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PubMed2004

Correlated C-C and C-O bond conformations in saccharide hydroxymethyl groups: parametrization and application of redundant 1H-1H, 13C-1H, and 13C-13C NMR J-couplings.

Methyl alpha- and beta-pyranosides of d-glucose and d-galactose 1-4 were prepared containing single sites of (13)C-enrichment at C4, C5, and C6 (12 compounds), and (1)H and (13)C[(1)H] NMR spectra were obtained to determine a complete set of J-couplings ((1)J, (2)J, and (3)J) involving the labeled carbon and nearby protons and carbons within the exocyclic hydroxymethyl group (CH(2)OH) of each compound. In parallel theoretical studies, the dependencies of (1)J, (2)J, and (3)J involving (1)H and (13)C on the C5-C6 (omega) and C6-O6 (theta;) torsion angles in aldohexopyranoside model compounds were computed using density functional theory (DFT) and a special basis set designed to reliably recover the Fermi contact contribution to the coupling. Complete hypersurfaces for (1)J(C5,C6), (2)J(C5,H6)(R), (2)J(C5,H6)(S), (2)J(C6,H5), (2)J(C4,C6), (3)J(C4,H6)(R), (3)J(C4,H6)(S), and (3)J(C6,H4), as well as (2)J(H6)(R)(,H6)(S), (3)J(H5,H6)(R), and (3)J(H5,H6)(S), were obtained and used to parametrize new equations correlating these couplings to omega and/or theta;. DFT-computed couplings were also tested for accuracy by measuring J-couplings in (13)C-labeled 4,6-O-ethylidene derivatives of d-glucose and d-galactose in which values of omega and theta; were constrained. Using a new computer program, Chymesa, designed to utilize multiple J-couplings sensitive to exocyclic CH(2)OH conformation, the ensemble of experimental couplings observed in 1-4 were analyzed to yield preferred rotamer populations about omega and theta;. Importantly, due to the sensitivity of some couplings, most notably (2)J(H6)(R)(,H6)(S), (2)J(C5,H6)(R), and (2)J(C5,H6)(S), to both omega and theta;, unique information on correlated conformation about both torsion angles was obtained. The latter treatment represents a means of evaluating correlated conformation in 1,6-linked oligosaccharides, since psi and theta; are redundant in these linkages. In the latter regard, multiple, redundant scalar couplings originating from both sides of the glycosidic linkage can be used collectively to evaluate conformational correlations between psi/theta; and C5-C6 bond rotamers.

Carbohydrate Conformation↗

PubMed2004

Theoretical calculation of the NMR spin-spin coupling constants and the NMR shifts allow distinguishability between the specific direct and the water-mediated binding of a divalent metal cation to guanine.

The calculated intermolecular and intramolecular indirect NMR spin-spin coupling constants and NMR shifts were used for the discrimination between the inner-shell and the outer-shell binding motif of hydrated divalent cations Mg(2+) or Zn(2+) with a guanine base. The intermolecular coupling constants (1)J(X,O6) and (1)J(X,N7) (X = Mg(2+), Zn(2+)) can be unambiguously assigned to the specific inner-shell binding motif of the hydrated cation either with oxygen O6 or with nitrogen N7 of guanine. The calculated coupling constants (1)J(Mg,O6) and (1)J(Zn,O6) were 6.2 and -17.5 Hz, respectively, for the inner-shell complex of cation directly interacting with oxygen O6 of guanine. For the inner-shell coordination of the cation at nitrogen N7, the calculated coupling constants (1)J(Mg,N7) and (1)J(Zn,N7) were 5.6 and -36.5 Hz, respectively. When the binding of the cation is water-mediated, the coupling constant is zero. To obtain reliable shifts in NMR parameters, hydrated guanine was utilized as the reference state. The calculated change of NMR spin-spin coupling constants due to the hydration and coordination of the cation with guanine is caused mainly by the variation of Fermi-contact coupling contribution while the variation of diamagnetic spin-orbit, paramagnetic spin-orbit, and spin-dipolar coupling contributions is small. The change of s-character of guanine sigma bonding, sigma antibonding, and lone pair orbitals upon the hydration and cation coordination (calculated using the Natural Bond Orbital analysis) correlates with the variation of the Fermi-contact term. The calculated NMR shifts delta(N7) of -15.3 and -12.2 ppm upon the coordination of Mg(2+) and Zn(2+) ion are similar to the NMR shift of 19.6 ppm toward the high field measured by Tanaka for N7 of guanine upon the coordination of the Cd(2+) cation (Tanaka, Y.; Kojima, C.; Morita, E. H.; Kasai. Y.; Yamasaki, K.; Ono, A.; Kainosho, M.; Taira, K. J. Am. Chem. Soc. 2002, 124, 4595-4601). The present data indicate that measurements of NMR intermolecular coupling constants may be used to discriminate between the specific inner- and outer-shell binding of divalent cations to nucleobases in DNA and RNA.

Cations, Divalent↗

PubMed2005

Strong effect of hydrodynamic coupling on the electric dichroism of bent rods.

The effect of hydrodynamic coupling on the spatial orientation of rigid bent rods in electric fields has been analyzed by Brownian dynamics simulations. Bead models for smoothly bent rods were constructed with dimensions of DNA double helices, and established simulation procedures were used to calculate their diffusion tensor, including the translational-rotational coupling tensor. The electric and optical parameters were assigned on the basis of known properties of double helices. Brownian dynamics simulations of the orientation of these models in electric fields showed that both transients and amplitudes of the calculated dichroism are very strongly dependent on translational-rotational coupling over a wide range of electric field strengths. For example, the stationary dichroism of a smoothly bent 179 bp DNA fragment calculated at low field strengths is positive in the presence and negative in the absence of hydrodynamic coupling. The transients are converted from a biphasic to a monophasic shape, when hydrodynamic coupling is turned off. The large changes resulting from hydrodynamic coupling were controlled by calculations based on analytical expressions derived for electrooptical response curves in the limit of low electric field strengths; the results obtained by this independent approach are in very satisfactory agreement with our Brownian dynamics simulations. The effect is strongly dependent on the electric dipole and on its direction. In the absence of any dipole the coupling effect was not observed. The coupling effect increases with the size of the bent rods. Because most macromolecular structures are known to have induced and/or permanent dipole moments, large effects of hydrodynamic coupling on both the amplitudes and the transients of the electric dichroism/birefringence must be expected in general for structures with nonsymmetric shape.

Journal Article↗

PubMed2003

Overall structure and sugar dynamics of a DNA dodecamer from homo- and heteronuclear dipolar couplings and 31P chemical shift anisotropy.

The solution structure of d(CGCGAATTCGCG)(2) has been determined on the basis of an exceptionally large set of residual dipolar couplings. In addition to the heteronuclear (13)C-(1)H and (15)N-(1)H and qualitative homonuclear (1)H-(1)H dipolar couplings, previously measured in bicelle medium, more than 300 quantitative (1)H-(1)H and 22 (31)P-(1)H dipolar restraints were obtained in liquid crystalline Pf1 medium, and 22 (31)P chemical shift anisotropy restraints. High quality DNA structures can be obtained solely on the basis of these new restraints, and these structures are in close agreement with those calculated previously on the basis of (13)C-(1)H and (15)N-(1)H dipolar couplings. In the newly calculated structures, (31)P-(1)H dipolar and (3)JsubH3(')Psub couplings and (31)P CSA data restrain the phosphodiester backbone torsion angles. The final structure represents a quite regular B-form helix with a modest bending of approximately 10 degrees, which is essentially independent of whether or not electrostatic terms are used in the calculation. Combined, the number of homo- and heteronuclear dipolar couplings significantly exceeds the number of degrees of freedom in the system. Results indicate that the dipolar coupling data cannot be fit by a single structure, but are compatible with the presence of rapid equilibria between C2(')-endo and C3(')-endo deoxyribose puckers (sugar switching). The C2(')-H2(')/H2(") dipolar couplings in B-form DNA are particularly sensitive to sugar pucker and yield the largest discrepancies when fit to a single structure. To resolve these discrepancies, we suggest a simplified dipolar coupling analysis that yields N/S equilibria for the ribose sugar puckers, which are in good agreement with previous analyses of NMR J(HH) couplings, with a population of the minor C3(')-endo form higher for pyrimidines than for purines.

Anisotropy↗

PubMed1986

Interaction of sex role self-concept with relationship quality and relationship beliefs in married, heterosexual cohabiting, gay, and lesbian couples.

The relation between sex role self-concept (masculine, feminine, undifferentiated, and androgynous) and both relationship quality and dysfunctional relationship beliefs was examined in 370 monogamous partners who represented four types of couples (married, heterosexual cohabiting, gay, and lesbian). Analyses used both the individual partner and the couple as the unit of analysis. The individual partner analyses revealed that relationship quality and relationship beliefs differed by subjects' sex role self-concept. Androgynous and feminine subjects reported higher relationship quality than masculine and undifferentiated subjects; androgynous subjects had fewer "disagreement is destructive" beliefs than feminine subjects; and androgynous subjects had fewer "partner cannot change" beliefs than undifferentiated subjects. The couple analyses revealed a relation between partners' sex role self-concept only for the heterosexual cohabiting couples. For these couples, masculine men tended to pair with feminine or undifferentiated women, and androgynous partners tended to pair together. Relative to other couples, couples in which one or both partners were androgynous or feminine reported the highest relationship quality, whereas couples in which one or both partners were undifferentiated or masculine reported the lowest relationship quality. These effects did not vary by type of couple. The study concluded that sex role self-concept is a robust factor in appraisals of relationship quality. Relative to masculine and undifferentiated individuals, androgynous and feminine individuals report greater positive relationship functioning.

Adult↗

PubMed2001

Activation of store-mediated calcium entry by secretion-like coupling between the inositol 1,4,5-trisphosphate receptor type II and human transient receptor potential (hTrp1) channels in human platelets.

Physical coupling between inositol 1,4,5-trisphosphate (IP(3)) receptors and transient receptor potential (Trp) channels has been demonstrated in both transfected and normal cells as a candidate mechanism for the activation of store-mediated Ca(2+) entry (SMCE). We have investigated the properties of the coupling between the type II IP(3) receptor and naturally expressed human Trp1 (hTrp1) in human platelets. Treatment with xestospongin C, an inhibitor of IP(3) receptor function, abolished SMCE and coupling between the IP(3) receptor and hTrp1. The coupling was activated by depletion of the intracellular Ca(2+) stores, and was reversed by refilling of the stores. We have also examined the role of actin filaments in the activation and maintenance of the coupling. Stabilization of the cortical actin network with jasplakinolide prevented the coupling, indicating that, as with secretion, the actin filaments at the cell periphery act as a negative clamp which prevents constitutive coupling. In addition, the actin cytoskeleton plays a positive role, since disruption of the actin network inhibited the coupling when the Ca(2+) stores were depleted. These results provide strong evidence for the activation of SMCE by a secretion-like coupling mechanism involving a reversible association between IP(3) receptors and hTrp1 in normal human cells.

Actins↗

PubMed2006

The allosteric enhancer PD81,723 increases chimaeric A1/A2A adenosine receptor coupling with Gs.

PD81,723 {(2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)-phenyl]methanone} is a selective allosteric enhancer of the G(i)-coupled A1 AR (adenosine receptor) that is without effect on G(s)-coupled A2A ARs. PD81,723 elicits a decrease in the dissociation kinetics of A1 AR agonist radioligands and an increase in functional agonist potency. In the present study, we sought to determine whether enhancer sensitivity is dependent on coupling domains or G-protein specificity of the A1 AR. Using six chimaeric A1/A2A ARs, we show that the allosteric effect of PD81,723 is maintained in a chimaera in which the predominant G-protein-coupling domain of the A1 receptor, the 3ICL (third intracellular loop), is replaced with A2A sequence. These chimaeric receptors are dually coupled with G(s) and G(i), and PD81,723 increases the potency of N6-cyclopentyladenosine to augment cAMP accumulation with or without pretreatment of cells with pertussis toxin. Thus PD81,723 has similar functional effects on chimaeric receptors with A1 transmembrane sequences that couple with G(i) or G(s). This is the first demonstration that an allosteric regulator can function in the context of a switch in G-protein-coupling specificity. There is no enhancement by PD81,723 of G(i)-coupled A2A chimaeric receptors with A1 sequence replacing A2A sequence in the 3ICL. The results suggest that the recognition site for PD81,723 is on the A1 receptor and that the enhancer acts to directly stabilize the receptor to a conformational state capable of coupling with G(i) or G(s).

Adenosine↗

PubMed2003

Coupling and innervation patterns of interstitial cells of Cajal in the deep muscular plexus of the guinea-pig.

Interstitial cells in the deep muscular plexus (ICC-DMP) are thought to be essential for neurotransmission in the circular muscle. There is evidence for gap junctions within the ICC-DMP network and between ICC-DMP and muscle cells; however, there is no evidence for functional coupling via these gap junctions. In addition, the innervation of individual ICC-DMP has not been studied. We investigated these questions by injecting the dye Lucifer yellow into ICC-DMP of guinea-pig ileum. Nerves were labelled immunohistochemically for protein gene product 9.5. Cells were imaged by confocal microscopy. Most (79%) of the dye-injected ICC-DMP were coupled to one to five other ICC-DMP, and 86% of them were coupled to one to five circular muscle cells. Octanol effectively blocked all coupling. Incubation in pH 6.8-7.0 reduced ICC-ICC coupling to 49% and ICC-muscle coupling to 32%. In contrast, pH 7.8-7.9 increased ICC-ICC and ICC-muscle coupling to 100%. Most ICC somata (95%) and processes (60%) were in close proximity with both nerve fibres and smooth muscle cells. These results provide direct evidence for functional coupling within the ICC-DMP network, and between this network and cells of the outer circular muscle layer and showed that coupling can be affected by pH.

Animals↗

PubMed2001

Translation at higher than an optimal level interferes with coupling at an intercistronic junction.

In pairs of adjacent genes co-transcribed on bacterial polycistronic mRNAs, translation of the first coding region frequently functions as a positive factor to couple translation to the distal coding region. Coupling efficiencies vary over a wide range, but synthesis of both gene products at similar levels is common. We report the results of characterizing an unusual gene pair, in which only about 1% of the translational activity from the upstream gene is transmitted to the distal gene. The inefficient coupling was unexpected because the upstream gene is highly translated, the distal initiation site has weak but intrinsic ability to bind ribosomes, and the AUG is only two nucleotides beyond the stop codon for the upstream gene. The genes are those in the filamentous phage IKe genome, which encode the abundant single-stranded DNA binding protein (gene V) and the minor coat protein that caps one tip of the phage (gene VII). Here, we have used chimeras between the related phage IKe and f1 sequences to localize the region responsible for inefficient coupling. It mapped upstream from the intercistronic region containing the gene V stop codon and the gene VII initiation site, indicating that low coupling efficiency is associated with gene V. The basis for inefficient coupling emerged when coupling efficiency was found to increase as gene V translation was decreased below the high wild-type level. This was achieved by lowering the rate of elongation and by decreasing the efficiency of suppression at an amber codon within the gene. Increasing the strength of the Shine-Dalgarno interaction with 16S rRNA at the gene VII start also increased coupling efficiency substantially. In this gene pair, upstream translation thus functions in an unprecedented way as a negative factor to limit downstream expression. We interpret the results as evidence that translation in excess of an optimal level in an upstream gene interferes with coupling in the intercistronic junction.

Bacteriophage IKe↗

PubMed1998

Effects of C-terminal truncation of the recombinant delta-opioid receptor on phospholipase C and adenylyl cyclase coupling.

Opioid receptors belong to the superfamily of guanine nucleotide binding (G) protein-coupled receptors. There is now growing evidence in support of a stimulatory coupling of opioid receptors to phospholipase C (PLC), via a pertussis toxin-sensitive G protein, leading to the generation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. We have generated two C-terminal truncation mutants of the delta-opioid receptor lacking the final 15 or 37 amino acids and examined their coupling to PLC and adenylyl cyclase. D-[Pen(2,5)]-enkephalin (DPDPE) mediated Ins(1,4,5)P3 formation and cyclic AMP inhibition was measured in whole cells and assayed using radioreceptor mass assays. DPDPE produced a time- and dose-dependent increase in Ins(1,4,5)P3 mass formation in Chinese hamster ovary (CHO) cells expressing the delta(wt), delta15, and delta37 receptors. As the C terminus was truncated, the time to maximum stimulation (15 s in CHO delta(wt), 60 s in CHO delta15, and 120 s in CHO delta37) increased and removal of the C terminus resulted in a prompt return to basal Ins(1,4,5)P3 levels. Whereas the dose-response curves to Ins(1,4,5)P3 formation and cyclic AMP inhibition remained largely unaffected by C-terminal truncation, there were large differences in the pEC/IC50 values, with cyclic AMP inhibition being the more potent, perhaps indicating G(i alpha) coupling to adenylyl cyclase and G(i beta/gamma) coupling to PLC. Collectively, these data indicate that the C terminus of the delta-opioid receptor is unimportant in the acute coupling to adenylyl cyclase but may have a role to play in PLC coupling. We hypothesize that an intact C terminus is required to allow normal "strong" coupling of receptor to Gi and that truncation weakens this link as reflected in an increased time to peak. In addition, if the coupling is weak, the acute response to agonist stimulation rapidly uncouples.

Adenylyl Cyclase Inhibitors↗

PubMed2001

Tethering of the platelet-derived growth factor beta receptor to G-protein-coupled receptors. A novel platform for integrative signaling by these receptor classes in mammalian cells.

Here we provide evidence to show that the platelet-derived growth factor beta receptor is tethered to endogenous G-protein-coupled receptor(s) in human embryonic kidney 293 cells. The tethered receptor complex provides a platform on which receptor tyrosine kinase and G-protein-coupled receptor signals can be integrated to produce more efficient stimulation of the p42/p44 mitogen-activated protein kinase pathway. This was based on several lines of evidence. First, we have shown that pertussis toxin (which uncouples G-protein-coupled receptors from inhibitory G-proteins) reduced the platelet-derived growth factor stimulation of p42/p44 mitogen-activated protein kinase. Second, transfection of cells with inhibitory G-protein alpha subunit increased the activation of p42/p44 mitogen-activated protein kinase by platelet-derived growth factor. Third, platelet-derived growth factor stimulated the tyrosine phosphorylation of the inhibitory G-protein alpha subunit, which was blocked by the platelet-derived growth factor kinase inhibitor, tyrphostin AG 1296. We have also shown that the platelet-derived growth factor beta receptor forms a tethered complex with Myc-tagged endothelial differentiation gene 1 (a G-protein-coupled receptor whose agonist is sphingosine 1-phosphate) in cells co-transfected with these receptors. This facilitates platelet-derived growth factor-stimulated tyrosine phosphorylation of the inhibitory G-protein alpha subunit and increases p42/p44 mitogen-activated protein kinase activation. In addition, we found that G-protein-coupled receptor kinase 2 and beta-arrestin I can associate with the platelet-derived growth factor beta receptor. These proteins play an important role in regulating endocytosis of G-protein-coupled receptor signal complexes, which is required for activation of p42/p44 mitogen-activated protein kinase. Thus, platelet-derived growth factor beta receptor signaling may be initiated by G-protein-coupled receptor kinase 2/beta-arrestin I that has been recruited to the platelet-derived growth factor beta receptor by its tethering to a G-protein-coupled receptor(s). These results provide a model that may account for the co-mitogenic effect of certain G-protein-coupled receptor agonists with platelet-derived growth factor on DNA synthesis.

Cell Line↗

PubMed2002

ERK activation by G-protein-coupled receptors in mouse brain is receptor identity-specific.

In transfected cells and non-neuronal tissues many G-protein-coupled receptors activate p44/42 MAP kinase (ERK), a kinase involved in both hippocampal synaptic plasticity and learning and memory. However, it is not clear to what degree these receptors couple to ERK in brain. G(s)-coupled beta-adrenergic receptor activation of ERK in neurons is critical in the regulation of synaptic plasticity in area CA1 of the hippocampus. In addition, alpha(1)- and alpha(2)-adrenergic receptors, present in CA1, could potentially activate ERK. We find that, like the beta-adrenergic receptor, the G(q)-coupled alpha(1)AR activates ERK in adult mouse CA1. However, activation of the G(i/o)-coupled alpha(2)AR does not activate ERK, nor does activation of a homologous G(i/o)-coupled receptor enriched in adult mouse CA1, the 5HT(1A) receptor. In contrast, the nonhomologous G(i/o)-coupled gamma-aminobutyric acid type B receptor does activate ERK in adult mouse CA1. Surprisingly, activation of alpha(2)ARs in CA1 from immature animals where basal phospho-ERK is low induces ERK phosphorylation. These data suggest that although most G-protein-coupled receptor subtypes activate ERK in non-neuronal cells, the coupling of G(i/o) to ERK is tightly regulated in brain.

Animals↗

PubMed2003

Structural requirements of the dihydropyridine receptor alpha1S II-III loop for skeletal-type excitation-contraction coupling.

Residues Leu720-Leu764 within the II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha1S subunit represent a critical domain for the orthograde excitation-contraction coupling as well as for retrograde DHPR L-current-enhancing coupling with the ryanodine receptor (RyR1). To better understand the molecular mechanism underlying this bidirectional DHPR-RyR1 signaling interaction, we analyzed the critical domain to the single amino acid level. To this end, constructs based on the highly dissimilar housefly DHPR II-III loop in an otherwise skeletal DHPR as an interaction-inert sequence background were expressed in dysgenic (alpha1S-null) myotubes for simultaneous recordings of depolarization-induced intracellular Ca2+ transients (orthograde coupling) and whole-cell Ca2+ currents (retrograde coupling). In the minimal skeletal II-III loop sequence (Asp734-Asp748 required for full bidirectional coupling, eight amino acids heterologous between skeletal and cardiac DHPR were exchanged for the corresponding cardiac residues. Four of these skeletal-specific residues (Ala739, Phe741, Pro742, and Asp744) turned out to be essential for orthograde and two of them (Ala739 and Phe741) for retrograde coupling, indicating that orthograde coupling does not necessarily correlate with retrograde signaling. Secondary structure predictions of the critical domain show that an alpha-helical (cardiac sequence-type) conformation of a cluster of negatively charged residues (Asp744-Glu751 of alpha1S) corresponds with significantly reduced Ca2+ transients. Conversely, a predicted random coil structure (skeletal sequence-type) seems to be prerequisite for the restoration of skeletal-type excitation-contraction coupling. Thus, not only the primary but also the secondary structure of the critical domain is an essential determinant of the tissue-specific mode of EC coupling.

Amino Acid Sequence↗

PubMed2003

A region of the third intracellular loop of the short form of the D2 dopamine receptor dictates Gi coupling specificity.

The D2 dopamine receptor has two isoforms, the short form (D2s receptor) and the long form (D2l receptor), which differ by the presence of a 29-amino acid insert in the third cytoplasmic loop. Both the D2s and D2l receptors have been shown to couple to members of the G alpha(i) family of G proteins, but whether each isoform couples to specific G alpha(i) protein(s) remains controversial. In previous studies using G alpha(i) mutants resistant to modification by pertussis toxin (G alpha(i)PT), we demonstrated that the D2s receptor couples selectively to G alpha(i2)PT and that the D2l receptor couples selectively to G alpha(i3)PT (Senogles, S. E. (1994) J. Biol. Chem. 269, 23120-23127). In this study, two point mutations of the D2s receptor were created by random mutagenesis (R233G and A234T). The two mutant D2s receptors demonstrated pharmacological characteristics comparable with those of the wild-type D2s receptor, with similar agonist and antagonist binding affinities. We used human embryonic kidney 293 cells stably transfected with G alpha(i1)PT, G alpha(i2)PT, or G alpha(i3)PT to measure agonist-mediated inhibition of forskolin-stimulated cAMP accumulation before and after pertussis toxin treatment. The two mutant D2s receptors demonstrated a change in G(i) coupling specificity compared with the wild-type D2s receptor. Whereas the wild-type D2s receptor coupled predominantly to G alpha(i2)PT, mutant R233G coupled preferentially to G alpha(i3)PT, and mutant A234T coupled preferentially to G alpha(i1)PT. These results suggest that this region of the third cytoplasmic loop is crucial for determining G(i) protein coupling specificity.

Amino Acid Sequence↗

PubMed2005

Coupled translation of the respiratory syncytial virus M2 open reading frames requires upstream sequences.

We have investigated the mechanism of the translation of the second open reading frame (ORF) of the respiratory syncytial virus M2 transcript that uses a novel coupled translation process requiring prior translation of the upstream ORF. The second M2-2 ORF sequences play no role in the coupling process and can be replaced with other gene sequences. Surprisingly, the overlap region of the two ORFs alone was not sufficient for coupled translation to occur. An analysis of the sequences required for the coupling process showed that portions of the transcript located along the length of the first ORF M2-1, upstream of the ORF overlap region, were essential for coupled translation to occur. A critically important region for this process was centered approximately 150 nucleotides upstream of the ORF2 initiation codons. This region was shown to contain a significant degree of secondary structure, and mutation of this sequence to remove predicted areas of base pairing significantly reduced coupled translation, confirming that the secondary structure was important for the coupling process. Additional sequences further upstream increased the efficiency of the coupled translation process. These data indicate that upstream sequences act in conjunction with the M2-1/M2-2 overlap region to promote coupled translation.

Base Sequence↗

PubMed1993

Relationship conflict management patterns among sex dysfunction, sex offender, and satisfied couples.

A substantial amount of research has distinguished specific behaviors of distressed from nondistressed couples. Among this evidence are differences in relationship conflict management styles. This investigation examined differences in relationship satisfaction and conflict management styles among 30 sex dysfunction, 25 sex offender, and 26 satisfied couples assessed by the Dyadic Adjustment Scale (DAS) and the Styles of Conflict Inventory (SCI). Results indicate significantly different levels of marital adjustment and distinctive conflict management patterns among the three couples groups. The two sex problem groups were significantly more distressed than the satisfied couples, and sex offender couples appeared more conflicted than sex dysfunction couples. Compared to satisfied couples, sex dysfunction couples address relationship conflict with somewhat polarized roles with an "avoid vs. engage" pattern, while sex offender couples employ a more chaotic "engage-avoid vs. engage-avoid" style. These different patterns of reactivity to conflict suggest that individualized treatment approaches to this dimension of the clinical picture is warranted.

Adaptation, Psychological↗

PubMed2005

Couples' happiness and its relationship to functioning in everyday life after brain injury.

The objective of this survey was to identify couples' joint perception of their satisfaction with life as a whole when one of the persons in the couple had acquired brain injury between one and five years earlier. The focus was on the influence that functioning and disability in everyday life have on the couple's joint life satisfaction after brain injury. The sample consisted of 55 couples, and the mean age of the brain-injured persons was 51 years. Both persons in the couple answered a mailed questionnaire encompassing questions concerning perceived impairments, activity limitations, participation restrictions, and life satisfaction. The results showed that in 16 of the 55 couples both partners were satisfied with life as a whole. The joint experience of life satisfaction was significantly related to the couple's functioning in everyday life, and specifically to perceived participation in leisure time and in their social life, and in their ability to wash clothes. Important implications from this study, showing that only one-third of the couples were satisfied, are that the partners should be included to a greater extent in the rehabilitation process and the couple's perspective of what they find difficult to deal with should serve as a guide during rehabilitation.

Activities of Daily Living↗

PubMed1976

Analysis of the thylakoid outer surface. Coupling factor is limited to unstacked membrane regions.

The structure of the spinach thylakoid outer surface has been examined by deepetching, a technique which exposes the true surfaces of biological membranes by sublimination of frozen dilute buffer. The membrane surface is covered with large (150 A average diameter) and small (90 A average diameter) particles. Approximately 30% of the large particles can be removed under conditions reported to selectively remove carboxydismutase from the membrane surface. The remaining large particles can be removed only under conditions which cause a loss of coupling factor activity. When purified coupling factor is readded to membranes from which all coupling factor activity has been removed, large particles reappear, indicating that they represent coupling factor molecules. Since the number of particles and the amount of ATPase activity in the reconstituted and control membranes were the same, coupling factor molecules may be attached to specific binding sites. Analysis of antibody labeling experiments, enzyme assays, and experiments involving the unstacking and restacking of thylakoid membranes indicate that coupling factor is excluded from regions of membrane stacking (grana) and is present only in unstacked membrane regions. The exclusion of coupling factor from grana, which are known to be centers of intense photosynthetic activity, strongly suggests that the mechanism coupling electron transport to photophosphorylation is indirect. In addition to the large and small particles, in some cases regularly spaced ridges are visible on the outer surface after unstacking. Coupling factor binding sites seem to be excluded from regions where these structures occur.

Cell Membrane↗