A mean field model of ligand-protein interactions: implications for the structural assessment of human immunodeficiency virus type 1 protease complexes and receptor-specific binding.

We propose a general mean field model of ligand-protein interactions to determine the thermodynamic equilibrium of a system at finite temperature. The method is employed in structural assessments of two human immuno-deficiency virus type 1 protease complexes where the gross effects of protein flexibility are incorporated by utilizing a data base of crystal structures. Analysis of the energy spectra for these complexes has revealed that structural and thermo-dynamic aspects of molecular recognition can be rationalized on the basis of the extent of frustration in the binding energy landscape. In particular, the relationship between receptor-specific binding of these ligands to human immunodeficiency virus type 1 protease and a minimal frustration principle is analyzed.

The BCL2 major breakpoint region is a sequence- and cell-cycle-specific binding site of the Ku antigen.

The majority of translocations involving BCL2 are very narrowly targeted to three breakpoint clusters evenly spaced over a 100-bp region of the gene's terminal exon. We have recently shown that the immediate upstream boundary of this major breakpoint region (mbr) is a specific recognition site for single-strand DNA (ssDNA) binding proteins on the sense and antisense strands. The downstream flank of the mbr is a helicase binding site. In this report we demonstrate that the helicase and ssDNA binding proteins show reciprocal changes in binding activity over the cell cycle. The helicase is maximally active in G1 and early S phases; the ssDNA binding proteins are maximally active in late S and G2/M phases. An inhibitor of helicase binding appears in late S and G2/M. Finally, at least one component of the helicase binding complex is the Ku antigen. Thus, a protein with helicase activity implicated in repair of double-strand breaks, variable (diversity) joining recombination, and, potentially, cell-cycle regulation is targeted to the BCL2 mbr.

Specific binding of RNA polymerase II to the human immunodeficiency virus trans-activating region RNA is regulated by cellular cofactors and Tat.

The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression in response to Tat is dependent on an element downstream of the HIV-1 transcriptional initiation site designated the trans-activating region (TAR). TAR forms a stable stem-loop RNA structure in which a 3-nt bulge structure and a 6-nt loop structure are important for Tat activation. In the absence of Tat, the HIV-1 promoter generates so-called short or nonprocessive transcripts terminating at +60, while in the presence of Tat the synthesis of these short transcripts is markedly decreased and transcripts that extend through the 9.0-kb HIV-1 genome are synthesized. Tat effects on transcriptional elongation are likely due to alterations in the elongation properties of RNA polymerase II. In this study we demonstrated that a set of cellular cofactors that modulate the binding of the cellular protein TRP-185 to the TAR RNA loop sequences also functioned to markedly stimulate the specific binding of hypophosphorylated (IIa) and hyperphosphorylated (IIo) RNA polymerase II to TAR RNA. The concentrations of RNA polymerase II required for this interaction with TAR RNA were similar to those required to initiate in vitro transcription from the HIV-1 long terminal repeat. RNA gel retardation analysis with wild-type and mutant TAR RNAs indicated that the TAR RNA loop and bulge sequences were critical for the binding of RNA polymerase II. The addition of wild-type but not mutant Tat protein to gel retardation analysis with TAR RNA and RNA polymerase II resulted in the loss of binding of RNA polymerase II binding to TAR RNA. These results suggest that Tat may function to alter RNA polymerase II, which is paused due to its binding to HIV-1 TAR RNA with resultant stimulation of its transcriptional elongation properties.

Depolarisation and suppression of burst firing activity in the mouse subthalamic nucleus by dopamine D1/D5 receptor activation of a cyclic-nucleotide gated non-specific cation conductance

Neuronal burst firing in the subthalamic nucleus (STN) is one of the hallmarks of dopamine depletion in Parkinson's disease. Here, we have determined the postsynaptic effects of dopamine in the STN and the functional consequences of dopamine receptor modulation on burst firing in vitro. STN cells displayed regular spiking activity at a rate of 7.9 +/- 0.5 Hz. Application of dopamine (30 mu M) induced membrane depolarisations accompanied by an increase in firing rate of mean 12.0 +/- 0.6 Hz in all 69 cells. The dopamine effect was mimicked by the dopamine D1/D5 receptor agonist SKF38393 (10 mu M, 17 cells) and the dopamine D2-like receptor agonist quinpirole (10 mu M, 35 cells), partly reduced by D1/D5 antagonist SCH23390 (2 mu M, seven cells), but unaffected by the D2 antagonists sulpiride (10 mu M, seven cells) or eticlopride (10 mu M, six cells). Using voltage ramps, dopamine induced an inward current of 69 +/- 9.4 pA at a holding potential of -60 mV (n = 17). This current was accompanied by an increase in input conductance of 1.55 +/- 0.35 nS which reversed at -30.6 +/- 2.3 mV, an effect mimicked by SKF38393 (10 AM, nine cells). Similar responses were observed when measuring instantaneous current evoked by voltage steps and in the presence of the I-h blocker, ZD7288, indicating effects independent of I-h. The increase in conductance was blocked by SCH23390 (2 mu M, n = 4), mimicked by the activator of adenylyl cyclase forskolin (10 mu M, n = 7) and blocked by H-89, an inhibitor of cyclic AMP dependent protein kinase A (10 PM, n = 6). These results indicate that the dopamine depolarisation is in part mediated by D1/D5 receptor mediated activation of a cyclic-nucleotide gated (CNG) non-specific cation conductance. This conductance contributes to the membrane depolarisation that changes STN neuronal bursting to more regular activity by significantly increasing burst duration and number of spikes per burst.

Exercise Prescription for Patients with Non-specific Chronic Low Back Pain: a Qualitative Study of Patients' Experiences in Decision Making

Background The culture of current clinical practice calls for collaboration between therapists and patients, sharing power and responsibility. This paper reports on the findings of a qualitative study of exercise prescription for patients with NSCLBP, taking into account issues such as decision making and how this accords with patient preferences and experiences. Objective To understand the treatment decision making experiences, information and decision support needs of patients with NSCLBP who have been offered exercise as part of their management plan. Design A qualitative study using a philosophical hermeneutic approach. Methods Semi-structured interviews with eight patients (including use of brief patient vignettes) was undertaken to explore their personal experiences of receiving exercise as part of the management of their NSCLBP, and their involvement in decisions regarding their care. Findings The findings provide a detailed insight into patients’ perceptions and experiences of receiving exercise-based management strategies. Four themes were formed from the texts: (1) patients’ expectations and patients’ needs are not synonymous, (2) information is necessary but often not sufficient, (3) not all decisions need to be shared, and (4) wanting to be treated as an individual. Conclusions Shared decision making did not appear to happen in physiotherapy clinical practice, but equally may not be what every patient wants. The overall feeling of the patients was that the therapist was dominant in structuring the interactions, leaving the patients feeling disempowered to question and contribute to the decision making.

Psychosocial Risk Factors, Interventions, and Comorbidity in Patients with Non-Specific Low Back Pain in Primary Care: Need for Comprehensive and Patient-Centered Care

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Non-specific transient mutualism between the plant parasitic nematode, Bursaphelenchus xylophilus , and the opportunistic bacterium Serratia quinivorans BXF1, a plant-growth promoting pine endophyte with antagonistic effects

The aim of this study is to understand the biological role of Serratia quinivorans BXF1, a bacterium commonly found associated with Bursaphelenchus xylophilus, the plant parasitic nematode responsible for pine wilt disease. Therefore, we studied strain BXF1 effect in pine wilt disease. We found that strain BXF1 promoted in vitro nematode reproduction. Moreover, the presence of bacteria led to the absence of nematode chitinase gene (Bxcht-1) expression, suggesting an effect for bacterial chitinase in nematode reproduction. Nevertheless, strain BXF1 was unable to colonize the nematode interior, bind to its cuticle with high affinity or protect the nematode from xenobiotic stress. Interestingly, strain BXF1 was able to promote tomato and pine plant-growth, as well as to colonize its interior, thus, acting like a plant-growth promoting endophyte. Consequently, strain BXF1 failed to induce wilting symptoms when inoculated in pine shoot artificial incisions. This bacterium also presented strong antagonistic activities against fungi and bacteria isolated from Pinus pinaster. Our results suggest that B. xylophilus does not possess a strict symbiotic community capable of inducing pine wilt disease symptoms as previously hypothesized. We show that bacteria like BXF1, which possess plant-growth promoting and antagonistic effects, may be opportunistically associated with B. xylophilus, possibly acquired from the bacterial endophytic community of the host pine.

Validity of (-)-[H-3]-CGP 12177A as a radioligand for the 'putative beta(4)-adrenoceptor' in rat atrium

1 We have recently suggested the existence in the heart of a 'putative beta(4)-adrenoceptor' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block beta(1)- and Bz-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (-)-[H-3]-CGP 12177A ((-)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (-)-[H-3]-CGP 12177A for this purpose for two reasons, because it is a nonconventional partial agonist and also because it is a hydrophilic radioligand. 2 Increasing concentrations of(-)-[H-3]-CGP 12177A, in the absence or presence of 20 mu M (-)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to beta(1)- and beta(2)-adrenoceptors (pK(D) 9.4+/-0.1, B-max 26.9+/-3.1 fmol mg(-1) protein) and higher concentrations bound to the 'putative beta(4)-adrenoceptor' (pK(D) 7.5+/-0.1, B-max 47.7+/-4.9 fmol mg(-1) protein). In other experiments designed to exclude beta(1)- and beta(2)-adrenoceptors, (-)-[H-3]-CGP 12177A (1-200 nM) binding in the presence of 500 nM (-)-propranolol was also saturable (pK(D) 7.6+/-0.1, B-max 50.8+/-7.4 fmol mg(-1) protein). 3 The non-conventional partial agonists (-)-CGP 12177A (pK(i) 7.3+/-0.2), (+/-)-cyanopindolol (pK(i) 7.6+/-0.2), (-)-pindolol (pK(i) 6.6+/-0.1) and (+)-carazolol (pk(i), 7.2+/-0.2) and the antagonist (-)-bupranolol (pK(i) 6.6+/-0.2), all competed for (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol at the 'putative beta(4)-adrenoceptor', with affinities closely similar to potencies and affinities determined in organ bath studies. 4 The catecholamines competed with (-)-[H-3]-CGP 12177A at the 'putative beta(4)-adrenoceptor' in a stereoselective manner, (-)-noradrenaline (pK(iH) 6.3 +/- 0.3, pK(i), 3.5 +/- 0.1), (-)-adrenaline (pK(iH) 6.5 +/- 0.2, pK(iL) 2.9 +/- 0.1), (-)-isoprenaline (pK(iH) 6.2 +/- 0.5, pK(iL) 3.3 +/- 0.1), (+)-isoprenaline (pK(i) < 1.7), (-)-R0363 ((-)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy)-2-propranol)oxalate, pK(i) 5.5 +/- 0.1). 5 The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (-)-CGP 12177A or (-)-isoprenaline to the 'putative beta(4)-adrenoceptor'. In competition binding studies, (-)-CGP 12177A competed with (-)-[H-3]-CGP 12177A for one receptor state in the absence (pK(i) 7.3 +/- 0.2) or presence of GTP (pK(i) 7.3 +/- 0.2). (-)-Isoprenaline competed with (-)-[H-3]-CGP 12177A for two states in the absence (pK(iH) 6.6 +/- 0.3, pK(iL) 3.5 +/- 0.1; % H 25 +/- 7) or presence of GTP (pK(iH) 6.2 +/- 0.5, pK(iL) 3.4 +/- 0.1; % H 37 +/- 6). In contrast, at beta(1)-adrenoceptors, GTP stabilized the low affinity state of the receptor for (-)-isoprenaline. 6 The specificity of binding to the 'putative beta(4)-adrenoceptor' was tested with compounds active at other receptors. High concentrations of the beta(4)-adrenoceptor agonists, BRL 37344 ((RR + SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy -ethyl]amino]propyl]phenoxy]acetic acid, 6 mu M), SR 58611A (ethyl((7S)-7-[(2R)-2-(3-chlorophenyl)-2-hydroxyethylamino]-5,6,7,8-tetrahydronaphtyl-2-yloxy) acetate hydrochloride, 6 mu M), ZD 2079 ((+/-)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)ethan-1-ol, 60 mu M), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 mu M) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 mu M) caused less than 22% inhibition of (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol. Histamine (1 mM), atropine (1 mu M), phentolamine (10 mu M), 5-HT(100 mu M) and the 5-HT4 receptor antagonist SE 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1 ,4-benzodioxan-5-carboxylate, 10 nM) caused less than 26% inhibition of binding. 7 Non-conventional partial agonists, the antagonist (-)-bupranolol and catecholamines all competed for (-)-[H-3]-CGP 12177A binding in the absence of (-)-propranolol at beta(1)-adrenoceptors, with affinities (pK(i)) ranging from 1.6-3.6 log orders greater than at the 'putative beta(4)-adrenoceptor'. 8 We have established and validated a radioligand binding assay in rat atrium for the 'putative beta(4)-adrenoceptor' which is distinct from beta(1)-, beta(2)- and beta(3)-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as 'putative beta(4)-adrenoceptor'.

Voltammetric immunosensor for the diagnosis of celiac disease based on the quantification of anti-gliadin antibodies

Antibodies against gliadin are used to detect celiac disease (CD) in patients. An electrochemical immunosensor for the voltammetric detection of human anti-gliadin antibodies (AGA) IgA and AGA IgG in real serum samples is proposed. The transducer surface consists of screen-printed carbon electrodes modified with a carbon nanotube/gold nanoparticle hybrid system, which provides a very useful surface for the amplification of the immunological interactions. The immunosensing strategy is based on the immobilization of gliadin, the antigen for the autoantibodies of interest, onto the nanostructured surface. The antigen–antibody interaction is recorded using alkaline phosphatase labeled anti-human antibodies and a mixture of 3-indoxyl phosphate with silver ions (3-IP/Ag+) was used as the substrate. The analytical signal is based on the anodic redissolution of the enzymatically generated silver by cyclic voltammetry. The electrochemical behavior of this immunosensor was carefully evaluated assessing aspects as sensitivity, non-specific binding and matrix effects, and repeatability and reproducibility. The results were supported with a commercial ELISA test.