Re: Bladder histological changes associated with chronic indwelling urinary catheter.
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Catheters, Indwelling↗
Biomedical subjects
S C Harrison
Publications and source records attributed to S C Harrison.
Crystal structures of c-Src reveal features of its autoinhibitory mechanism.
Src family kinases are maintained in an assembled, inactive conformation by intramolecular interactions of their SH2 and SH3 domains. Full catalytic activity requires release of these restraints as well as phosphorylation of Tyr-416 in the activation loop. In previous structures of inactive Src kinases, Tyr-416 and flanking residues are disordered. We report here four additional c-Src structures in which this segment adopts an ordered but inhibitory conformation. The ordered activation loop forms an alpha helix that stabilizes the inactive conformation of the kinase domain, blocks the peptide substrate-binding site, and prevents Tyr-416 phosphorylation. Disassembly of the regulatory domains, induced by SH2 or SH3 ligands, or by dephosphorylation of Tyr-527, could lead to exposure and phosphorylation of Tyr-416.
Adenosine Triphosphate↗
Functional organization of clathrin in coats: combining electron cryomicroscopy and X-ray crystallography.
The sorting of specific proteins into clathrin-coated pits and the mechanics of membrane invagination are determined by assembly of the clathrin lattice. Recent structures of a six-fold barrel clathrin coat at 21 A resolution by electron cryomicroscopy and of the clathrin terminal domain and linker at 2.6 A by X-ray crystallography together show how domains of clathrin interact and orient within the coat and reveal the strongly puckered shape and conformational variability of individual triskelions. The beta propeller of the terminal domain faces the membrane so that recognition segments from adaptor proteins can extend along its lateral grooves. Clathrin legs adapt to different coat environments in the barrel by flexing along a segment at the knee that is free of contacts with other molecules.
Clathrin↗
Selection of gp41-mediated HIV-1 cell entry inhibitors from biased combinatorial libraries of non-natural binding elements.
The trimeric, alpha-helical coiled-coil core of the HIV-1 gp41 ectodomain is thought to be part of a transient, receptor-triggered intermediate in the refolding of the envelope glycoprotein into a fusion-active conformation. In an effort to discover small organic inhibitors that block gp41 activation, we have generated a biased combinatorial chemical library of non-natural binding elements targeted to the gp41 core. From this library of 61,275 potential ligands, we have identified elements that, when covalently attached to a peptide derived from the gp41 outer-layer alpha-helix, contribute to the formation of a stable complex with the inner core and to inhibition of gp41-mediated cell fusion.
Amino Acid Sequence↗
Reactive oxygen species (ROS) production and the outcome of diagnostic tests of sperm function.
Washed sperm suspensions from 64 out of 89 (72%) randomly selected infertility patients produced detectable reactive oxygen species (ROS) compared to 17 out of 67 (25%) prospective semen donors (p < 0.01, Chi-square test). Among patients, the median sperm concentration in ejaculates which yielded sperm suspensions that generated detectable levels of ROS was lower than in those which did not: 36.2 (15.63-57.64) vs. 71.5 (22-108) x 10(6)/mL, respectively (median (interquartile range), p < 0.05, Kruskal-Wallis test). In samples that produced ROS, the basal rate of production and the rates after stimulation with 50 mumol N-formyl met leu phe (N-FMLP) l-1 or with 100 nmol phorbol 12-myristate 13-acetate (PMA) l-1 were significantly and inversely correlated with sperm concentration in the ejaculate (r = -0.43, -0.41 and -0.35, respectively, p < 0.01 Spearman's rank correlation). The rate of ROS production showed no relationship to the motility of spermatozoa in semen, whether evaluated visually or via computer assisted semen analysis. However, there was a significant negative correlation (r = -0.370) between the motile, normal sperm concentration (MNSC) and basal ROS production, and when stimulated with N-FMLP (r = -0.311) or with PMA (r = -0.249) (all p < 0.05). In patient samples that generated detectable ROS, the ability of the spermatozoa to retain motility for 24 h after preparation on a 40/80% Percoll gradient was negatively correlated with basal ROS production (r = -0.310, p < 0.05). ROS production was also related to the outcome of in vitro sperm mucus penetration tests. Unstimulated levels of ROS production showed a significant (p < 0.05), negative correlation with the number of progressively motile spermatozoa present in mucus after 15 (r = -0.379) and 60 (r = -0.362) min. These results suggest that sperm samples with increased ROS tend to have poor semen quality and reduced performance in a number of routine, diagnostic sperm function tests.
Adult↗
Natural-fill urodynamics in chronically catheterized patients with spinal-cord injury.
OBJECTIVE: To determine whether an indwelling catheter on free drainage provides a constantly low intravesical pressure in patients with a neuropathic bladder. PATIENTS AND METHODS: Thirty patients with complete spinal-cord injury (SCI) whose bladders were managed exclusively with an indwelling catheter were assessed urodynamically using natural-fill urodynamics (ambulatory monitoring) while their catheters were left on free drainage. Their upper urinary tracts were assessed using plain X-rays and ultrasonography. RESULTS: Detrusor contractions causing intravesical pressure rises of >40 cmH2O for up to 4.5 min were observed in 11 patients. Renal scarring was observed in nine patients; of these, six were in the group with contractions of > 40 cmH2O, whereas only five of 21 patients with normal kidneys had such pressure rises. CONCLUSION: An indwelling catheter on free drainage is no guarantee of a constantly low intravesical pressure. This study provides evidence to suggest that there is an association between phasic bladder contractions which occur despite catheter drainage and upper urinary tract damage in permanently catheterized patients with SCI.
Adult↗
Signal integration by transcription-factor assemblies: interactions of NF-AT1 and AP-1 on the IL-2 promoter.
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Animals↗
Peptide ligands to human immunodeficiency virus type 1 gp120 identified from phage display libraries.
We have used phage-displayed peptide libraries to identify novel ligands to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120. Screening of libraries of random 12-mers, 7-mers, and cyclic 9-mers produced two families of gp120 binding peptides. Members of a family with the prototype sequence RINNIPWSEAMM (peptide 12p1) inhibit the interaction between gp120 and both four-domain soluble CD4 (4dCD4) and monoclonal antibody (MAb) 17b, a neutralizing antibody that covers the chemokine receptor binding surface on gp120. Peptide 12p1 inhibits the interaction of 4dCD4 with gp120 from three different HIV strains, implying that it binds to a conserved site on gp120. Members of a second family of peptides, with the prototype sequence TSPYEDWQTYLM (peptide 12p2), bind more weakly to gp120. They do not detectably affect its interaction with 4dCD4, but they enhance its binding to MAb 17b. A common sequence motif in the two peptide families and cross-competition for gp120 binding suggest that they have overlapping contacts. Their divergent effects on the affinity of gp120 for MAb 17b may indicate that their binding stabilizes distinct conformational states of gp120. The functional properties of 12p1 suggest that it might be a useful lead for the development of inhibitors of HIV entry.
Amino Acid Sequence↗
Discrimination between sialic acid-containing receptors and pseudoreceptors regulates polyomavirus spread in the mouse.
Variations in the polyomavirus major capsid protein VP1 underlie important biological differences between highly pathogenic large-plaque and relatively nonpathogenic small-plaque strains. These polymorphisms constitute major determinants of virus spread in mice and also dictate previously recognized strain differences in sialyloligosaccharide binding. X-ray crystallographic studies have shown that these determinants affect binding to the sialic acids. Here we report results of further experiments designed to test the importance of specific contacts between VP1 and the carbohydrate moieties of the receptor. With minor exceptions, substitutions at positions predicted from crystallography to be important in binding the terminal alpha-2,3-linked sialic acid or the penultimate sugar (galactose) destroyed the ability of the virus to replicate in cell culture. Substitutions that prevented binding to a branched disialyloligosaccharide were found to result in viruses that were both viable in culture and tumorigenic in the mouse. Conversely, substitutions that allowed recognition and binding of the branched carbohydrate chain inhibited spread in the mouse, though the viruses remained viable in culture. Mice of five different inbred strains, all highly susceptible to large-plaque virus, showed resistance to the spread of polyomavirus strains bearing the VP1 type which binds the branched-chain receptor. We suggest that glycoproteins bearing the appropriate O-linked branched sialyloligosaccharide chains are effective pseudoreceptors in the host and that they block the spread of potentially tumorigenic or virulent virus strains.
3T3 Cells↗
Structure of an IkappaBalpha/NF-kappaB complex.
The inhibitory protein, IkappaBalpha, sequesters the transcription factor, NF-kappaB, as an inactive complex in the cytoplasm. The structure of the IkappaBalpha ankyrin repeat domain, bound to a partially truncated NF-kappaB heterodimer (p50/ p65), has been determined by X-ray crystallography at 2.7 A resolution. It shows a stack of six IkappaBalpha ankyrin repeats facing the C-terminal domains of the NF-kappaB Rel homology regions. Contacts occur in discontinuous patches, suggesting a combinatorial quality for ankyrin repeat specificity. The first two repeats cover an alpha helically ordered segment containing the p65 nuclear localization signal. The position of the sixth ankyrin repeat shows that full-length IkappaBalpha will occlude the NF-kappaB DNA-binding cleft. The orientation of IkappaBalpha in the complex places its N- and C-terminal regions in appropriate locations for their known regulatory functions.
Amino Acid Sequence↗
Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance.
A combinatorial disulfide cross-linking strategy was used to prepare a stalled complex of human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase with a DNA template:primer and a deoxynucleoside triphosphate (dNTP), and the crystal structure of the complex was determined at a resolution of 3.2 angstroms. The presence of a dideoxynucleotide at the 3'-primer terminus allows capture of a state in which the substrates are poised for attack on the dNTP. Conformational changes that accompany formation of the catalytic complex produce distinct clusters of the residues that are altered in viruses resistant to nucleoside analog drugs. The positioning of these residues in the neighborhood of the dNTP helps to resolve some long-standing puzzles about the molecular basis of resistance. The resistance mutations are likely to influence binding or reactivity of the inhibitors, relative to normal dNTPs, and the clustering of the mutations correlates with the chemical structure of the drug.
Anti-HIV Agents↗
Atomic structure of clathrin: a beta propeller terminal domain joins an alpha zigzag linker.
Clathrin triskelions form the lattice that organizes recruitment of proteins to coated pits and helps drive vesiculation of the lipid bilayer. We report the crystal structure at 2.6 A resolution of a 55 kDa N-terminal fragment from the 190 kDa clathrin heavy chain. The structure comprises the globular "terminal domain" and the linker that joins it to the end of a triskelion leg. The terminal domain is a seven-blade beta propeller, a structure well adapted to interaction with multiple partners, such as the AP-1 and AP-2 sorting adaptor complexes and the nonvisual arrestins. The linker is an alpha-helical zigzag emanating from the propeller domain. We propose that this simple motif may extend into the rest of the clathrin leg.
Amino Acid Sequence↗
A Zn2+ ion links the cytoplasmic tail of CD4 and the N-terminal region of Lck.
Lck is a lymphoid-specific, Src family protein-tyrosine kinase that is known to interact with the T-cell coreceptors, CD4 and CD8. This interaction, which is critical for proper T-cell function, is mediated by the N-terminal unique region of Lck and the C-terminal cytoplasmic tail of the coreceptors. A pair of cysteines on each molecule is essential for association, suggesting that CD4 or CD8 may interact with Lck by jointly coordinating a metal ion. We describe here experiments in which a maltose-binding protein fusion protein bearing the CD4 tail has been coexpressed in Escherichia coli with an N-terminal fragment of Lck. The proteins associate in the expressing cells, forming a complex that can be affinity-purified. Formation of this complex, like the in vivo interaction, depends upon the two pairs of cysteines. Biochemical and biophysical experiments show that the complex dissociates in the presence of EDTA and that it contains a single Zn2+ ion. These results are consistent with the proposal that Lck and CD4 associate by thiol-mediated co-coordination of zinc.
Amino Acid Sequence↗
Structural similarities between topoisomerases that cleave one or both DNA strands.
Type IA and type II DNA topoisomerases are distinguished by their ability to cleave one or two strands, respectively, of a DNA duplex. Both types have been proposed to use an "enzyme-bridging" mechanism, in which a break is formed in a DNA strand and a gap is opened between the broken pieces to allow passage of a second DNA strand or duplex segment. Although the type IA and type II topoisomerase structures appear overall quite different from one another, unexpected similarities between several structural elements suggest that members of the two subfamilies may use comparable mechanisms to bind and cleave DNA.
Binding Sites↗
Interaction of polyomavirus internal protein VP2 with the major capsid protein VP1 and implications for participation of VP2 in viral entry.
A complex of the polyomavirus internal protein VP2/VP3 with the pentameric major capsid protein VP1 has been prepared by co-expression in Escherichia coli. A C-terminal segment of VP2/VP3 is required for tight association, and a crystal structure of this segment, complexed with a VP1 pentamer, has been determined at 2.2 A resolution. The structure shows specific contacts between a single copy of the internal protein and a pentamer of VP1. These interactions were not detected in the previously described structure of the virion, but the location of VP2 in the recombinant complex is consistent with features in the virion electron-density map. The C-terminus of VP2/VP3 inserts in an unusual, hairpin-like manner into the axial cavity of the VP1 pentamer, where it is anchored strongly by hydrophobic interactions. The remainder of the internal protein appears to have significant flexibility. This structure restricts possible models for exposure of the internal proteins during viral entry.
Amino Acid Sequence↗
Differing roles for zinc fingers in DNA recognition: structure of a six-finger transcription factor IIIA complex.
The crystal structure of the six NH2-terminal zinc fingers of Xenopus laevis transcription factor IIIA (TFIIIA) bound with 31 bp of the 5S rRNA gene promoter has been determined at 3.1 A resolution. Individual zinc fingers are positioned differently in the major groove and across the minor groove of DNA to span the entire length of the duplex. These results show how TFIIIA can recognize several separated DNA sequences by using fewer fingers than necessary for continuous winding in the major groove.
Amino Acid Sequence↗
Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA.
The nuclear factor of activated T cells (NFAT) and the AP-1 heterodimer, Fos-Jun, cooperatively bind a composite DNA site and synergistically activate the expression of many immune-response genes. A 2.7-A-resolution crystal structure of the DNA-binding domains of NFAT, Fos and Jun, in a quaternary complex with a DNA fragment containing the distal antigen-receptor response element from the interleukin-2 gene promoter, shows an extended interface between NFAT and AP-1, facilitated by the bending of Fos and DNA. The tight association of the three proteins on DNA creates a continuous groove for the recognition of 15 base pairs.
Amino Acid Sequence↗
Construction and characterization of a radio-iodinatable mutant of recombinant human CD4.
Recombinant soluble human CD4 (rsCD4) has been used in iodinated form to study the interaction of CD4 with its ligands. However, the utility of [125I]-rsCD4 is limited because rsCD4 is inefficiently iodinated and the iodinated protein is poorly active. The iodination properties of rsCD4 most likely reflect the poor accessibility of the tyrosine residues, apparent from the available X-ray structures. We have generated an iodinatable mutant of rsCD4 by substituting Tyr for Phe(179) in the flexible, solvent-exposed C-terminal region of rsCD4(183), a truncated form of CD4 that consists of the first 183 residues of CD4 and includes the binding sites for HIV-1 gp120 and MHC class II molecules. When F179Y rsCD4(183) is iodinated under trace-labeling conditions, the efficiency of 125I incorporation and the percentage of iodinated molecules that are active are much enhanced compared with WT rsCD4. Moreover, trace-labeled [125I]-F179Y rsCD4(183) has the same affinity for HIV-1 rgp120 as unlabeled WT rsCD4. The improved activity of trace-labeled [125I]-F179Y rsCD4(183) appears to be due to effective competition by Y179 for reactive iodine species that, in WT rsCD4, react with traces of denatured protein and/or with residues critical for activity or conformational integrity. The incorporation of accessible tyrosine residues may improve the iodinatibility of a protein both by introducing a readily iodinatable residue and by protecting sensitive proteins from adverse reactions.
Animals↗