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Biomedical subjects

A Gibson

Publications and source records attributed to A Gibson.

At least 19 recordsLinked to original sources
PubMed2001

GTPase activity of dynamin and resulting conformation change are essential for endocytosis.

Dynamin is a large GTPase with a relative molecular mass of 96,000 (Mr 96K) that is involved in clathrin-mediated endocytosis and other vesicular trafficking processes. Although its function is apparently essential for scission of newly formed vesicles from the plasma membrane, the nature of dynamin's role in the scission process is still unclear. It has been proposed that dynamin is a regulator (similar to classical G proteins) of downstream effectors. Here we report the analysis of several point mutants of dynamin's GTPase effector (GED) and GTPase domains. We show that oligomerization and GTP binding alone, by dynamin, are not sufficient for endocytosis in vivo. Rather, efficient GTP hydrolysis and an associated conformational change are also required. These data argue that dynamin has a mechanochemical function in vesicle scission.

Amino Acid Sequence↗
PubMed2001

Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes.

Adaptor protein 180 (AP180) and its homolog, clathrin assembly lymphoid myeloid leukemia protein (CALM), are closely related proteins that play important roles in clathrin-mediated endocytosis. Here, we present the structure of the NH2-terminal domain of CALM bound to phosphatidylinositol-4,5- bisphosphate [PtdIns(4,5)P2] via a lysine-rich motif. This motif is found in other proteins predicted to have domains of similar structure (for example, Huntingtin interacting protein 1). The structure is in part similar to the epsin NH2-terminal (ENTH) domain, but epsin lacks the PtdIns(4,5)P2-binding site. Because AP180 could bind to PtdIns(4,5)P2 and clathrin simultaneously, it may serve to tether clathrin to the membrane. This was shown by using purified components and a budding assay on preformed lipid monolayers. In the presence of AP180, clathrin lattices formed on the monolayer. When AP2 was also present, coated pits were formed.

Adaptor Protein Complex 2↗
PubMed2001

Phosphodiesterase 5 inhibitors and nitrergic transmission-from zaprinast to sildenafil.

Phosphodiesterase 5 terminates the cellular actions of the second messenger molecule cyclic GMP; inhibitors of phosphodiesterase 5 will therefore increase and prolong the actions of endogenous substances that signal via the cyclic GMP pathway, including nitric oxide released as a neurotransmitter from nitrergic nerves. To date, the most widely used phosphodiesterase 5 inhibitors, zaprinast and sildenafil, have proved vital in the elucidation of the widespread role of cyclic GMP in nitrergic transmission and, specifically in the case of sildenafil, have provided a major breakthrough in the treatment of erectile dysfunction in men. Although still a matter of debate, early evidence indicates that sildenafil may also be of benefit in some forms of sexual dysfunction in women. The remarkable clinical success of sildenafil has prompted the search for further novel phosphodiesterase 5 inhibitors which might be used to enhance nitrergic function in other disease states.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2001

Three-dimensional electrical impedance tomography of human brain activity.

Regional cerebral blood flow and blood volume changes that occur during human brain activity will change the local impedance of that cortical area, as blood has a lower impedance than that of brain. Theoretically, such impedance changes could be measured from scalp electrodes and reconstructed into images of the internal impedance of the head. Electrical Impedance Tomography (EIT) is a newly developed technique by which impedance measurements from the surface of an object are reconstructed into impedance images. It is fast, portable, inexpensive, and noninvasive, but has a relatively low spatial resolution. EIT images were recorded with scalp electrodes and an EIT system, specially optimized for recording brain function, in 39 adult human subjects during visual, somatosensory, or motor activity. Reproducible impedance changes of about 0.5% occurred in 51/52 recordings, which lasted from 6 s after the stimulus onset to 41 s after stimulus cessation. When these changes were reconstructed into impedance images, using a novel 3-D reconstruction algorithm, 19 data sets demonstrated significant impedance changes in the appropriate cortical region. This demonstrates, for the first time, that significant impedance changes, which could form the basis for a novel neuroimaging technology, may be recorded in human subjects with scalp electrodes. The final images contained spatial noise and strategies to reduce this in future work are presented.

Adolescent↗
PubMed2001

Inhibition of capacitative calcium entry is not obligatory for relaxation of the mouse anococcygeus by the NO/cyclic GMP signalling pathway.

1. The object of this study was to determine whether inhibition of capacitative calcium entry is essential for relaxation of the mouse anococcygeus via the NO/cyclic GMP signalling pathway. 2. In intact muscles, thapsigargin (Tg; 100 nM)-induced tone was relaxed by NO, sodium nitroprusside (SNP), 8-Br-cyclic GMP, and nitrergic field stimulation. The relaxations were similar in magnitude to those observed against carbachol (50 microM) tone and, with the exception of those to 8-Br-cyclic GMP, were reduced by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 microM). 3. In single smooth muscle cells, loaded with Fura-2, both carbachol and Tg produced sustained elevations in cytoplasmic calcium levels ([Ca2+]i). SNP inhibited the rise in [Ca2+]i produced by carbachol, an effect attenuated by ODQ. In contrast, neither SNP nor 8-Br-cyclic GMP reduced the elevated [Ca2+]i associated with Tg. 4. In beta-escin skinned preparations, NO had no effect on tone induced by calcium (1 microM in the presence of 100 microM GTP). Carbachol and Tg produced further increases in calcium/GTP-induced tone and, in both cases, this additional tone was relaxed by NO and 8-Br-cyclic GMP. 5. The results support the hypothesis that the NO/cyclic GMP pathway inhibits capacitative calcium entry by refilling the internal stores, since reduction in [Ca2+]i was not observed in the presence of Tg. However, as muscle relaxation was still observed, impairment of capacitative calcium entry cannot be considered obligatory for relaxation. Results from skinned tissues suggest that inhibition of calcium sensitization processes, perhaps associated with store-depletion, may be an important mechanism of NO/cyclic GMP-induced relaxation.

Animals↗
PubMed2001

Functional glucocorticoid receptors in the mesonephros of the ovine fetus.

BACKGROUND: At 27 days of gestation in the ovine fetus (term = 145 to 150 days), the only kidney is the mesonephros, and allantoic fluid represents fetal urine. The hypothesis tested in this study was that functional glucocorticoid receptors (GRs) are present in this early mesonephric kidney. METHODS: Pregnant ewes, between 26 and 30 days, were infused with saline, dexamethasone (0.48 mg/hour), cortisol (5 mg/hour), or aldosterone (10 microg/hour) for 48 hours and were then killed for collection of fetuses and fetal fluids. GR mRNA was measured by real-time polymerase chain reaction in whole fetuses, and the location of gene expression was determined by hybridization histochemistry. RESULTS: Significant changes in allantoic fluid composition were produced by the exposure of the fetus to maternally infused synthetic (dexamethasone) and natural (cortisol) glucocorticoids, over a period of two days, compared with fetuses of ewes infused with vehicle (isotonic saline; N = 8) or aldosterone (N = 8). Volume of fluid was unchanged by any treatment, but both dexamethasone (N = 10) and cortisol (N = 8) caused significant (P < 0.05) decreases in sodium and chloride concentrations and increases in concentrations of potassium, urea, glucose, and fructose. GR mRNA was detected in equivalent concentrations in the whole fetuses of saline, dexamethasone, and cortisol treatments. The GR mRNA levels were significantly decreased in the aldosterone group. By hybridization histochemistry, GR mRNA was detected in most of the tubular cells of the mesonephros. CONCLUSION: These results suggest that functional GRs are present in the early ovine mesonephros.

Allantois↗
PubMed2001

Solving the forward problem in electrical impedance tomography for the human head using IDEAS (integrated design engineering analysis software), a finite element modelling tool.

If electrical impedance tomography is to be used as a clinical tool, the image reconstruction algorithms must yield accurate images of impedance changes. One of the keys to producing an accurate reconstructed image is the inclusion of prior information regarding the physical geometry of the object. To achieve this, many researchers have created tools for solving the forward problem by means of finite element methods (FEMs). These tools are limited, allowing only a set number of meshes to be produced from the geometric information of the object. There is a clear need for geometrical accurate FEM models to improve the quality of the reconstructed images. We present a commercial tool called IDEAS, which can be used to create FEM meshes for these models. The application of this tool is demonstrated by using segmented data from the human head to model impedance changes inside the head.

Algorithms↗
PubMed2001

Electrical impedance tomography of human brain activity with a two-dimensional ring of scalp electrodes.

Previously, electrical impedance tomography (EIT) has been used to image impedance decreases in the exposed cortex of rabbits during brain activity. These are due to increased blood volume at the site of the stimulated cortex; as blood has a lower impedance than brain, the impedance decreases. During human brain activity similar blood flow changes have been detected using positron emission tomography (PET) and functional magnetic resonance imaging (fMRI). If blood volume also changes then the impedance of human cortex will change during brain activity; this could theoretically be imaged with EIT. EIT data were recorded from a ring of 16 scalp electrodes in 34 recordings in 19 adult volunteers before, during and after stimulation with (1) a visual stimulus produced by an 8 Hz oscillating checkerboard pattern or (2) sensory stimulation of the median nerve at the wrist by a 3 Hz electrical square wave stimulus. Reproducible impedance changes, with a similar timecourse to the stimulus, were seen in all experiments. Significant impedance changes were seen in 21 +/- 5% (n = 16, mean +/- SEM) and 19 +/- 3% (n = 18) of the electrode measurements for visual and somatosensory paradigms respectively. The reconstructed 2D EIT images showed reproducible impedance changes in the approximate region of the stimulated cortex in 7/16 visual and 5/18 somatosensory experiments. This demonstrates that reproducible impedance changes can be measured during human brain activity. The final images contained spatial noise; the reasons for this and strategies to reduce this in future are discussed.

Adult↗
PubMed2001

Validation of a 3D reconstruction algorithm for EIT of human brain function in a realistic head-shaped tank.

Previous work has demonstrated that electrical impedance tomography can be used to image human brain activity during evoked responses, but two-thirds of the reconstructed images fail to localize an impedance change to the expected stimulated cortical area. The localization failure may be caused by modelling the head as a homogenous sphere in the reconstruction algorithm. This assumption may lead to errors when used to reconstruct data obtained from the human head. In this study a 3D reconstruction algorithm, based on a model of the head as a homogenous sphere, was characterized by simulating the algorithm model, the head shape and the presence of the skull in saline-filled tanks. EIT images of a sponge, 14 cm3 volume with a resistivity contrast of 12%, were acquired in three different positions in tanks filled with 0.2% saline. In a hemispherical tank, 19 cm in diameter, the sponge was localized to within 3.4-10.7% of the tank diameter. In a head-shaped tank, the errors were between 3.1 and 13.3% without a skull and between 10.3 and 18.7% when a real human skull was present. A significant increase in localization error therefore occurs if an algorithm based on a homogeneous sphere is used on data acquired from a head-shaped tank. The increased error is due to the presence of the skull, as no significant increase in error occurred if a head-shaped tank was used without the skull present, compared to the localization error within the hemispherical tank. The error due to the skull significantly shifted the impedance change within the skull towards the centre of the image. Although the increased localization error due to the skull is not sufficient to explain the localization errors of up to 50% of the image diameter present in the images of some human subjects, the future use of a realistic head model in the reconstruction algorithm is likely to reduce the localization error in the human images due to the presence of the skull.

Algorithms↗
PubMed2000

Sildenafil citrate on nitrergic transmission in anococcygeus muscles from the urogenital system of male and female mice.

The effects of sildenafil on nitrergic relaxations were compared in anococcygeus muscles from male and female mice. In muscles from both sexes, sildenafil (10-300 nM) produced a weak, direct relaxation of carbachol-induced tone, and increased both the amplitude and duration of nitrergic relaxations. The most marked effect was on nitrergic duration (300-400% increase with 300 nM sildenafil); no differences in potency were observed between male (EC(50), 30 nM) and female (EC(50), 25 nM). The rate of onset for potentiation of nitrergic duration was similar in both sexes; but, on washout, the effects of sildenafil declined more slowly in the male muscle. Relaxations to both nitric oxide (NO) and sodium nitroprusside were also increased in amplitude and duration by 50 nM sildenafil, while those to forskolin and papaverine were unaffected. The results demonstrate that sildenafil causes a similar, potent and selective potentiation of nitrergic transmission in urogenital smooth muscle from both male and female mice.

Animals↗
PubMed2000

Quantitative evaluation of simian immunodeficiency virus infection using NASBA technology.

The most commonly used animal model for the study of HIV-1 infection in humans is the infection of non-human primates by simian immunodeficiency virus (SIV). The animal hosts used most frequently are different species of macaques, which are readily infected with SIV, and can therefore be used to study natural infection, pathogenesis, therapy, and vaccine efficacy. The study of HIV-1 infection in humans relies heavily on the quantification of HIV-1 load (i.e. viral RNA) in patient plasma. Given the importance of HIV-1 RNA levels in humans, it follows that SIV RNA levels in animals are also relevant to the study of infection in this model system. This report describes the development of the isothermal amplification-based NASBA technology for the quantification of SIV RNA load in macaque plasma. Evaluation of the assay using model systems demonstrated that the assay is accurate and reproducible over nearly four orders of magnitude. Viral RNA load data were compared to other infection measurements in the macaque system. Further, the assay was used to provide copy number levels of SIV RNA in macaque plasma samples, permitting characterization of viral load during the course of SIV infection.

Animals↗
PubMed2000

Two-dimensional finite element modelling of the neonatal head.

Electrical impedance tomography (EIT) could allow the early diagnosis of infant brain injury following birth asphyxia. The purpose of this work was to determine the effect of variations in skull, scalp or cerebrospinal fluid (CSF) resistivity, as these vary in clinical conditions and could degrade image quality. These factors were investigated using finite element models of the adult and neonatal head. The results suggest that there is a wide range over which the resistivity of the neonatal skull has little effect on the sensitivity to a central impedance change. The scalp and CSF appear to shunt current away from the brain; when their resistivity was decreased from normal values, this shunting effect increased and caused a decrease in sensitivity to a central resistance change. The resistivity of neonatal skull has not, to our knowledge, been directly measured and will anyway vary within and between individuals; this work suggests that EIT will be relatively insensitive to variations in neonatal skull impedance.

Adult↗
PubMed1999

Characterisation of nitrergic transmission in the isolated anococcygeus muscle of the female mouse.

Field stimulation of anococcygeus muscles from female mice produced frequency-dependent relaxations of carbachol-induced tone, which were independent of the oestrus cycle but were abolished by the nitric oxide synthase (NOS) inhibitor L-N(G)-nitroarginine (L-NOARG; 100 microM) and the soluble guanylyl cyclase inhibitor 1 H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 microM); L-NOARG inhibition was reversed by L-, but not D-arginine. The selective phosphodiesterase V inhibitor zaprinast (1-130 microM) directly relaxed tone and enhanced both the amplitude and duration of field stimulation-induced relaxations; the effect on amplitude was greater at lower frequencies of stimulation, while increased duration dominated at higher frequencies. The duration, but not the amplitude, of relaxations to exogenous nitric oxide (NO; 15 microM) was also increased by zaprinast. The mouse anococcygeus provides a useful model for pharmacological investigation of nitrergic neurotransmission in female urogenital smooth muscle.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Correlation between store-operated cation current and capacitative Ca2+ influx in smooth muscle cells from mouse anococcygeus.

In mouse anococcygeus cells, simultaneous measurements of membrane currents and changes in intracellular Ca2+ were obtained using "perforated-patch" whole-cell recordings and Fura-2 microfluorimetry. Carbachol (50 microM) and cyclopiazonic acid (10 microM) produced a biphasic inward current; a transient Ca2+-dependent chloride current (I(ClCa)), followed by a smaller, sustained current (I(DOC)) This response was mirrored by a biphasic increase in the intracellular Ca2+ concentration. SKF96365 (1-{beta-[3-(4-methoxyphenyl) propoxyl]-4-methoxyphenethyl}-1H-imidazole; 10 microM) and Cd2+ (100 microM) inhibited both I(DOC) and the sustained increase in intracellular Ca2+; La3+ (400 microM) inhibited neither response. The results confirm that the non-selective cation current I(DOC) underlies capacitative Ca2+ influx supporting sustained contractions in this tonic smooth muscle.

Animals↗