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Biomedical subjects

A Gibson

Publications and source records attributed to A Gibson.

At least 73 records · Page 4Linked to original sources
PubMed1998

In polarized MDCK cells basolateral vesicles arise from clathrin-gamma-adaptin-coated domains on endosomal tubules.

Human transferrin receptors (TR) and receptors for polymeric immunoglobulins (pIgR) expressed in polarized MDCK cells maintain steady-state, asymmetric distributions on the separate basolateral and apical surfaces even though they are trafficking continuously into and across these cells. The intracellular mechanisms required to maintain these asymmetric distributions have not been located. Here we show that TR and pIgR internalize from both surfaces to a common interconnected endosome compartment that includes tubules with buds coated with clathrin lattices. These buds generate vesicles that carry TR to the basolateral border. The lattices contain gamma-adaptin and are dispersed by treatment with brefeldin A (BFA). Since BFA treatment abrogates the vectorial trafficking of TR in polarized MDCK cells, we propose that the clathrin-coated domains of the endosome tubules contain the polarized sorting mechanism responsible for their preferential basolateral distribution.

Adaptor Protein Complex gamma Subunits↗
PubMed1998

Signal-dependent trafficking of beta-amyloid precursor protein-transferrin receptor chimeras in madin-darby canine kidney cells.

We have investigated the intracellular trafficking of a chimeric molecule consisting of the cytoplasmic domain of the beta-amyloid precursor protein (APP) and the transmembrane region and external domain of the human transferrin receptor (TR) in Madin-Darby canine kidney cells. Newly synthesized APP-TR chimeras are selectively targeted to the basolateral surface by a tyrosine-dependent sorting signal in the APP cytoplasmic tail. APP-TR chimeras are then rapidly internalized from the basolateral surface and a significant fraction ( approximately 20-30%) are degraded. Morphological studies show that APP-TR chimeras internalized from the basolateral surface are found in tubulo-vesicular endosomal elements, internal membranes of multivesicular bodies, and lysosomes. APP-TR chimeras are also found in 60-nm diameter vesicles previously shown to selectively deliver wild-type TR to the basolateral surface; this result is consistent with the fact that 90% of internalized chimeras that are not degraded are selectively recycled back to the basolateral surface. APP-TR chimeras internalized from the apical surface are selectively transcytosed to the basolateral surface underscoring the importance of basolateral sorting in the endocytic pathway for maintaining the polarized phenotype. Tyr-653, an important element of the YTSI internalization signal in the APP cytoplasmic domain, is required for basolateral sorting in the biosynthetic and endocytic pathways. However, the structural features for basolateral sorting differ from those required for internalization.

Amino Acid Sequence↗
PubMed1998

Depletion of either ryanodine- or IP3-sensitive calcium stores activates capacitative calcium entry in mouse anococcygeus smooth muscle cells.

The properties of the calcium stores coupled to a depletion-operated cation current (IDOC) proposed to underlie capacitative calcium entry were studied in single smooth muscle cells isolated from the mouse anococcygeus using the whole-cell patch-clamp technique. Both caffeine (10 mM) and carbachol (50 microM) activated an initial, large ( approximately 200 pA), transient, calcium-dependent chloride current (IClCa) followed by a smaller ( approximately 10 pA) sustained, non-selective cation current (IDOC). Intracellular application of heparin (5 mg ml-1) abolished the response to carbachol but potentiated that to caffeine. Ryanodine (3 microM) activated IDOC but not IClCa; ryanodine (30 microM) failed to produce any response. Both concentrations of ryanodine abolished the response to caffeine and prevented activation of IClCa by carbachol. In the presence of 30 microM, but not 3 microM, ryanodine, carbachol was able to activate IDOC. Cyclopiazonic acid (CPA; 10 microM) abolished the response to carbachol; however, caffeine was still able to activate IClCa. In whole-muscle tension recordings, ryanodine at both 3 and 30 microM produced contractions of the tissue but only that in response to the lower concentration was maintained. Thus, depletion of either inositol 1,4, 5-trisphosphate-(IP3-) sensitive or ryanodine-sensitive calcium stores is able to activate IDOC, and, by extension, capacitative calcium entry in this tissue.

Animals↗
PubMed1998

Capacitative Ca2+ entry and the regulation of smooth muscle tone.

In many non-excitable cells, activation of phospholipase C-linked receptors results in a biphasic increase in the cytosolic Ca2+ concentration; an initial transient increase, owing to the release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR), is followed by a much smaller but sustained elevation, which often involves capacitative Ca2+ entry, where depletion of Ca2+ within the ER signals the opening of store-operated Ca2+ channels in the plasma membrane. However, in excitable cells such as smooth muscle, the role of capacitative Ca2+ entry is less clear and the main Ca2+ entry mechanisms responsible for sustained cellular activation have been considered to be either voltage-operated or receptor-operated Ca2+ channels. Although store-regulated Ca2+ entry was known to occur following agonist activation of smooth muscle, it was believed to be important only for the re-filling of the depleted SR and not as a source of activator Ca2+ for the contractile mechanisms. Here, Alan Gibson, Ian McFadzean, Pat Wallace and Christopher Wayman review recent evidence that capacitative Ca2+ entry might indeed be important for the regulation of smooth muscle tone, and that it might provide an important for pharmacological intervention.

Animals↗
PubMed1998

Production of biologically active salmon calcitonin in the milk of transgenic rabbits.

Salmon calcitonin (sCT) is an example of one of the many bioactive peptides that require amidation of the carboxy terminus for full potency. We describe a method for the production of amidated sCT in the mammary gland of transgenic rabbits. Expression of a fusion protein comprising human alpha lactalbumin joined by an enterokinase cleavable linker to sCT was directed to the mammary gland under the control of the ovine beta lactoglobulin promoter. C-terminal amidation in vivo was achieved by extending the sCT by a single glycine residue that provides a substrate for endogenous amidating activity in the mammary gland. Full characterization of the released sCT demonstrated it to be equivalent to synthetic standard in terms of structure, purity, and potency.

Amides↗
PubMed1998

No evidence for a significant non-nitrergic, hyperpolarising factor contribution to field stimulation-induced relaxation of the mouse anococcygeus.

1. The aim of the study was to determine whether a nerve-derived hyperpolarizing factor (NDHF) might contribute to non-adrenergic, non-cholinergic (NANC) relaxations of the mouse anococcygeus when low concentrations of contractile agent are used to raise tone and low frequencies of field stimulation applied; such a non-nitrergic NDHF has been proposed to contribute to NANC relaxations of the rat anococcygeus and guinea-pig taenia coli. 2. Phenylephrine (0.1-100 microM) produced concentration-related contractions of the mouse isolated anococcygeus muscle; 0.2 microM phenylephrine (EC26) was used to raise tone in subsequent experiments. 3. Field stimulation (0.5, 1.0 and 5.0 Hz) produced frequency-dependent relaxations of phenylephrine-induced tone. In the presence of the nitric oxide synthase inhibitor L-NG-nitro-arginine (L-NOARG; 100 microM), the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 microM), or a combination of these two drugs, relaxations to field stimulation were abolished at all frequencies studied. Relaxations to sodium nitroprusside (0.01-5 microM) were unaffected by L-NOARG but strongly inhibited by ODQ; neither enzyme inhibitor affected relaxations to 8-Br-cyclic GMP (10 microM). 4. Nifedipine (1 microM) reduced the contractile response to 0.2 microM phenylephrine by 38%; however, it had no effect on NANC relaxations. 5. It is concluded that NANC relaxations of the mouse anococcygeus are purely nitrergic and that there is no significant contribution from a putative NDHF.

Adrenergic alpha-Agonists↗
PubMed1998

Renpenning syndrome maps to Xp11.

Mutations in genes on the X chromosome are believed to be responsible for the excess of males among individuals with mental retardation. Such genes are numerous, certainly >100, and cause both syndromal and nonsyndromal types of mental retardation. Clinical and molecular studies have been conducted on the Mennonite family with X-linked mental retardation (XLMR) reported, in 1962, by Renpenning et al. The clinical phenotype includes severe mental retardation, microcephaly, up-slanting palpebral fissures, small testes, and stature shorter than that of nonaffected males. Major malformations, neuromuscular abnormalities, and behavioral disturbances were not seen. Longevity is not impaired. Carrier females do not show heterozygote manifestations. The syndrome maps to Xp11.2-p11.4, with a maximum LOD score of 3.21 (recombination fraction 0) for markers between DXS1039 and DXS1068. Renpenning syndrome (also known as "MRXS8"; gene RENS1, MIM 309500) shares phenotypic manifestations with several other XLMR syndromes, notably the Sutherland-Haan syndrome. In none of these entities has the responsible gene been isolated; hence, the possibility that two or more of them may be allelic cannot be excluded at present.

Abnormalities, Multiple↗
PubMed1998

The effect of a teledermatology program on rural referral patterns to dermatologists and the management of skin disease.

Teledermatology can be defined as the use of imaging and telecommunications technologies to provide skin care services at a distance. The potential value of teledermatology is especially great in rural and medically underserved areas that do not have, or cannot support, providers specializing in the diagnosis and management of skin diseases. Rural patients and primary care providers should be able to use teledermatology as a greatly simplified and potentially less expensive means of referral to an urban dermatologist. In an effort to gauge the impact of a simple teledermatology system on referral patterns and the management of rural patients with skin disorders, we studied baseline rates of referral to dermatologists from five primary care clinics in rural Oregon. Economical, easy-to-use teledermatology systems were subsequently installed, and the effects on patient referral and management were recorded over time. The interim results suggest that primary care providers (PCPs) are reluctant to refer patients with skin conditions, even when the primary care providers confidence in the correct diagnosis and treatment plan for that condition are relatively low. The installation of a teledermatology system increases the number of patients referred for specialist evaluation dramatically, even while the number of in-person visits to specialists fell. Although diagnostic agreement between dermatologists and primary care providers was mixed, a marked difference was found in their recommended treatment plans. A number of cases were found in which use of the telemedicine technology system resulted in reversing conditions that had been poorly controlled for a number of years prior to teleconsultation. This work is important as an indicator that referral rates to dermatologists may be inappropriately low in rural areas of the U.S., and that use of teledermatology may improve this trend.

Dermatology↗
PubMed1997

Case-control study of sudden infant death syndrome in Scotland, 1992-5.

OBJECTIVE: To investigate the relation between routine infant care practices and the sudden infant death syndrome in Scotland. METHODS: National study of 201 infants dying of the sudden infant death syndrome (cases) and 276 controls by means of home interviews comparing methods of infant care and socioeconomic factors. RESULTS: Sleeping prone (odds ratio 6.96 (95% confidence interval 1.51 to 31.97) and drug treatment in the previous week (odds ratio 2.33 (1.10 to 4.94)) were more common in the cases than controls on multivariate analysis. Smoking was confirmed as a significant risk factor (odds ratio for mother and father both smoking 5.19 (2.26 to 11.91)). The risk increased with the number of parents smoking (P < 0.0001), with the number of cigarettes smoked by mother or father (P = 0.0001), and with bed sharing (P < 0.005). A new finding was an increased risk of dying of the syndrome for infants who slept at night on a mattress previously used by another infant or adult (odds ratio 2.51 (1.39 to 4.52)). However, this increased risk was not established for mattresses totally covered by polyvinyl chloride. CONCLUSIONS: Sleeping prone and parental smoking are confirmed as modifiable risk factors for the sudden infant death syndrome. Sleeping on an old mattress may be important but needs confirmation before recommendations can be made.

Bedding and Linens↗
PubMed1997

The use of fluorescent probes to characterize conformational changes in the interaction between vitronectin and plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator and urokinase, is known to convert readily to a latent form by insertion of the reactive center loop into a central beta-sheet. Interaction with vitronectin stabilizes PAI-1 and decreases the rate of conversion to the latent form, but conformational effects of vitronectin on the reactive center loop of PAI-1 have not been documented. Mutant forms of PAI-1 were designed with a cysteine substitution at either position P1' or P9 of the reactive center loop. Labeling of the unique cysteine with a sulfhydryl-reactive fluorophore provides a probe that is sensitive to vitronectin binding. Results indicate that the scissile P1-P1' bond of PAI-1 is more solvent exposed upon interaction with vitronectin, whereas the N-terminal portion of the reactive loop does not experience a significant change in its environment. These results were complemented by labeling vitronectin with an arginine-specific coumarin probe which compromises heparin binding but does not interfere with PAI-1 binding to the protein. Dissociation constants of approximately 100 nM are calculated for the vitronectin/PAI-1 interaction from titrations using both fluorescent probes. Furthermore, experiments in which PAI-1 failed to compete with heparin for binding to vitronectin argue for separate binding sites for the two ligands on vitronectin.

Fluorescent Dyes↗
PubMed1997

Multifocal motor neuropathy.

We present a review of the literature on multifocal motor neuropathy (MMN), a rare neurological disorder which has features in common with both chronic inflammatory demyelinating neuropathy and lower motor neuron disease. Clinically, MMN is characterised by slowly progressive asymmetrical limb weakness, usually most prominent in the forearms. Weakness may be associated with muscle wasting, fasciculations and decreased tendon reflexes. Serum anti-GM1 ganglioside antibody titres may be increased. The diagnostic hallmark of MMN is the electrophysiological demonstration of persistent localised motor conduction blocks, with otherwise normal or near-normal motor and sensory conduction velocities. The pathogenesis of MMN has not been elucidated completely. There is, however, substantial evidence for an autoimmune mechanism. Based on the possible involvement of the immune system in the pathogenesis of MMN the therapeutic efficacy of several immunomodulatory drugs has been tested. Treatment of MMN patients with human immunoglobulin or cyclophosphamide is usually followed by a marked improvement of strength. The finding that MMN is a potentially treatable disorder underscores the importance of distinguishing MMN from lower motor neuron disease, for which no effective therapy is currently available.

Biopsy↗
PubMed1997

Superoxide anions, free-radical scavengers, and nitrergic neurotransmission.

1. There is now strong evidence that the L-arginine/nitric oxide (NO) pathway generates the transmitter released from certain nonadrenergic, noncholinergic nerves that mediate smooth-muscle relaxation in the respiratory, gastrointestinal, and urogenital tracts. In particular, nitric oxide synthase (NOS) has been detected in these nitrergic nerves, and nerve-induced relaxation can be prevented by NOS inhibitors. Thus, free-radical NO has been considered the putative transmitter candidate. 2. Despite such evidence, a number of superoxide anion-generating compounds and direct NO scavengers have been found to abolish relaxations to exogenous NO, but to have very little effect on relaxations in response to nitrergic field stimulation. A number of hypotheses have been put forward to explain this paradox: first, that the NO generated within the nerve is attached to a carrier molecule (such as a thiol) to form an adduct, that is released into the junctional gap and that is resistant to superoxide anions and other scavengers; second, that over short distances (up to 200 microns) the rapid diffusion characteristics of NO render it resistant to inhibition by scavengers; third, that NO is indeed released as a free radical, but that it is protected from radical scavengers by other substances present in the junctional region. 3. Recent experimental evidence supports the third explanation, because nitrergic relaxations, normally resistant to inhibition by superoxide anions, become sensitive following inactivation of copper/ zinc superoxide dismutase (Cu/Zn SOD); the inhibition can be reversed by adding exogenous Cu/Zn SOD (or ascorbate). In addition, the ability of two NO-scavenger compounds, hydroquinone and carboxy-PTIO, to inhibit relaxations to exogenous NO is prevented by certain physiological antioxidants (ascorbate and reduced glutathione in the case of hydroquinone, and ascorbate and alpha-tocopherol in the case of carboxy-PTIO). 4. Thus, it is possible that the presence of integrated antioxidant mechanisms within the tissue protects neuronally- released NO from attack by scavenging molecules; exogenous NO would be vulnerable before reaching the protection of the tissue, thus explaining the paradoxical effects mentioned. Organ antioxidant status may therefore be very important in preserving the potency of nitrergic transmission and in preventing NO from reacting with other compounds to produce cytotoxic metabolites (eg., with superoxide anions to form peroxynitrite).

Animals↗
PubMed1997

Hypothermia and the use of space blankets: a literature review.

Developed in the 1960s, the 'space blanket' subsequently became a popular tool in the management of the hypothermic patient. Recent articles addressing the issue of the management of hypothermia have tended to offer a variety of suggestions for combating hypothermia, but mention of the space blanket as a tool in this aim appears to have diminished. A review of 20 articles concerned with the management of hypothermia (and identified using a computerized search strategy) indicates that the space blanket has fallen from popularity in treating hypothermic patients and supports the notion that A & E departments should employ alternative methods for rewarming the patient with hypothermia.

Bedding and Linens↗
PubMed1997

Positional cloning of the gene associated with X-linked juvenile retinoschisis.

X-linked juvenile retinoschisis(RS) is a recessively inherited vitreo-retinal degeneration characterized by macular pathology and intraretinal splitting of the retina. The RS gene has been localized to Xp22.2 to an approximately 1 Mb interval between DXS418 and DXS999/DXS7161. Mapping and expression analysis of expressed sequence tags have identified a novel transcript, designated XLRS1, within the centromeric RS locus that is exclusively expressed in retina. The predicted XLRS1 protein contains a highly conserved motif implicated in cell-cell interaction and thus may be active in cell adhesion processes during retinal development. Mutational analyses of XLRS1 in affected individuals from nine unrelated RS families revealed one nonsense, one frameshift, one splice acceptor and six missense mutations segregating with the disease phenotype in the respective families. These data provide strong evidence that the XLRS1 gene, when mutated, causes RS.

Amino Acid Sequence↗
PubMed1997

Cellular mechanisms underlying carbachol-induced oscillations of calcium-dependent membrane current in smooth muscle cells from mouse anococcygeus.

1. At a holding potential of -40 mV, carbachol (50 microM) produced a complex pattern of inward currents in single smooth muscle cells freshly isolated from the mouse anococcygeus. Membrane currents were monitored by the whole-cell configuration of the patch-clamp technique. Previous work has identified the first, transient component as a calcium-activated chloride current (ICl(Ca)) and the second sustained component as a store depletion-operated non-selective cation current (I(DOC)). The object of the present study was to examine the cellular mechanisms underlying the third component, a series of inward current oscillations (I(oscil)) superimposed on I(DOC). 2. Carbachol-induced I(oscil) (amplitude 97 +/- 11 pA; frequency 0.26 +/- 0.02 Hz) was inhibited by the chloride channel blocker anthracene-9-carboxylic acid (A-9-C; 1 mM), and by inclusion of 1 mM EGTA in the patch-pipette filling solution. 3. In calcium-free extracellular medium (plus 1 mM EGTA), carbachol produced an initial burst of oscillatory current which lasted 94 s before decaying to zero; I(oscil) could be restored by re-admission of calcium. The frequency, but not the amplitude, of I(oscil) increased with increasing concentrations of extracellular calcium (0.5-10 mM). 4. Inclusion of the inositol triphosphate (IP3) receptor antagonist heparin (5 mg ml(-1) in the patch-pipette filling solution, or pretreatment of cells with the sarcoplasmic reticulum (SR) calcium ATPase inhibitor cyclopiazonic acid (CPA; 10 microM), prevented the activation of I(oscil) by carbachol. Caffeine (10 mM) activated both ICl(Ca) and I(DOC) and prevented the induction of I(oscil) by carbachol. Caffeine and CPA also abolished I(oscil) in the presence of carbachol, as did both a low (3 microM) and a high (30 microM) concentration of ryanodine. 5. Carbachol-induced I(oscil) was abolished by the general calcium entry blocker SKF 96365 (10 MM) and by Cd2+ (100 microM), but was unaffected by La3+ (400 microM). As found previously, I(DOC) was also blocked by SKF 96365 and Cd2+, but not La3+; the inhibition of I(DOC) preceded the abolition of I(oscil) by 27 s with SKF 96365 and by 30 s with Cd2+. Nifedipine (1 microM) produced a partial inhibition of the carbachol-induced I(oscil) frequency at holding potentials of -20 mV and -60 mV and, in addition, reduced I(DOC) at -60 mV by 18%. 6. It is concluded that carbachol-induced inward current oscillations in mouse anococcygeus cells are due to a calcium-activated chloride current, and reflect oscillatory changes in cytoplasmic calcium ion concentration. These calcium oscillations are derived primarily from the SR stores, but entry of calcium into the cell is necessary for store replenishment and maintenance of the oscillations. Capacitative calcium entry (via I(DOC) appears to be important not only for sustained contraction of this tissue, but also as a route for re-filling of the SR and, therefore, represents an important target for the development of novel and selective drugs.

Animals↗
PubMed1997

Release of the antioxidants ascorbate and urate from a nitrergically-innervated smooth muscle.

1. The main object of the present study was to determine whether ascorbate, an antioxidant which has been shown to protect nitric oxide (NO) from attack by scavenger molecules, might be released from nitrergically-innervated smooth muscle; ascorbate release from the rat anococcygeus was measured by use of h.p.l.c. with electrochemical detection. 2. Incubation of rat anococcygeus muscles in normal physiological salt solution (PSS; 30 min) resulted in release of ascorbate into the bathing medium (7.7 +/- 0.9 nmol g-1 tissue). This release was increased by 96% when muscles were incubated in high K+ (70 mM) PSS. The resting release of ascorbate was unaffected by tetrodotoxin (TTX; 1 microM), omega-conotoxin GVIA (10 nM) or omission of calcium ions from the PSS (with addition of 0.2 mM EGTA), but all three procedures attenuated the increased release observed under depolarizing conditions. Resting release of ascorbate was unaffected by glutamate (100 microM), aspartate (100 microM), gamma-aminobutyric acid (100 microM) or carbachol (50 microM). 3. A second h.p.l.c. peak, which always preceded the ascorbate peak, was identified as urate. Urate release from the anococcygeus, following 30 min incubation in normal PSS, was 64.6 +/- 12.7 nmol g-1 tissue but, unlike ascorbate, urate release was unchanged in high K+ PSS. In functional experiments, urate (100-400 microM) partially protected NO (15 microM)-induced relaxations of the rat anococcygeus from inhibition by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; 50 microM), but not from inhibition by hydroquinone or duroquinone (both 100 microM). 4. Muscles chemically sympathectomized with 6-hydroxydopamine (6-OHDA, 500 microM; 2 h) still exhibited release of ascorbate (2.5 +/- 0.4 nmol g-1 tissue) and urate (22.2 +/- 2.9 nmol g-1 tissue); in both cases the release was similar to that observed in time-matched control tissues not exposed to 6-OHDA. High K+ PSS produced a TTX-sensitive increase in release of ascorbate, but not urate, from 6-OHDA-treated muscles. 5. The results demonstrate that significant amounts of ascorbate and urate are released from the rat anococcygeus muscle. Ascorbate, but not urate, release appears to be enhanced by activation of nerves which are resistant to 6-OHDA pretreatment. Since both antioxidants can protect NO from attack by scavenger molecules, their release in nitrergically-innervated tissues may be important for the provision of the correct redox environment to allow NO to fulfill its proposed neurotransmitter role.

Adrenergic Agents↗