Targeting motifs and functional parameters governing the assembly of connexins into gap junctions
Explore the source record for details and available documents.
Journal Article↗
Biomedical subjects
C Wilson
Publications and source records attributed to C Wilson.
2.8 A crystal structure of the malachite green aptamer.
Previous in vitro selection experiments identified an RNA aptamer that recognizes the chromophore malachite green (MG) with a high level of affinity, and which undergoes site-specific cleavage following laser irradiation. To understand the mechanism by which this RNA folds to recognize specifically its ligand and the structural basis for chromophore-assisted laser inactivation, we have determined the 2.8 A crystal structure of the aptamer bound to tetramethylrosamine (TMR), a high-affinity MG analog. The ligand-binding site is defined by an asymmetric internal loop, flanked by a pair of helices. A U-turn and several non-canonical base interactions stabilize the folding of loop nucleotides around the TMR. The aptamer utilizes several tiers of stacked nucleotides arranged in pairs, triples, and a novel base quadruple to effectively encapsulate the ligand. Even in the absence of specific stabilizing hydrogen bonds, discrimination between related fluorophores and chromophores is possible due to tight packing in the RNA binding pocket, which severely limits the size and shape of recognized ligands. The site of laser-induced cleavage lies relatively far from the bound TMR ( approximately 15 A). The unusual backbone conformation of the cleavage site nucleotide and its high level of solvent accessibility may combine to allow preferential reaction with freely diffusing hydroxyl radicals generated at the bound ligand. Several observations, however, favor alternative mechanisms for cleavage, such as conformational changes in the aptamer or long-range electron transfer between the bound ligand and the cleavage site nucleotide.
Base Pairing↗
The quality of systematic reviews. Cochrane Collaboration should ensure equitable participation in management and policy.
Explore the source record for details and available documents.
Asthma↗
A water channel in the core of the vitamin B(12) RNA aptamer.
BACKGROUND: The 3.0 A crystal structure of the vitamin B(12) RNA aptamer revealed an unusual tertiary structure that is rich in novel RNA structural motifs. Important details of the interactions that stabilize noncanonical base pairing and the role of solvent in the structure were not apparent owing to the limited resolution. RESULTS: The structure of the vitamin B(12) RNA aptamer in complex with its ligand has been determined at 2.3 A resolution by X-ray crystallography. The crystallographic asymmetric unit contains five independent copies of the aptamer-vitamin B(12) complex, making it possible to accurately define well-conserved features. The core of the aptamer contains an unusual water-filled channel that is buried between the three strands of an RNA triplex. Well-ordered water molecules positioned within this channel form bridging hydrogen bonds and stabilize planar base triples that otherwise lack significant direct base-base contacts. The water channel terminates at the interface between the RNA and the bound ligand, leaving a pair of water molecules appropriately positioned to hydrogen bond with the highly polarized cyanide nitrogen of vitamin B(12). Analysis of the general solvation patterns for each nucleotide suggests that water molecules are not precisely positioned, as observed in previous RNA duplex structures, but instead might adjust in response to the varying local environment. Unusual intermolecular base pairing contributes to the formation of three different dimerization contacts that drive formation of the crystal lattice. CONCLUSIONS: The structure demonstrates the important role of water molecules and noncanonical base pairing in driving the formation of RNA tertiary structure and facilitating specific interactions of RNAs with other molecules.
Computer Simulation↗
Targeting motifs and functional parameters governing the assembly of connexins into gap junctions.
To study the assembly of gap junctions, connexin--green-fluorescent-protein (Cx--GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26-- and Cx32--GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32--GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx--GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.
Amino Acid Motifs↗
The 1.3 A crystal structure of a biotin-binding pseudoknot and the basis for RNA molecular recognition.
A pseudoknot-containing aptamer isolated from a pool of random sequence molecules has been shown previously to represent an optimal RNA solution to the problem of binding biotin. The affinity of this RNA molecule is nonetheless orders of magnitude weaker than that of its highly evolved protein analogs, avidin and streptavidin. To understand the structural basis for biotin binding and to compare directly strategies for ligand recognition available to proteins and RNA molecules, we have determined the 1.3 A crystal structure of the aptamer complexed with its ligand. Biotin is bound at the interface between the pseudoknot's stacked helices in a pocket defined almost entirely by base-paired nucleotides. In comparison to the protein avidin, the aptamer packs more tightly around the biotin headgroup and makes fewer contacts with its fatty acid tail. Whereas biotin is deeply buried within the hydrophobic core in the avidin complex, the aptamer relies on a combination of hydrated magnesium ions and immobilized water molecules to surround its ligand. In addition to demonstrating fundamentally different approaches to molecular recognition by proteins and RNA, the structure provides general insight into the mechanisms by which RNA function is mediated by divalent metals.
Avidin↗
Evidence for evolutionarily conserved secondary structure in the H19 tumor suppressor RNA.
The molecular basis for function of the mammalian H19 as a tumor suppressor is poorly understood. Large, conserved open reading frames (ORFs) are absent from both the human and mouse cDNAs, suggesting that it may act as an RNA. Contradicting earlier reports, however, recent studies have shown that the H19 transcript exists in polysomal form and is likely translated. To distinguish between possible functional roles for the gene product, we have characterized the sequence requirements for H19-mediated in vitro suppression of tumor cell clonogenicity and analyzed the sequence of the gene cloned from a range of mammals. A cDNA version of the human gene, lacking the unusually short introns characteristic of imprinted genes, is as effective as a genomic copy in blocking anchorage-independent growth by G401 cells. The first 710 nucleotides of the gene can be deleted with no effect on in vitro activity. Further truncations from either the 5'- or 3'-end, however, cause a loss of suppression of clonogenicity. Using conserved sequences within the H19 gene as PCR primers, genomic DNA fragments were amplified from a range of mammalian species that span the functional domain defined by deletion analysis. Sequences from cat, lynx, elephant, gopher and orangutan complement the previous database of sequences from human, mouse, rat and rabbit. Hypothetical translation of the resulting sequences shows an absence of conserved ORFs of any size. Free energy and covariational analysis of the RNA sequences was used to identify potential helical pairings within the H19 transcript. A set of 16 helices are supported by covariation (i.e. conservation of base pairing potential in the absence of primary sequence conservation). The predicted RNA pairings consist largely of local hairpins but also include several long range interactions that bridge the 5'- and 3'-ends of the functional domain. Given the evolutionary conservation of structure at the RNA level and the absence of conservation at the protein level, we presume that the functional product of the H19 gene is a structured RNA.
Animals↗
T-lymphocyte subsets in HIV-infected and high-risk HIV-uninfected adolescents: retention of naive T lymphocytes in HIV-infected adolescents. The Adolescent Medicine HIV/AIDS Research Network.
BACKGROUND: The capacity of the immune system of adolescents to generate and repopulate naive and memory cell populations under conditions of normal homeostasis and human immunodeficiency virus (HIV) infection is largely unknown. OBJECTIVE: To assess lymphocyte subsets in HIV-infected and high-risk HIV-negative adolescents. DESIGN: The Reaching for Excellence in Adolescent Care and Health Project of the Adolescent Medicine HIV/AIDS Research Network recruits a cohort of HIV-infected and high-risk HIV-uninfected adolescents, aged 13 to 18 years 364 days, into a study of biomedical and behavioral features of HIV infection as seen in the context of full availability of primary care and HIV-related consultative services. Lymphocyte phenotypes were determined using standard 3-color flow cytometry. SETTING: The Reaching for Excellence in Adolescent Care and Health Project is carried out at 16 clinical sites in 14 urban areas. PARTICIPANTS: T-lymphocyte subsets are reported in 192 HIV-positive and 78 HIV-negative youths. RESULTS: For HIV-positive subjects, the total CD4+ cell count and the percentage of CD4+ cells are decreased when compared with those of the HIV-negative controls (P<.001). The reduction in total CD4+ cells reflects a loss of naive, and memory, CD4+ cells compared with HIV-negative youths. Human immunodeficiency virus-infected adolescents, many of whom have been infected recently (ie, those with CD4+ cell counts > or =0.500 x 10(9)/L [500/microL]), have a significant increase in naive CD8+ cells compared with HIV-negative youths (P<.01). There also is a significant increase in memory CD8+ cells at all strata of total CD4+ cells compared with HIV-negative youths (P<.01). The increase in naive CD8+ cells in those subjects with CD4+ cell counts of 0.500 x 10(9)/L or greater is a unique finding in this cohort. CONCLUSIONS: This study demonstrates high levels of naive CD8+ cells in response to HIV infection in adolescents with CD4+ cell counts of 0.500 X 10(9)/L or greater. The presence of high levels of naive CD8+ cells suggests functioning thymic tissue in some adolescents infected with HIV. Furthermore, the normal level of naive CD4+ cells in adolescents with CD4+ levels of 0.500 x 10(9)/L or greater provides additional support for the concept of a more robust immune system in HIV-infected adolescents compared with HIV-infected adults. These observations suggest that the immune system of HIV-infected adolescents may be capable of better responses to neoantigens and cytotoxic T-lymphocyte responses to HIV than the immune system of infected children or adults. Human immunodeficiency virus-infected adolescents may have an immune system that is capable of reconstitution following highly active antiretroviral therapy.
Adolescent↗
A thermal spin transition in [Co(bpy)3][LiCr(ox)3] (ox = C2O4(2-); bpy = 2,2'-bipyridine).
In the three-dimensional oxalate network structures [M(II)(bpy)3][M(I)-M(III)(ox)3] (ox= C2O4(2-); bpy = 2,2'-bipyridine) the negatively charged oxalate backbone provides perfect cavities for tris-bipyridyl complex cations. The size of the cavity can be adjusted by variation of the metal ions of the oxalate backbone. In [Co(bpy)3][NaCr(ox)3], the [Co(bpy)3]2 + complex is in its usual 4T1(t2g5e(g)2) high-spin ground state. Substituting Na+ by Li+ reduces the size of the cavity. The resulting chemical pressure destabilises the high-spin state of [Co(bpy)3]2+ to such an extent that the 2E(t2g6e(g)1) low-spin state becomes the actual ground state. As a result. [Co(bpy)3][LiCr(ox)3] becomes a spin-crossover system, as shown by temperature-dependent magnetic susceptibility measurements and single-crystal optical spectroscopy, as well as by an X-ray structure determination at 290 and 10 K.
Journal Article↗
MAP kinases in pollen.
The male gametophyte of flowering plants has a highly regulated developmental programme to ensure efficient fertilization of the ovule and the faithful transmission of genetic material to the offspring. Cell cycle control mechanisms dictate the formation of the vegetative and generative (sperm) cells, while an increase in transcriptional/translational activity and the accumulation of stored proteins and mRNA is followed by a quiescent state at maturation. A switch to a new developmental programme occurs after the pollen tube lands on the stigma with the formation of the pollen tube, growth through the style, and subsequent fertilization. Apart from the internal control mechanisms involved in this developmental programme, pollen grains must cope with physical changes during development within the anther (desiccation) and subsequently during germination on the stigma (rehydration). The metabolic and structural changes that occur throughout these processes should require signaling mechanisms to co-ordinate the appropriate response, and recent data demonstrate the presence in pollen of an array of molecules belonging to diverse signalling pathways, including mitogen-activated protein (MAP) kinases. The role of MAP kinases in pollen is discussed in the context of the various developmental and physical changes that occur throughout pollen maturation and germination.
MAP Kinase Signaling System↗
Differential cytokeratin and glycoconjugate expression by the surface and crypt epithelia of human palatine tonsils.
Human palatine tonsils are clinically important due to their susceptibility to tonsillitis and association with other local and systemic diseases. Paradoxically, the tonsils function as antigen sampling sites of the mucosal immune system and, consequently, the tonsil epithelia perform both protective and antigen sampling roles. These epithelia are divided into stratified squamous epithelium overlying the tonsil surface and crypt epithelium lining the tonsil crypts, the latter of which includes reticular areas which are infiltrated by lymphocytes and are responsible for antigen sampling. In this study we characterised cytokeratin and glycoconjugate expression by healthy epithelia of human palatine tonsils. We identified pan-epithelial tonsil markers and also demonstrated that the surface and reticular crypt epithelia are differentiated by the expression of multiple cytokeratins. The latter finding supports the hypothesis that these epithelia undergo alternate differentiation pathways and possess different functional roles. In addition, we identified cell subpopulations in the tonsil epithelia which may represent distinct cell subtypes including specialised antigen sampling cells. These findings establish a basis for future studies to investigate histochemical changes in tonsil epithelia that are associated with or predispose to local and/or systemic disease.
Adolescent↗
Effects of serotonergic agents on the transport response in rats.
In two experiments, researchers investigated the effects of manipulating serotonin systems on the transport response and dorsal immobility response in developing rats. In Experiment 1, administration of ketanserin and cinanserin, but not metergoline, suppressed the transport response in 23-day-old rats. These agents were without effect on dorsal immobility durations. In Experiment 2, administration of quipazine to 30-, 40-, and 50-day-old rats resulted in significant increases in transport response intensities and dorsal immobility durations. Results are discussed with respect to the nature of the transport response.
Age Factors↗
Sexual Assault Nurse Examiner programs in the United States.
STUDY OBJECTIVE: We sought to provide a descriptive study of the Sexual Assault Nurse Examiner (SANE) programs and their characteristics in the United States. METHODS: A confidential survey addressing patient and staff demographics, administration attributes, examination procedures, and medical and legal issues was mailed to SANE programs in the United States. RESULTS: Sixty-one (66%) of 92 programs responded. More than half of the programs (32/58 [55%]) had been in operation for less than 5 years. Thirty (52%) of the 58 programs performed the initial sexual assault examination in hospital emergency departments. Written consent (57/59 [97%]) was obtained for the initial examination, and most (51/59 [86%]) programs used preprepared commercial sexual assault kits. Program directors were predominately registered nurses. All but one program mandated specific training requirements for their staff, with a median requirement of 80 hours. Procedures used for initial examinations varied; most offered pregnancy testing (56/58 [97%]), pregnancy prophylaxis (57/59 [97%]), and sexually transmitted disease (STD) prophylaxis (53/59 [90%]). HIV testing was not offered in 32 (54%) of 59 programs. Almost all programs used Wood's lamp (51/59 [86%]), colposcopes (42/59 [71%]), and photographs (46/59 [78%]) for documentation. Median time required per patient for initial examination and evidence collection was 3 hours (range, 1 to 8 hours). Follow-up is consistently offered to the survivor. Most programs (45/61 [74%]) could report the number of survivors treated, but few could provide information on survivor medical follow-up or the number of prosecutions by survivors and their outcomes. CONCLUSION: This survey provided an overview of SANE programs. SANE programs are similar across the country with regard to staffing, training, STD and pregnancy prophylaxis, and documentation techniques. They are inconsistent in the use of STD cultures, HIV testing, and alcohol and drug screening. SANE programs were unable to provide data regarding survivor follow-up and legal outcomes. This information is essential to evaluate the programs' effectiveness and to improve performance. The need for better outcome data should be addressed to define success or failure of SANE programs.
Emergency Service, Hospital↗
Interaction of alpha-melanotropin (alpha-MSH) and noradrenaline in the median eminence in the control of female sexual behavior.
In the present study, we examined the effects of the injection of alpha-melanotropin (alpha-MSH), noradrenaline (NA), and dopamine in the median eminence of ovariectomized-adrenalectomized rats on female sexual behavior. The animals were primed with l0 microg of estradiol benzoate, and 52-54 h later they were injected into the median eminence with either 1 microl of artificial cerebrospinal fluid, 1 microg/rat alpha-MSH, 200 ng/rat NA, 200 ng or 2 microg/rat dopamine, in 1 microl of artificial cerebrospinal fluid. Both alpha-MSH and NA significantly stimulated sexual behavior. This effect was antagonized by two beta-adrenergic antagonists: propranolol (500 ng/rat) and metoprolol (400 ng/rat) applied 15 min before the alpha-MSH or NA. The alpha-adrenergic antagonist prazosine (500 ng/rat) was ineffective in reducing the effect of alpha-MSH. The vehicle and dopamine at both doses had no effect on sexual activity. These results indicate that alpha-MSH and NA in the median eminence stimulate female sexual behavior and that NA mediates the action of alpha-MSH via beta-receptors.
Adrenalectomy↗
Collagenous fibroma (desmoplastic fibroblastoma): a unique presentation as a goiter in an 88-year-old man.
Collagenous fibroma (desmoplastic fibroblastoma) is a recently described entity in the medical literature. This entity has been reported in various locations, including the upper extremities, posterior neck, upper back, lower extremities, abdominal wall, and hip. We report an interesting case of an 88-year-old man who presented with an apparent goiter involving the right anterolateral neck. Histologic studies revealed a well-circumscribed, paucicellular lesion composed of stellate and spindle-shaped fibroblasts separated by bundles of collagen. No mitotic figures, necrosis, or calcification was observed. The stellate and spindle-shaped cells were positive for vimentin and focally positive for desmin, indicating myofibroblastic differentiation. Our case exemplifies the diagnostic difficulties that these tumors may pose from the clinical and radiologic standpoint when they clinically present as a goiter.
Aged↗
Molecular mimicry between HLA-DR alleles associated with rheumatoid arthritis and Proteus mirabilis as the Aetiological basis for autoimmunity.
Molecular mimicry is one of the pathological mechanisms proposed to explain the association between microorganisms and autoimmune diseases. This review deals with the association between bacteria and rheumatic diseases with a special emphasis on rheumatoid arthritis where upper urinary tract infection by Proteus mirabilis is the possible cause of this severe, arthritic condition. Prospective trials involving anti-Proteus therapy should be carried out.
Alleles↗
The effect of ablation sequence and duration on lesion shape using rapidly pulsed radiofrequency energy through multiple electrodes.
Sequences of energy application to multiple electrodes and a study of ablation duration with distal tip and multi-electrode ablations were explored with a radiofrequency controller that distributes energy from a generator to up to 4 electrodes with various duty cycles. In vitro ablations were performed on bovine left ventricle in circulating blood and lesions in goats were performed to verify the in vitro results. All of the ablation sequences with simultaneous electrode activation of contiguous electrodes resulted in deeper lesions than those created in sequence. There was also no scalloping of the lesion if contiguous electrodes were activated simultaneously. During all distal tip ablations, lesion volume and depth was greater after 3 minutes of energy delivery than after 1 minute, but did not increase from 3 minutes to 5 minutes. There was a significant increase in multi-electrode ablation lesion depth with each additional minute in the ablation cycle. The in vivo ablations verified these results at 120 and 300 second ablations. Pulsed energy distal tip ablations resulted in deeper lesions than continuous only if power amplitudes over 50 W were employed. In conclusion, contiguous electrodes in simultaneous use create lesions that resemble one large lesion rather than two lesions positioned next to each other. Multi-electrode ablation lesions continue to grow at ablation durations of up to 5 minutes compared to distal tip lesions which reach steady-state between 1 and 3 minutes. Pulsed energy delivery to distal tips may result in deeper lesions than conventional if high powers are employed.
Animals↗
The structural basis for molecular recognition by the vitamin B 12 RNA aptamer.
Previous solution structures of ligand-binding RNA aptamers have shown that molecular recognition is achieved by the folding of an initially unstructured RNA around its cognate ligand, coupling the processes of RNA folding and binding. The 3 A crystal structure of the cyanocobalamin (vitamin B12) aptamer reported here suggests a different approach to molecular recognition in which elements of RNA secondary structure combine to create a solvent-accessible docking surface for a large, complex ligand. Central to this structure is a locally folding RNA triplex, stabilized by a novel three-stranded zipper. Perpendicular stacking of a duplex on this triplex creates a cleft that functions as the vitamin B12 binding site. Complementary packing of hydrophobic surfaces, direct hydrogen bonding and dipolar interactions between the ligand and the RNA appear to contribute to binding. The nature of the interactions that stabilize complex formation and the possible uncoupling of folding and binding for this RNA suggest a strong mechanistic similarity to typical protein-ligand complexes.
Base Pairing↗