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D Newgreen

Publications and source records attributed to D Newgreen.

18 recordsLinked to original sources
PubMed2003

Impedance measurements and connexin expression in human detrusor muscle from stable and unstable bladders.

UNLABELLED: Three of this month's Scientific Discovery papers highlight the importance of collaboration in delivering high quality scientific research. As scientific technology increases in power and cost, and specific areas of interest become more specialized, it is becoming more difficult to cover all aspects of a completeresearch story. Collaborating with other experts in the field or other fields, including industry, allows strong scientific proof to be generated for the hypothesis and aims. Building strong collaborative,inter-disciplinary, multi-institutional, international groups with academic and industrial partners is the way forward for all discovery. We look forward to publishing more of these collaborative papersin future issues of the BJU International. OBJECTIVES: To test the hypothesis that intercellular electrical coupling is altered in human detrusor smooth muscle from patients with unstable bladders. MATERIALS AND METHODS: Human detrusor biopsy samples were obtained from patients with stable and unstable bladders. Intracellular electrical impedance was measured with alternating current (20 Hz-300 kHz) across the ends of detrusor strips in an oil-gap, after correcting for extracellular space resistance. Gap junctions were identified by localization of connexins (Cx), specifically Cx45, Cx43 and Cx40 transcripts, using immunoconfocal microscopy. RESULTS: Total intracellular resistivity was greater in strips from unstable than from stable bladders (median 1246 vs 817 Omega.cm). The increase was attributed to an increase in junctional resistance; cytoplasmic resistance was unchanged. Cx43 was localized to a submucosal layer and to connective tissue; Cx40 label was confined to endothelial cells of blood vessels. Cx45 labelling was localized to detrusor bundles and appeared to be less marked in samples from unstable bladders. Semi-quantitative analysis of Northern blots showed that Cx45 expression in unstable was less than that in stable bladders. CONCLUSIONS: These data suggest that intercellular coupling is reduced in detrusor from unstable bladders. Cx45 was localized to the detrusor layer, with Cx 43 more evident in the suburothelial mucosa. Cx45 labelling was less intense in detrusor samples from unstable bladders. These results are consistent with reduced gap junction coupling in detrusor from unstable bladders.

Blotting, Northern↗
PubMed2002

The contractile potency of adenosine triphosphate and ecto-adenosine triphosphatase activity in guinea pig detrusor and detrusor from patients with a stable, unstable or obstructed bladder.

PURPOSE: We compared the potency of adenosine triphosphate (ATP) and its nonhydrolyzable analogue alpha,beta-methylene ATP for generating contractions in human detrusor smooth muscle from patients with a stable, unstable and obstructed bladders. The different ATP potencies were compared with the ecto-adenosine triphosphatase (ATPase) of these samples. MATERIALS AND METHODS: Contractile experiments were done in vitro by superfusing samples with purines and dose-response curves were generated. Ecto-ATPase activity was measured from the rate of ATP hydrolysis sensitive to the ecto-ATPase inhibitor ARL 67156 with a luciferin-luciferase assay. RESULTS: ATP generated contractions with a mean EC50 of 933 microM. in tissue from stable bladders and was significantly more potent in tissue from unstable and obstructed bladders (EC50 141 and 172 microM., respectively). alpha,beta-methylene ATP was more potent in tissue from stable and unstable bladders (mean combined EC50 3 microM.). In guinea pig detrusor the mean EC50 for ATP and alpha,beta-methylene ATP was 138 and 5.5 microM., respectively. Mean total ATPase activity in unstable bladder biopsies plus or minus standard deviation was about 50% of that in stable bladder biopsies (2.54 +/- 1.50 versus 1.37 +/- 0.46 nmol. per second per mg. protein ). The ARL 67156 sensitive fraction was also significantly less in samples from unstable compared with stable bladders (mean 0.94 +/- 0.41 versus 0.36 +/- 0.26 nmol. per second mg. protein ). CONCLUSIONS: The greater potency of ATP for generating contractions in detrusor from unstable bladders may be due to reduced extracellular hydrolysis, allowing purine greater access to detrusor smooth muscle. This finding may explain atropine resistant purine based contractions in detrusor from unstable bladders.

Adenosine Triphosphatases↗
PubMed2001

Ypel1: a novel nuclear protein that induces an epithelial-like morphology in fibroblasts.

BACKGROUND: Embryonic development depends on the regulation of cell morphology and behaviour to carry out morphogenesis. One example of this is the development of the face, which is constructed from independent blocks of tissue that must grow in a coordinated way and then fuse to form a continuous tissue. RESULTS: We have isolated a novel gene of unknown function from mouse and quail embryos in a search for genes involved in craniofacial development. Sequence analysis of this gene, known as Ypel1, demonstrates a striking level of amino acid conservation between vertebrates and invertebrates but no significant homology with any other characterized genes. Ypel1 is expressed in the ventral half of early embryos including the branchial arches from which the face derives. Ypel1 localizes to the nucleus, and transfection into fibroblasts induces an epithelial-like transition, which is accompanied by alterations to the cytoskeleton and cell adhesion machinery. In addition, human YPEL1 localizes to chromosome 22q11.2, a region associated with a number of syndromes involving malformation of the craniofacial complex. CONCLUSION: These data suggest a role in regulation of cellular morphology and behaviour that is important for development of the craniofacial complex.

Amino Acid Sequence↗
PubMed2000

Effects of NS1608 on MaxiK channels in smooth muscle cells from urinary bladder.

Using the patch-clamp technique, we have characterized membrane currents in single detrusor smooth muscle cells from rat and human urinary bladder. From the voltage- and Ca(2+)-dependence of the current as well as the single channel conductance we conclude that rat and human urinary bladder smooth muscle cells express MaxiK channels. In smooth muscle cells from rat urinary bladder we tested the action of NS1608 on current through these MaxiK channels. Application of 10 microm NS1608 increased the amplitude of the current and this increase could be explained by a shift in the activation voltage of the MaxiK channels approximately 100 mV towards more negative potentials. Charybdotoxin as well as paxilline, well known blockers of MaxiK channels, were able to reduce current through MaxiK channels in our cell preparation. In addition, application of 10 microm NS1608 hyperpolarized the membrane potential of the investigated cells. This hyperpolarization could be antagonized by the application of paxilline. We conclude that application of NS1608 results in the opening of MaxiK channels under physiological conditions that leads to a hyperpolarization of the cells. This hyperpolarization in turn could relax urinary bladder smooth muscle cells. MaxiK channels in these cells could therefore play a role in directly controlling muscle tone by regulating the membrane potential. This opens up the possibility of MaxiK channels being targets for the treatment of urge incontinence.

Animals↗
PubMed2000

Lumbo-sacral neural crest contributes to the avian enteric nervous system independently of vagal neural crest.

Most of the avian enteric nervous system is derived from the vagal neural crest, but a minority of the neural cells in the hindgut, and to an even lesser extent in the midgut, are of lumbo-sacral crest origin. Since the lumbo-sacral contribution was not detected or deemed negligible in the absence of vagal cells, it had been hypothesised that lumbo-sacral neural crest cells require vagal crest cells to contribute to the enteric nervous system. In contrast, zonal aganglionosis, a rare congenital human bowel disease led to the opposite suggestion, that lumbo-sacral cells could compensate for the absence of vagal cells to construct a complete enteric nervous system. To test these notions, we combined E4 chick midgut and hindgut, isolated prior to arrival of neural precursors, with E1. 7 chick vagal and/or E2.7 quail lumbo-sacral neural tube as crest donors, and grafted these to the chorio-allantoic membrane of E9 chick hosts. Double and triple immuno-labelling for quail cells (QCPNA), neural crest cells (HNK-1), neurons and neurites (neurofilament) and glial cells (GFAP) indicated that vagal crest cells produced neurons and glia in large ganglia throughout the entire intestinal tissues. Lumbo-sacral crest contributed small numbers of neurons and glial cells in the presence or absence of vagal cells, chiefly in colorectum, but not in nearby small intestinal tissue. Thus for production of enteric neural cells the avian lumbo-sacral neural crest neither requires the vagal neural crest, nor significantly compensates for its lack. However, enteric neurogenesis of lumbo-sacral cells requires the hindgut microenvironment, whereas that of vagal cells is not restricted to a particular intestinal region.

Animals↗
PubMed1999

A paraxial exclusion zone creates patterned cranial neural crest cell outgrowth adjacent to rhombomeres 3 and 5.

Cranial neural crest cell migration is patterned, with neural crest cell-free zones adjacent to rhombomere (R) 3 and R5. These zones have been suggested to result from death of premigratory neural crest cells via upregulation of BMP-4 and Msx-2 in R3 and R5, consequent to R2-, R4-, and R6-derived signals. We reinvestigated this model and found that cell death detected by acridine orange staining in avian embryos varied widely numerically and in pattern, but with a tendency for an elevated zone centered at the R2/3 boundary. In situ hybridization of BMP-4 mRNA resolved to centers at R3 and R5 but Msx-2 resolved to the R2/3 border with only a faint smear from R5 to R6. Outgrowth of neural crest cells was less in isolated R3 cultures than in R1+2, R2, and R4 cultures, but R3 showed neither a decrease in outgrowth of neural crest cells nor an increase in cell death when cocultured with R1+2, R2, or R4. In addition, in serum-free culture, exogenous BMP-4 strikingly reduced neural crest cell outgrowth from R1+2 and R4 as well as R3. Thus we cannot confirm the role of intraneural cell death in patterning rhombomeric neural crest outgrowth. However, grafting quail R2 or R4 adjacent to the chick hindbrain demonstrated a neural crest cell exclusion zone next to R3 and R5. We suggest that one important pattern determinant for rhombomeric neural crest cell migration involves the microenvironment next to the neural tube.

Animals↗
PubMed1999

Catenary cultures of embryonic gastrointestinal tract support organ morphogenesis, motility, neural crest cell migration, and cell differentiation.

The embryonic gastrointestinal tract develops from a simple tube into a coiled, flexed, and regionalized structure. The changes in gut morphology coincide with the differentiation of multiple cell types in concentric layers, and include colonization by migratory neuron precursors, and the development of gastrointestinal motility. We describe a reliable method for growing embryonic mouse intestine in vitro by the attachment of segments of intestinal tract by their cut ends, with the intervening region suspended in the culture medium. These are termed "catenary cultures." E11-E11.5 mouse midgut, hindgut, or mid- plus hindgut segments were grown in catenary culture for up to 10 days and their growth, morphology, cell differentiation, ability to support neural precursor migration, and contractile activity were assessed. The increase in size of the cultured explants was not large, but morphogenesis proceeded, best exemplified by elongation of the caecum. Cell differentiation also proceeded. In the mucosa, goblet cells differentiated. Muscle layers, characterized by desmin expression, and kit-positive interstitial cells of Cajal differentiated in the correct positions. Where segments initially included neural precursors in a small sub-region, these migrated and proliferated to form uniform neuronal networks throughout the entire explant, and the cells expressed the neuron markers nitric oxide synthase and neuron specific enolase. Gut motility was attained 5-6 days into the culture period, and both contractile- and mixing-type movements were observed. Thus, cell types representative of all three germ layer contributions developed, and in addition, the gut, being mainly free, was able to elongate and bend (unlike on solid support cultures), while retaining its rostrocaudal identity.

Animals↗
PubMed1999

A quantitative study of atropine-resistant contractile responses in human detrusor smooth muscle, from stable, unstable and obstructed bladders.

PURPOSE: The objective of the study was to quantify in vitro the magnitude of atropine-resistant contractions using human detrusor samples and to determine the cellular processes underlying these contractions. MATERIALS AND METHODS: Isometric contractile responses were measured in isolated strips of human detrusor muscle obtained from patients with i) stable, ii) unstable or iii) obstructed bladders. Preparations were electrically stimulated or exposed to carbachol and ATP in the superfusate. RESULTS: Force-frequency curves were shifted to the right in samples from unstable and obstructed bladders. These same tissue groups also showed significant atropine-resistant contractions which were abolished by the neurotoxin TTX, or the non-hydrolysable ATP analog, alpha,beta-methylene ATP, suggesting that these contractions were mediated by neurally released ATP. Sub-division of the patient group with unstable bladders demonstrated that those with neuropathic instability did not show atropine-resistance, whereas those with idiopathic instability or secondary instability after obstruction did show atropine-resistant contractions. The potency of carbachol in generating a contracture was significantly greater than ATP (mean EC50 0.65 microM and 151 microM respectively) however, for each agonist there was no difference in potency between the three patient groups. Direct muscle excitability was similar in all three patient groups. CONCLUSIONS: It is concluded that purinergic, atropine-resistant contractions are present in some types of dysfunctional bladder, and these are not caused by a differential sensitivity of the muscle to ATP and cholinergic agonists.

Adenosine Triphosphate↗
PubMed1999

A comparison of the mode of action of ATP and carbachol on isolated human detrusor smooth muscle.

PURPOSE: The objectives of the study were: i) to examine the ability of carbachol and ATP to raise intracellular [Ca2+] in isolated detrusor myocytes; ii) to determine the origin of the intracellular Ca2+ and iii) to address the question of whether the appearance of purinergic contractions in detrusor from unstable and obstructed human bladders is reflected in the sensitivity of the cell to the two agonists. MATERIALS AND METHODS: Intracellular Ca2+ transients generated by extracellular ATP and carbachol were recorded from isolated human detrusor myocytes. Cells were dissociated by collagenase disruption of the biopsy. Intracellular Ca2+ was measured by epifluorescence microscopy using Fura-2 and electrophysiological recordings were made with patch electrodes. RESULTS: In cells from stable bladder biopsies the half-maximal concentrations (EC50) for ATP and carbachol to generate Ca2+ transients were 0.10 and 0.25 microM respectively. With cells from unstable bladders the EC50 values for both agonists and the magnitude of the Ca2+ transients were not significantly different from those obtained in cells from normal bladders. The transient in ATP was preceded by a transient depolarisation generated by a large inward current. The carbachol-Ca2+ transient was independent of changes to membrane potential, except in a subset of cells where complex membrane potential changes followed the rise of intracellular Ca2+. The ATP-Ca2+ transient was partially abolished by nicardipine and completely abolished by zero-Ca solutions, the carbachol-Ca2+ transient was unaffected by nicardipine and less completely attenuated by zero-Ca solutions. Prior exposure to caffeine suggested that the carbachol-Ca2+ transient, but not the ATP-Ca2+ transient, originated from intracellular stores. CONCLUSIONS: It is concluded that both agonists are equipotent in increasing intracellular Ca2+, but by different routes. The generation of purinergic contractions in detrusor from unstable bladder is not due to altered sensitivities of the detrusor myocyte to ATP or cholinergic agonists.

Adenosine Triphosphate↗
PubMed1998

GDNF and ET-3 differentially modulate the numbers of avian enteric neural crest cells and enteric neurons in vitro.

Vagal (hindbrain) neural crest cells migrate rostrocaudally in the gut to establish the enteric nervous system. Glial-derived neurotrophic factor (GDNF) and its receptor(s), and endothelin-3 (ET-3) and its receptor, are crucial for enteric nervous system development. Mutations interrupting either of these signaling pathways cause aganglionosis in the gut, termed Hirschsprung's disease in humans. However, the precise functions of GDNF and ET-3 in enteric neurogenesis are still unknown. We isolated precursor cells of the enteric nervous system from the vagal level neural crest of E1.7 quail embryos prior to entry into the gut and from the developing midgut at stages corresponding to migrating (E4.7) and longer resident differentiating cells (E7) using HNK-1 immunoaffinity and magnetic beads. These cells were tested for their response to GDNF and ET-3 in culture. ET-3 and GDNF had little effect in vitro on the growth, survival, migration, or neurogenesis of E1.7 vagal neural crest cells. In contrast, GDNF increased the proliferation rate and numbers of enteric neural precursors isolated from the E4.7 and E7 gut. Also, many more neurons and neurites developed in cultures treated with GDNF, disproportionately greater than the effect on cell numbers. At high cell density and in the presence of serum, ET-3, and GDNF had an additive effect on proliferation of neuron precursor cells. In defined medium, or low cell density, ET-3 reduced cell proliferation, overriding the proliferative effect of GDNF. Regardless of the culture condition, the stimulatory effect of GDNF on neuron numbers was strikingly diminished by the simultaneous presence of ET-3. We propose first that GDNF promotes the proliferation in the migratory enteric neural precursor cell population once the cells have entered the gut and is especially crucial for the differentiation of these cells into nonmigrating, nonproliferating enteric neurons. Second, we suggest that ET-3 modulates the action of GDNF, inhibiting neuronal differentiation to maintain the precursor cell pool, so ensuring sufficient population numbers to construct the entire enteric nervous system. Third, we suggest that generalized defects in enteric neural precursor cell numbers and differentiation due to mutations in the ET-3 and GDNF systems are converted to distal gut neural deficiencies by the rostrocaudal migration pattern of the precursors. Fourth, we suggest that additional factors such as those found in serum and produced by the enteric neural cells themselves are likely also to be involved in enteric nervous system development and consequently in Hirschsprung's disease.

Animals↗
PubMed1995

Epithelium-mesenchyme transition during neural crest development.

The neural crest is the organ system whose presence defines vertebrates. The onset of migration of neural crest cells is an archetypal epithelium to mesenchyme transition (EMT), and this event identifies the cell lineage. Little is known yet of the establishment of the neural crest, although the zinc finger gene Slug seems to be involved in specifying EMT competence. The details, especially the temporal order of events in neural crest EMT, vary between different species and between different axial levels, but several important features have emerged from observations in situ and experiments in vitro and in vivo. EMT seems to be strongly associated with decrease in cell-cell adhesion, and particularly with loss of N-cadherin on the surface of neural crest cells at the time of onset of migration. The related adhesion molecule T-cadherin is also present, but correlated changes have not yet been described, while the unrelated adhesion molecule N-CAM also declines on neural crest cells, but with a time course unrelated to EMT. The extracellular matrix is also important: EMT-related changes in matrix receptor (i.e. integrin) activity are recorded in avian crest cells, while the nature of the matrix itself changes in urodele amphibians. Changes in cell shape and in cell motility also occur at the time of EMT, consistent with changes in the cytoskeleton. These concerted changes can be triggered by TGF-beta family growth factors, of which dorsalin-I appears particularly important. These may act through pathways involving controlled alterations in phosphorylation to effect the complex of responses that make up EMT. Although much remains to be understood, the spatiotemporal definability of this system makes it a very useful model for studying EMTs in general.

Animals↗
PubMed1985

Do cells show an inverse locomotory response to fibronectin and laminin substrates?

Sixteen cell types from a variety of tissues and from primary and secondary cell cultures and established cell lines were tested for their ability to distinguish between fibronectin and laminin substrates during locomotion in vitro. Laminin and fibronectin were presented to the cells as directly adjacent tracks. Most cells, regardless of origin, showed no preference for one substrate over the other. Only two of the cell types tested showed a strong preference for one or other other substrate molecule. Cells were responding to the local substrate, since antibodies directed against one substrate molecule only interfered with locomotion on tracks coated with that molecule. We conclude that many cells simultaneously express functionally active receptors for fibronectin and laminin, and that differential locomotory response to these two molecules cannot be assumed without experimental confirmation.

Animals↗
PubMed1984

Spreading of explants of embryonic chick mesenchymes and epithelia on fibronectin and laminin.

Tissues from 2.5-day chick embryos were explanted onto glass coated with adsorbed fibronectin or laminin, or extracellular matrices (ECM) of deoxycholate-extracted chick embryo cells. Spreading of somitic and trunk neural-crest mesenchyme cells was equally rapid and extensive on fibronectin, laminin and on the fibronectin-rich, laminin-poor ECM produced by mesenchymal cells. No preference for fibronectin over laminin was displayed by these two mesenchymes when a choice of mutually exclusive alternating tracks was provided. Epithelial cells did not spread from explants of the neural tube on any substrate tested up to 24 h in vitro, but adhesion of the explant and outgrowth of axons was greatest on laminin. Explants of endodermal epithelium spread rapidly on or near ECM formed by endoderm cells. This ECM was deficient in laminin but contained dense fibronectin fibers. Spreading was less rapid on fibronectin, and even more retarded on laminin. Ectodermal epithelium explants spread rapidly on and near fibronectin-rich, laminin-poor ECM produced by ectoderm cells, and almost as rapidly on laminin, but spreading was strongly delayed and reduced on fibronectin. The observations suggest that the mesenchymal nature of somite and neural crest cells does not correspond to a lowered responsiveness to laminin relative to fibronectin, while the relationship between laminin and superior epithelial cell spreading should not be generalized. The spreading of the epithelia on complex ECM also indicates the presence of a component(s) other than fibronectin or laminin, which strongly promote(s) spreading. In addition, the methods used indicate that plasma fibronectin and laminin do not specifically bind to each other, and that bovine serum albumen may be inadequate in preventing the attachment of proteins, especially laminin, to cell culture substrates.

Animals↗
PubMed1982

Factors controlling the time of onset of the migration of neural crest cells in the fowl embryo.

Transmission electron microscopy of fowl embryos during the 7-10 h preceding migration of trunk-level neural crest (NC) cells revealed extracellular material near the NC-cells. In contrast to the cells of the neural tube, the basal surfaces of NC-cells possessed projections, and were neither contiguous nor covered by a complete basal lamina. The apical zones of NC-cells showed intercellular junctions at the stage of neural-fold fusion, but such junctions were absent in some NC-cells 5 h before migration. The basal laminae of the neural tube and the ectoderm were fused lateral to the NC before migration. In vitro, NC-cell migration commenced immediately when neural anlagen were explanted onto fibronectin-rich matrices, but only when the neural anlagen were from a level where migration had commenced in vivo. Migration was delayed 4-8 h when premigratory-level explants were used. Short-term cell-adhesion assays showed that NC-cells of both premigratory and migratory levels could adhere to fibronectin-rich matrices and to collagen gels, but only migratory NC-cells could be detached from the neural anlage. The results suggest that the precise schedule of the onset of NC-cell migration correlates with a decrease in the intercellular adhesion of NC-cells.

Animals↗
PubMed1980

Fibronectin in early avian embryos: synthesis and distribution along the migration pathways of neural crest cells.

Immunoperoxidase labelling for fibronectin (FN) in chick embryos showed FN-positive basement membranes surrounding the neural crest cell population prior to crest-cell migration. At cranial levels, crest cells migrated laterally into a large cell-free space. Initially they moved as a tongue of cells contacting the FN-positive basement membrane of the ectoderm but later the crest cell population expanded into space further from the ectoderm, until eventually the entire cranial cell-free space was occupied by mesenchyme cells. This was accompanied by the appearance of FN among the crest cells. At trunk levels, crest cells entered a relatively small space already containing FN-positive extracellular material. At later stages the migration of trunk crest cells broadly matched the distribution of FN. In vitro, chick and quail embryo ectoderm, endoderm, somites, notochord and neural tube synthesized and organized fibrous FN-matrices, as shown by immunofluorescence. Ectoderm and endoderm deposited this matrix only on the substrate face. The FN content of endoderm and neural tube matrices was transient, the immunofluorescence intensity declining after 1-2 days in culture. Some crest cells of cranial and sacral axial levels synthesized FN. Our data suggests that these were the earliest crest cells to migrate from these levels. This ability may be the first expression of mesenchymal differentiation in these crest cells, and in vivo enable them to occupy a large space. Almost all crest cells from cervico-lumbar axial levels were unable to synthesize FN. In vivo, this inability may magnify the response of these crest cells to FN provided by the neighbouring embryonic tissues.

Animals↗