAc-(1----6)]-alpha-D-Manp-(1- ---6)-beta-D-ManpO(CH2)8CO2Me and beta-D-GlcpNAc-(1----2)-alpha-D-Manp-(1----3)-beta-D-ManpO(C H2)8CO2Me with the glycosyl halide and imidate methods were investigated. 3,4,6-Tri-O-acetyl-2-deoxy-(2,2,2-trichloroethoxycarbonylamino)-al pha-D-glucopyranosyl bromide or trichloroacetimidate are suitable glycosyl donors for beta-D-glycoside coupling with secondary hydroxyl groups.

Carbohydrate Sequence↗
PubMed1990

Mucinous cystic neoplasm of the pancreas. Estimation of grade of malignancy with imaging techniques and its surgical implications.

The results of diagnostic imaging procedures, macroscopic and microscopic findings and clinical follow up data of 20 mucinous cystic neoplasms of the pancreas were retrospectively reviewed to determine the grade of malignancy and its surgical implications. The largest mean diameter was 8.9 cm for 10 malignant cases, 5.0 cm for two premalignant cases, and 3.1 cm for eight benign tumours. Ultrasonography or computed tomography or both, corresponded well with macroscopy. The 10 malignant and two premalignant lesions had intracystic mural nodules or extracystic solid components, while the eight benign specimens had neither nodules nor solid portions. Structural complexity seen on ultrasonography or computed tomography, which reflected the irregularity in size and shape of the cysts, internal septa, walls, and solid components, was severe in the 10 malignant and two borderline tumours and mild or moderate in the eight benign lesions. Angiography showed that five of the nine malignant tumours were hypervascular, while the two premalignant and four benign lesions were all avascular. Five of 10 patients with malignant tumours died of local recurrence or remote hematogenous metastases, and one with malignant disease was still alive with liver metastases at the time of writing. We propose that a careful preoperative estimate of the malignant potential of such cysts should be made based on the size of the tumour, the presence of mural nodules and solid areas, and the structural complexity on ultrasonography or computed tomography, as well as the amount of vascularity seen on angiography. Attention should be paid at follow up to the presence of local recurrence and haematogenous metastases in cases of malignant disease.

Adenocarcinoma, Mucinous↗
PubMed1991

Candida albicans mycelial wall structure: supramolecular complexes released by zymolyase, chitinase and beta-mercaptoethanol.

Different techniques released from the wall of Candida albicans mycelial cells high molecular weight mannoprotein materials with different levels of complexity. SDS solubilized among others one protein of 180 kDa which reacted with a monoclonal antibody (MAb) specific of a O-glycosylated protein secreted by regenerating mycelial protoplasts [Elorza et al. (1989) Biochem Biophys Res Commun 162:1118-1125]. Zymolyase, chitinase and beta-mercaptoethanol, released different types of high molecular highly polydisperse mannoprotein materials (greater than 180 kDa) that also reacted with the same MAb. These materials had N-glycosidically linked sugar chains, in addition to the O-glycosidically bonded sugars, as their molecular masses were significantly reduced by Endo H digestion. Besides, the specific materials released by either zymolyase or chitinase seemed to be the same throughout the process of germ tube formation. Transmission electron microscopy of thin sections of cells and walls showed that mannoproteins and chitin are evenly distributed throughout the entire cell wall structure.

Blotting, Western↗
PubMedDate not supplied

[DNA interaction with low molecular ligands of different structure. II. Complexes of DNA with actinomine and its analogs].

DNA-complexes with actinomine and its analogues containing omega-dialkylaminoalkyl groups at 1,9 positions of the phenoxazone moiety were studied by technique of spectrophotometry, viscometry and flow birefringence. In the process of spectrophotometry titration two groups of spectra corresponding to different DNA--ligand ratio in a complex were observed. According to the experimental data the investigated compounds are bounded to DNA by means of intercalation and external binding. There within a region of low degrees of binding the intercalation type of the ligand--DNA interaction prevails. In virtue of the spectrophotometry data the intercalation binding share was calculated. The intrinsic viscosity of a complex increases in the case of ligand intercalation and does not change as it joins to the DNA double-helix from outside. Optical anisotropy of DNA molecule increases linearly irrespective of the way of ligand binding. Data on the flow birefringence permits to conclude that under external binding the angle between the normal to the ligand chromophore plane and the axis of DNA double-helix is about zero. During ligand intercalation the equilibrium rigidity of DNA molecules increases.

Chemical Phenomena↗
PubMed1986

The sucrase-isomaltase complex: primary structure, membrane-orientation, and evolution of a stalked, intrinsic brush border protein.

The complete primary structure (1827 amino acids) of rabbit intestinal pro-sucrase-isomaltase (pro-SI) was deduced from the sequence of a nearly full-length cDNA. Pro-SI is anchored in the membrane by a single 20 amino acid segment spanning the bilayer only once. The amino-terminal, cytoplasmic domain consists of 12 amino acids and is not preceded by a cleaved leader sequence. This suggests a dual role for the membrane-spanning segment as an uncleaved signal for membrane insertion. This is followed by a 22 residue serine/threonine-rich, probably glycosylated, stretch, presumably forming the stalk on which the globular, catalytic domains are directed into the intestinal lumen. Following this is a high degree of homology between the isomaltase and sucrase portions (41% amino acid identity), indicating that pro-SI evolved by partial gene duplication.

Amino Acid Sequence↗
PubMed1991

Characterization of the memory gene dunce of Drosophila melanogaster.

The dunce (dnc) gene of Drosophila melanogaster encodes cAMP phosphodiesterase (PDEase) and is required for learning/memory and female fertility. The gene is structurally complex, demonstrated in part by Northern blotting experiments which detected multiple RNAs ranging in size from 4.2 to 9.6 kb (1 kb = 10(3) bases or base-pairs). To characterize these RNAs and to understand their sequence heterogeneity, we isolated and analyzed 29 new and independent cDNA clones representing the dnc RNAs. Restriction mapping, hybridization analysis and sequence determination of these cDNA clones and the corresponding genomic exons resolved these into six different classes. Exons defined by the cDNA clones are distributed over more than 148 kb of genomic DNA, with some exons being used alternatively among the RNAs. The RNAs are transcribed from at least three initiation sites: two of these were mapped by parallel S1-nuclease and primer extension experiments. In addition, some of the heterogeneity is generated by using varying lengths of a 3'-untranslated trailer sequence. Altogether, the results indicate that the size and sequence heterogeneity of dnc transcripts results from transcription initiation at multiple sites, alternative splicing, and processes which generate different 3' ends. The existence of multiple protein products is suggested by the alternative use of exons which code for portions of the open reading frame. The protein variation potentially includes N-terminal differences coded for by transcript-specific 5' exons and internal differences arising from the optional inclusion of a 39 base-pair exon and from the alternative use of two 3' splice sites separated by six base-pairs. Expression of a cDNA clone in yeast containing a large portion of the open reading frame produced cAMP PDEase activity identical in properties to the Drosophila enzyme affected by the dnc mutation. The results suggest that the remarkable structural complexity of dnc may reflect an intricate control of the spatial and/or temporal expression of various isoforms of cAMP PDEase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

HLA-A2 antigen phosphorylation in vitro by cyclic AMP-dependent protein kinase. Sites of phosphorylation and segmentation in class i major histocompatibility complex gene structure.

The heavy chain of the HLA-A2 antigen is phosphorylated by cyclic AMP-dependent protein kinase at two serine residues of the intracellular region. Limited proteolysis was performed on purified [32P]HLA-A2 antigens in order to define the sites of phosphorylation. Both of the phosphorylated serine residues are located in the carboxyl terminus of the heavy chain; one is encoded by exon 5, while the other is encoded by exon 6. The phosphoserine encoded by exon 5 is part of the conserved sequence Arg-Arg-Lys-Ser-Ser. This protein sequence contains the proper arrangement of amino acids for recognition and phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. In the murine class I antigens (H-2), exon 5 encodes a similar sequence of basic residues followed by one intervening residue and a threonine rather than a serine residue in the last amino acid position. A composite figure is presented that compares the carboxyl-terminal sequences of human and murine class I antigens and illustrates the known sites of phosphorylation recognized by various kinases. Each site of phosphorylation in the carboxyl terminus is contained within a conserved protein sequence encoded by one of the three exons. A separation and preservation of unique sites of phosphorylation could explain why there is segmentation in the genomic arrangement of class I molecules.

Amino Acid Sequence↗
PubMedDate not supplied

Imaging of complex NMR spectra.

The Point Spread Function (PSF) in NMR imaging is the result of both the line broadening due to magnet field inhomogeneity and the intrinsic spectrum of the nucleus at resonance. In the case of proton imaging, the line broadening dominates the small chemical shifts and the spectral lines are not resolved. This is not generally the case with other nuclei having strong chemical shifts and the PSF then has a complex structure. During imaging, the complex PSF is convolved with the spatial distribution of the nucleus at resonance and this leads to halo artifacts which are dependent on the imaging technique employed. The images due to the ensemble of spectral lines can be separated in principle by deconvolution of the data with the PSF before reconstruction. In the special case where the complex PSF is spatially independent, it can be obtained from the Free Induction Decay (FID) data produced in the absence of a spatially encoding gradient field. This technique has been successfully applied to in-vivo imaging of exogenous perfluorocarbon material.

Animals↗
PubMed1992

Drug binding to higher ordered DNA structures: netropsin complexation with a nucleic acid triple helix.

We have used a combination of spectroscopic and calorimetric techniques to characterize how netropsin, a ligand that binds in the minor groove of DNA, influences the properties of a DNA triple helix. Specifically, our data allow us to reach the following conclusions: (i) netropsin binds to the triplex without displacing the major-groove-bound third strand; (ii) netropsin binding to the triplex exhibits a lower saturation binding density (7.0 base triplets per netropsin bound) than netropsin binding to the corresponding duplex (5.5 base pairs per netropsin bound); (iii) the netropsin-free and the netropsin-bound triplexes each melt in two well-resolved transitions, initial conversion of the triplex to the duplex state followed by duplex melting to the component single-stranded states; (iv) netropsin remains bound to DNA as the triplex melts to the duplex state; (v) netropsin binding thermally destabilizes the triplex in equilibrium with duplex equilibrium dramatically, while thermally stabilizing the duplex to single-strand equilibrium; (vi) netropsin binding to the triplex is enthalpically 4 times more favorable (more exothermic) than netropsin binding to the corresponding duplex; (vii) netropsin binding to the triplex decreases the cooperativity of the triplex----duplex melting event. These results demonstrate that occupancy of the minor groove of a triplex by a ligand such as netropsin can exert a profound impact on the properties of the host triplex, particularly with regard to the equilibrium in which the third strand is expelled from the major groove. Thus, our results reveal considerable major groove/minor groove crosstalk. Such knowledge may prove of practical importance by providing an approach for modulating the affinity and specificity of major-groove-binding third strands in triplex-forming protocols designed to target specific duplex domains. Fundamentally, our results provide insights into the crosstalk that can result when ligands bind to the two major receptor sites of duplex DNA--namely, the major and minor grooves.

Calorimetry, Differential Scanning↗
PubMed1983

Solution conformation of asparagine-linked oligosaccharides: alpha(1-6)-linked moiety.

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of the alpha(1-6)-linked moiety is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions and to draw conclusions about the behavior in solution of these molecules. For all classes, identical conformations were found for the 6-arm except for the torsional angle, omega, about the C5-C6 bond of the alpha 1-6 linkage. For high mannose and hybrid structures omega was found to be -60 degrees, for bisected biantennary complex structures omega was 180 degrees, and for complex biantennary structures averaging between -60 degrees and 180 degrees occurs.

Asparagine↗
PubMed1979

Complex factorial structure of Ellis' Irrational Beliefs.

Previous research with a newly developed children's scale for measuring Ellis' constructs with regard to irrational beliefs showed that while the scale clearly reflected theoretically predicted developmental trends, the internal consistency reliability of the overall 11-item scale was relatively low (.56). This low internal consistency suggested that the 11 items were in fact heterogeneous and were not measuring a single, univocal, construct. An exploratory factor analysis was performed on the responses of 788 fifth-through thirteenth-grade students. Results of the Alpha factor and analysis clearly indicate that the scale is multifaceted, i.e., comprised of four orthogonal, interpretable factors. Estimates of internal consistency reliabilities for the separate factors, when corrected for length, revealed that each of the new factors was more reliable than the full scale score in this sample. Potential revisions of the scale, as well as cautions for its use in clinical applications on its present form are discussed.

Adolescent↗
PubMed1976

Fine structure of complex ocelli of a cubomedusan, Tamoya bursaria Haeckel.

The retina of the distal and proximal lens-bearing complex ocelli are composed of pigmented sensory cells and long pigmented cells. A ciliary sheath from each sensory cell, together with the processes of long pigmented cells, extends through the vitreous layer as far as the capsule that envelops the lens. Each ciliary sheath has several ballon-like swellings and the ciliary microtubules, arranged in the 9+2 pattern in the proximal part, are markedly disorganized distally in the swollen parts, out of which extends most of the microvilli in the vitreous layer. It is suggested that some of the microvilli may originate in vesicles that are constricted off from the surface of the pigmented sensory cells. Closely packed microvilli run in parallel in short bundles. In addition to characteristic junctions between sensory cells, junctions that are presumably synaptic and, of a new type in coelenterates, are observed between sensory cells and nerve endings.

Animals↗