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A J Mason

Publications and source records attributed to A J Mason.

At least 19 recordsLinked to original sources
PubMed2006

Separated local field NMR experiments on oriented samples rotating at the magic angle.

Biophysical studies on membrane proteins by solid state NMR (SSNMR) can be carried out directly in a membrane environment. Samples are usually prepared in form of multi-lamellar dispersions for magic angle sample spinning or as aligned multi-layers for orientation dependent NMR experiments without sample rotation. A new development is the application of MAS NMR to aligned samples (MAOSS; Magic Angle Oriented Sample Spinning). In combination with separated local field (SLF) experiments, size and orientation of heteronuclear dipolar couplings may be extracted from two-dimensional experiments which correlate dipolar couplings with isotropic chemical shifts. The orientation of these (1)H-X dipolar couplings can be directly related to the orientation of molecular groups in the sample. Here, we demonstrate the feasibility of these experiments on highly ordered polyethylene fibers which serve as model compound. Based on these data, the experiment is also applied to ordered multi-layers of bacteriorhodopsin (purple membrane) which is used as a model for aligned membrane proteins. We present a detailed analysis of different experimental designs with respect to angular sensitivity and the influence of residual sample disorder ("mosaic spread"). The results of the MAOSS-SLF experiment are discussed within the context of established solid state NMR experiments which are usually performed without sample rotation and we compare the data to orientation information obtained from X-ray diffraction.

Bacteriorhodopsins↗
PubMed2001

Directional motion asymmetry in infant VEPs--which direction?

Monocular viewing during early infancy reveals asymmetries in optokinetic nystagmus (OKN) and visual evoked potentials (VEPs). This study investigates the VEP asymmetry to see if it is consistent in direction with the OKN asymmetry. Steady-state VEPs were recorded from infants (5-21 weeks) viewing gratings that underwent successive displacements in the same direction, leftward or rightward. In addition, transient VEPs were recorded to the two directions of an oscillating stimulus. Both tests produced larger VEP amplitudes for nasal-to-temporal compared to temporal-to-nasal movement. Horizontal eye movements were monitored by EOG while viewing these stimuli to test whether the asymmetry was a consequence of eye movements. No difference in eye movements as a function of the stimulus was found, excluding differences in retinal slip as an explanation of the asymmetry. The stronger neural response for nasal-to-temporal displacements is opposite to the asymmetry of OKN. Oculomotor and VEP asymmetries may be related; however this relationship is not simply that the stronger neural response, indicated by the VEP, leads to a stronger optokinetic response.

Adult↗
PubMed2000

Observations of light-induced structural changes of retinal within rhodopsin.

Photo-isomerization of the 11-cis retinal chromophore activates the mammalian light-receptor rhodopsin, a representative member of a major superfamily of transmembrane G-protein-coupled receptor proteins (GPCRs) responsible for many cell signal communication pathways. Although low-resolution (5 A) electron microscopy studies confirm a seven transmembrane helix bundle as a principal structural component of rhodopsin, the structure of the retinal within this helical bundle is not known in detail. Such information is essential for any theoretical or functional understanding of one of the fastest occurring photoactivation processes in nature, as well as the general mechanism behind GPCR activation. Here we determine the three-dimensional structure of 11-cis retinal bound to bovine rhodopsin in the ground state at atomic level using a new high-resolution solid-state NMR method. Significant structural changes are observed in the retinal following activation by light to the photo-activated M(I) state of rhodopsin giving the all-trans isomer of the chromophore. These changes are linked directly to the activation of the receptor, providing an insight into the activation mechanism of this class of receptors at a molecular level.

Animals↗
PubMed2000

Are there separate first-order and second-order mechanisms for orientation discrimination?

In a series of experiments we compared orientation discrimination performance for Gabor stimuli in which the stimulus profile was either matched to the receptive field profile of single V1 simple cells ('simple'), or in which the carrier and envelope orientations were different ('tigertails'). In the first Experiment, using small, high spatial frequency, peripheral stimuli to minimise the number of detectors involved, we found that simple stimuli were more detectable than tigertails of the same contrast energy, and that orientation discrimination thresholds for simple stimuli were lower than for tigertails of equal detectability. In later experiments with larger stimuli we measured thresholds for detecting tilts of the envelope with the carrier fixed in orientation. Envelope thresholds were similar for different carrier orientations, but carrier orientation had a strong biasing effect upon perceived envelope orientation. When the orientation difference between envelope and carrier was small, the carrier orientation was attracted to that of the envelope; when the difference was large (>10 degrees ) repulsion was found. The biases were reduced by half-wave rectifying the stimuli, putatively making the envelope visible to a first-order filter (Experiment 2). Discrimination thresholds for envelope orientation were higher than those for carrier orientation, and this difference was greater for briefly-presented parafoveal stimuli than for long duration foveal stimuli (Experiments 3 and 4). We conclude from these results that there are separate mechanisms for envelope and carrier orientation discriminations for large stimuli, but that first- and second-order mechanisms are not independent in the discrimination of orientation.

Analysis of Variance↗
PubMed2000

Localization of activin beta(A)-, beta(B)-, and beta(C)-subunits in humanprostate and evidence for formation of new activin heterodimers of beta(C)-subunit.

Activin ligands are formed by dimerization of activin ss(A)- and/or ss(B)-subunits to produce activins A, AB, or B. These ligands are members of the transforming growth factor-ss superfamily and act as growth and differentiation factors in many cells and tissues. New additions to this family include activin ss(C)-, ss(D)-, and ss(E)-subunits. The aim of this investigation was to examine the localization of and dimerization among activin subunits; the results demonstrate that activin ss(C) can form dimers with activin ss(A) and ss(B) in vitro, but not with the inhibin alpha-subunit. Using a specific antibody, activin ss(C) protein was localized to human liver and prostate and colocalized with ss(A)- and ss(B)-subunits to specific cell types in benign and malignant prostate tissues. Activin C did not alter DNA synthesis of the prostate tumor cell line, LNCaP, or the liver tumor cell line, HepG2, in vitro when added alone or with activin A. Therefore, the capacity to form novel activin heterodimers (but not inhibin C) resides in the human liver and prostate. Activin A, AB, and B have diverse actions in many tissues, including liver and prostate, but there is no known biological activity for activin C. Thus, the evidence of formation of activin AC or BC heterodimers may have significant implications in the regulation of levels and/or biological activity of other activins in these tissues.

Activins↗
PubMed1999

A sensitive and specific in vitro bioassay for activin using a mouse plasmacytoma cell line, MPC-11.

A new in vitro bioassay for activin was developed using the mouse plasmacytoma cell line, MPC-11. Human recombinant (hr) activin A dose-dependently inhibited the proliferation of these cells, whereas a range of other factors, including inhibin, follistatin and transforming growth factor-beta1, -beta2 and -beta3 had no effect. Conditioned medium containing activin B induced an inhibition similar to hr-activin A. The inhibitory influence of activin A could be blocked by follistatin, but not by hr-inhibin A. This bioassay had a sensitivity for activin A of around 0.4 ng/ml, an ED50 response of 3.5 ng/ml, and an intra-assay coefficient of variation of <11%. It offers substantial advantages over existing in vitro activin bioassays in terms of ease of use, specificity and throughput. The utility of the MPC-11 bioassay was demonstrated in the purification of activin from amniotic fluid, where an almost identical profile of bioactive activin A was detected compared with the pituitary cell bioassay of activin. Bioactive activin could also be detected in unpurified ovine allantoic and amniotic fluids and bovine follicular fluid. Measuring activin in untreated and heat-treated human sera or seminal plasma was hampered by a non-specific inhibitory effect, so that several serum samples did not run parallel with the hr-activin A standard. This inhibitory effect by serum could not be overcome by addition of follistatin, suggesting it is not activin-like bioactivity. This new bioassay for activin demonstrates widespread applicability for monitoring of purified or partially purified samples during purification procedures, bioactivity measurements, receptor-binding studies and assays of cell culture medium.

Activins↗
PubMed1997

Differential expression of NADPH-diaphorase between electrophysiologically-defined classes of pyramidal neurons in rat ventral subiculum, in vitro.

The subiculum is the major output region of the hippocampal formation. We have studied pyramidal neurons in slices of rat ventral subiculum to determine if there is a correlation between nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity and electrophysiological phenotype. The majority of NADPH-d-positive pyramidal neurons were found in the superficial cell layer (i.e. nearest to the hippocampal fissure) of the subiculum and appreciable NADPH-d activity was absent from pyramidal neurons in area CA1. This distribution of NADPH-d activity was mimicked by that of immunoreactivity for the neuronal isoform of nitric oxide synthase. Subicular pyramidal neurons were classified, electrophysiologically, as intrinsically burst-firing or regular spiking. After electrophysiological characterization, neurons were filled with Neurobiotin and revealed using fluorescence immunocytochemistry. The slices containing these neurons were also processed for NADPH-d. NADPH-d activity was found in six out of eight regular spiking neurons but was not found in any of 13 intrinsically burst-firing neurons (P=0.0008, Fisher's Exact Test). We conclude that in rat ventral subiculum, NADPH-d activity is present in a proportion of pyramidal neurons and indicates the presence of the neuronal isoform of nitric oxide synthase. Furthermore, amongst pyramidal neurons, NADPH-d activity is distributed preferentially to those with the regular spiking phenotype. The distribution of regular spiking neurons suggests that they may not be present to the same extent in all subicular output pathways. Thus, the actions of nitric oxide may be relatively specific to particular hippocampal connections.

Animals↗
PubMed1996

Enzyme linked immunosorbent assay for detecting benzodiazepines in urine.

A relatively simple ELISA technique was developed for the detection of a range of benzodiazepines (BZs) in urine. The assay employs a mouse anti-oxazepam antibody that is highly specific for the BZs. The limit of detection using 10 microliters samples of urine was 0.3 microgram ml-1 oxazepam. N-Desmethyldiazepam showed equal cross-reactivity to oxazepam, 11 BZs cross-reacted weakly and flurazepam and chlordiazepoxide did not cross-react at levels reported to be found in urine. No cross-reactivity was observed with drugs of abuse and a range of therapeutic drugs commonly found in urine. The assay was used as a screen to detect the presence of BZs in urine from 88 addicts that had been screened by the EMIT technique and a radioreceptor assay (RRA) for BZs. The ELISA produced two false negatives that were EMIT and RRA positive whereas the EMIT produced four different false negatives that were positive by both ELISA and RRA. Thirty-three positives were common to all three assays. The ELISA was also used to monitor nitrazepam-like activity in the urine of a greyhound receiving 5 mg oral medication and the results were compared with those obtained by RRA. Both assays were able to detect nitrazepam-like activity for up to 10 h post-administration.

Animals↗
PubMed1996

Characterization and determination of the biological activities of noncleavable high molecular weight forms of inhibin A and activin A.

Recombinant expression of human alpha- and beta A-inhibin subunit cDNAs in mammalian 293 cells results in the secretion of 20-53K free alpha-subunit-derived products, 30-105K alpha beta A-inhibin dimers, and 24-110K beta A-activin dimers. The present study verifies that the wide variation in the size of these products is due to incomplete cleavage of the proteolytic processing sites and the differential glycosylation of the N-linked glycosylation site at amino acid number 302 in the alpha C-subunit. The identity of each of these products was established by mutagenesis of proteolytic processing sites and N-linked glycosylation sites, combined with the analysis of transfection products by immunoprecipitation and one- and two-dimensional SDS-PAGE (SDS/SDS-beta-ME). Transient expression of processing site mutants of the alpha- and beta A-subunits in 293 cells was used to generate microgram quantities of noncleavable 55K and 65K inhibin dimers, and noncleavable 110K activin A dimers. The 55K and 65K inhibin A forms were purified and found to be fully biologically active in a rat pituitary cell bioassay. The 110K high molecular weight (HMW) form of human activin A failed to show any FSH-releasing activity in the pituitary assay. Since radioactively labeled 55K and 65K inhibin A and 110K activin A remained intact after incubation with rat pituitary cells for 72 h, there appears to be no conversion of these dimers to lower molecular weight forms by proteolytic cleavage at additional sites. These results show for the first time that 55K and 65K inhibit A are intrinsically biologically active and do not require cleavage to the 32K form for activation. In contrast, cleavage of the 110K activin A precursor to the 24K form would appear to be necessary for activity.

Activins↗
PubMed1994

Functional analysis of the cysteine residues of activin A.

Site-directed mutagenesis and mammalian cell expression was used to analyze the function of each of the 13 cysteine residues in the human activin A beta-subunit precursor. Substitution of the four cysteine residues in the proregion with alanine residues did not affect the function of the proregion in facilitating the dimerization and secretion of activin A homodimers. A series of activin mutants were constructed in which the nine cysteine residues (amino acids 4, 11, 12, 40, 44, 80, 81, 113, and 115) in the mature 116-amino acid beta-subunit were individually altered to alanine residues. Alanine substitution at either cysteine residues 4 or 12 did not interfere with homodimer formation, but the mutant activin A molecules had reduced biological and receptor binding activity (2- to 3-fold). Activin A monomers were produced when cysteine mutants 44, 80, and 113 were expressed in tissue culture cells. Monomers of cys mutants 44 and 80 had approximately 2% of the biological and receptor binding activity of wild type activin A. Cys 113 monomers had undetectable levels of biological activity. No detectable activin monomers or dimers were secreted from cells transfected with plasmids containing cys mutants 11, 40, 81, and 115. The data presented here suggest that a low level of noncovalent dimer formation of cysteine mutant monomers 44 and 80 may explain their low level of biological activity. Therefore, dimer formation is suggested to be an essential prerequisite for high affinity receptor binding and biological potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Activins↗
PubMed1992

Effects in vivo of recombinant human inhibin on myelopoiesis in mice.

Inhibin, a protein dimer, has been implicated in negative regulation of human erythropoiesis in vitro. In this study, purified recombinant human (rhu) inhibin was assessed for its effect in vivo on the proliferation of hematopoietic progenitor cells in C3H/HeJ mice. Administration of single doses of inhibin i.v. to mice resulted 24 hrs later in significant decreases in cell cycling status of marrow and splenic granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) progenitors. While no apparent effect was observed on marrow cellularity or on absolute numbers of marrow CFU-GM and BFU-E, inhibin significantly reduced absolute numbers of marrow CFU-GEMM, spleen nucleated cellularity and also absolute numbers of CFU-GM, BFU-E and CFU-GEMM in the spleen. The results demonstrate in vivo myelosuppressive effects for inhibin and demonstrate that effects in vivo are not restricted to erythropoiesis.

Animals↗
PubMed1991

Yellow spectacles to improve vision in children with binocular amblyopia.

Yellow spectacles were given to 20 children who had binocular amblyopia. Immediately, their vision improved. Furthermore, in the younger children, after they had worn the yellow lenses for up to nine months, this improvement persisted even when they were not wearing the spectacles. Yellow filters may help these children with binocular amblyopia because they remove the blue fringes that are due to chromatic aberration of the eye.

Adolescent↗
PubMed1991

Effect of recombinant inhibin on androgen synthesis in cultured human thecal cells.

Effects of inhibin (recombinant human inhibin-A) on ovarian androgen synthesis were tested in vitro using serum-free monolayer cultures of human thecal cells. Treatment for 4 days with inhibin alone at doses between 10 and 100 ng/ml caused modest (approximately 2-fold) increases in production of androgen (androstenedione and dehydroepiandrosterone): similar to the maximal level of stimulation caused by luteinizing hormone (LH) (10 ng/ml) alone but only about one-third of that caused by insulin-like growth factor I (IGF-I) (30 ng/ml) alone. Combined treatment with LH and inhibin elicited additive effects on androgen production whereas LH and IGF-I were synergistic, giving rise to androgen production rates at least 40 times greater than control. Additional presence of inhibin caused up to 10-fold augmentation of the response to LH + IGF-I. Activin (recombinant human activin-A) was previously shown to inhibit LH + IGF-I-induced androgen synthesis in this human thecal cell culture system. In the present study we found that the additional presence of inhibin (greater than 1 ng/ml) completely neutralized this inhibitory action of activin (10 ng/ml). These effects of inhibin were dose-dependent (ED50 1-10 ng/ml) and maximal at approximately 100 ng/ml. Inhibin stimulation of androgen synthesis occurred in the absence of measurable effects on progesterone production, and cell numbers in cultured cell monolayers were unaltered by the protein. It is concluded that inhibin exerts potent and selective stimulation of human thecal cell androgen synthesis in vitro. These results a paracrine role for inhibin(s) in modulating follicular androgen biosynthesis in the human ovary.

Activins↗
PubMed1991

Prevention of hemodialysis subclavian vein catheter infections by topical povidone-iodine.

Subclavian catheter (SCC) related infections are a major cause of morbidity in hemodialysis patients, the vast majority due to staphylococci species. Povidone-iodine (PI) has proven anti-staphylococcal activity. Therefore, a randomized controlled trial of topical PI ointment was undertaken to evaluate the impact of this prophylactic intervention on the incidence of SCC related infections in hemodialysis patients. The role of S. aureus nasal carrier state in the acquisition of infection was also evaluated. Patients requiring SCC for temporary hemodialysis access were randomized to receive the treatment (T; N = 63) or sterile gauze dressings alone (C; N = 66). Catheter duration ranged from 2 to 210 days in both groups, with a mean of 38.6 days in T and 36.2 days in C (NS). Exit site (ES) infections were significantly less in T (5%) versus C (18%) (P less than 0.02); tip colonization (TC) was 17% in T versus 36% in C (P less than 0.01), while the incidence of septicemia (S) was also significantly less in T (2%) versus C (17%; P less than 0.01). S. aureus nasal carriers were at a threefold higher risk of SCC related septicemia (0.009/day) than noncarriers (0.003/day; P less than 0.05). The beneficial effect of PI ointment was most evident in this high risk group of S. aureus carriers: ES = 0% T versus 24% C, TC = 12% T versus 42% C, S = 0% T versus 29% C, P less than 0.05. There were no adverse effects of the treatment. The routine application of topical PI ointment to temporary hemodialysis catheter exit sites is effective in reducing SCC related infections.

Acute Kidney Injury↗
PubMed1991

Evidence for an autocrine role of activin B within rat anterior pituitary cultures.

Activins, dimers of inhibin beta subunits, are potent stimulators of FSH secretion in vivo and in vitro and of FSH beta mRNA expression in rat anterior pituitary cultures. In this study, we investigated the possibility that locally secreted activin B (beta B beta B) may function as an autocrine modulator of basal FSH secretion and expression based on the previous observation that beta B is expressed within gonadotropes. The incubation of cultured rat anterior pituitary cells with a m mouse monoclonal antibody specific for the activin B homodimer (MAb-activin B) significantly attenuated the basal secretion of FSH in a concentration- and time-dependent manner, without influencing LH secretion. Moreover, MAb-activin B selectively inhibited FSH beta mRNA accumulation without affecting either LH beta or alpha subunit mRNAs. The MAb-activin B completely blocked the stimulation of FSH secretion by exogenous activin B, but not by activin A, confirming its specificity. As previously shown, inhibin A and follistatin significantly suppressed basal FSH secretion in these cultures. This inhibitory effect, albeit of lower magnitude, was still evident even in the presence of the MAb-activin B which by itself suppressed basal FSH secretion. These data suggest that the secretion of activin B by the gonadotropes of the anterior pituitary may serve as an autocrine signal in the selective modulation of FSH expression and secretion. Furthermore, the inhibitory actions of inhibins and follistatins on gonadotropes may, in part, be explained by their ability to interfere with the actions of endogenous activin B.

Activins↗
PubMed1991

Effect of recombinant activin on androgen synthesis in cultured human thecal cells.

The effect of activin-A on ovarian androgen synthesis was tested in vitro using serum-free monolayer cultures of human thecal cells. Maximal rates of androgen (androstenedione and dehydroepiandrosterone) production were induced by treating the cells for 4 days with LH (10 ng/mL) in the presence of insulin-like growth factor-I (greater than or equal to 30 ng/mL). The additional presence of recombinant activin-A (1-100 ng/mL) in culture medium caused dose-dependent suppression of thecal cell androgen production, with 50% maximal inhibition occurring at an activin-A concentration of about 10 ng/mL. Progesterone production was only suppressed by high dose (100 ng/mL) activin-A, and inhibition of steroid production occurred without inhibition of DNA synthesis (tritiated thymidine uptake). These results reveal a potent and selective inhibitory action of activin-A on thecal cell androgen synthesis, consistent with a paracrine function for activin(s) in modulating follicular androgen biosynthesis in the human ovary.

Activins↗