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1.
World J Urol ; 35(5): 827-838, 2017 May.
Article in English | MEDLINE | ID: mdl-27514371

ABSTRACT

PURPOSE: This large dose-ranging study explored the benefits of different combinations of mirabegron and solifenacin on health-related quality of life (HRQoL), based on patient-reported outcomes (PROs), and patients ('responders') achieving clinically meaningful improvements in efficacy and HRQoL. METHODS: SYMPHONY (NCT01340027) was a Phase II, placebo- and monotherapy-controlled, dose-ranging, 12-week trial. Adult patients with overactive bladder (OAB) for ≥3 months were randomized to 1 of 12 groups: 6 combination (solifenacin 2.5/5/10 mg + mirabegron 25/50 mg), 5 monotherapy (solifenacin 2.5/5/10 mg, or mirabegron 25/50 mg), or placebo. Change from baseline to end of treatment was assessed, versus placebo and solifenacin 5 mg in: PROs (OAB-q [Symptom Bother/total HRQoL] and Patient Perception of Bladder Condition score), and responders achieving minimally important differences (MIDs) in PROs and predetermined clinically meaningful improvements in efficacy (e.g. <8 micturitions/24 h). Changes in PROs and responders were analysed using an ANCOVA model and logistic regression, respectively. RESULTS: The Full Analysis Set included 1278 patients. Combination therapy of solifenacin 5/10 mg + mirabegron 25/50 mg significantly improved PROs versus solifenacin 5 mg and placebo, and significantly more responders achieved MIDs in PROs and efficacy. Micturition frequency normalization was approximately twofold greater with 10 + 25 mg (OR 2.06 [95 % CI 1.11, 3.84; p = 0.023]) and 5 + 50 mg (OR 1.91 [95 % CI 1.14, 3.21; p = 0.015]) versus solifenacin 5 mg. CONCLUSION: Combining mirabegron 25/50 mg and solifenacin 5/10 mg improves objective and subjective efficacy outcomes compared with placebo or solifenacin 5 mg.


Subject(s)
Acetanilides/administration & dosage , Quality of Life , Solifenacin Succinate/administration & dosage , Thiazoles/administration & dosage , Urinary Bladder, Overactive/drug therapy , Urological Agents/administration & dosage , Adult , Aged , Double-Blind Method , Drug Therapy, Combination , Female , Health Status , Humans , Male , Middle Aged , Minimal Clinically Important Difference , Patient Reported Outcome Measures , Treatment Outcome
2.
Clin Pharmacol Ther ; 99(4): 442-51, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26422298

ABSTRACT

A multicriteria decision analysis (MCDA) approach was developed and used to estimate the benefit-risk of solifenacin and mirabegron and their combination in the treatment of overactive bladder (OAB). The objectives were 1) to develop an MCDA tool to compare drug effects in OAB quantitatively, 2) to establish transparency in the evaluation of the benefit-risk profile of various dose combinations, and 3) to quantify the added value of combination use compared to monotherapies. The MCDA model was developed using efficacy, safety, and tolerability attributes and the results of a phase II factorial design combination study were evaluated. Combinations of solifenacin 5 mg and mirabegron 25 mg and mirabegron 50 (5+25 and 5+50) scored the highest clinical utility and supported combination therapy development of solifenacin and mirabegron for phase III clinical development at these dose regimens. This case study underlines the benefit of using a quantitative approach in clinical drug development programs.


Subject(s)
Acetanilides/therapeutic use , Adrenergic beta-3 Receptor Agonists/therapeutic use , Decision Support Techniques , Muscarinic Antagonists/therapeutic use , Solifenacin Succinate/therapeutic use , Thiazoles/therapeutic use , Urinary Bladder, Overactive/drug therapy , Urological Agents/therapeutic use , Acetanilides/administration & dosage , Acetanilides/adverse effects , Adrenergic beta-3 Receptor Agonists/administration & dosage , Adrenergic beta-3 Receptor Agonists/adverse effects , Drug Dosage Calculations , Drug Monitoring , Drug Therapy, Combination , Female , Humans , Male , Muscarinic Antagonists/administration & dosage , Muscarinic Antagonists/adverse effects , Risk Assessment , Risk Factors , Solifenacin Succinate/administration & dosage , Solifenacin Succinate/adverse effects , Thiazoles/administration & dosage , Thiazoles/adverse effects , Treatment Outcome , Urinary Bladder, Overactive/physiopathology , Urological Agents/administration & dosage , Urological Agents/adverse effects
3.
Anat Rec (Hoboken) ; 294(7): 1249-60, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21618435

ABSTRACT

Gubernacular elongation during inguinoscrotal testicular descent and cremaster muscle development remains poorly described in mammals. The role of the genitofemoral nerve (GFN) remains elusive. We performed detailed histological analysis of testicular descent in normal rats to provide a comprehensive anatomical description for molecular studies. Fetuses and neonatal male offspring (5-10 per group) from time-mated Sprague-Dawley dams (embryonic days 15, 16, and 19; postnatal days 0, 2, and 8) were prepared for histology. Immunohistochemistry was performed for nerves (Class III tubulin, Tuj1) and muscle (desmin). At embryonic days 15 and 16, the gubernaculum and breast bud are adjacent and both supplied by the GFN. By embryonic day 19, the breast bud has regressed and the gubernacular swelling reaction is completed. Postnatally, the gubernacular core regresses, except for a cranial proliferative zone. The cremaster is continuous with internal oblique and transversus abdominis. By postnatal day 2 (P2), the gubernaculum has everted, locating the proliferative zone caudally and the residual mesenchymal core externally. Eversion creates the processus vaginalis, with the everted gubernaculum loose in subcutaneous tissue but still remote from the scrotum. By P8, the gubernaculum has nearly reached the scrotum with fibrous connections attaching the gubernaculum to the scrotal skin. A direct link between GFN, gubernaculum, and breast bud suggests that the latter may be involved in gubernacular development. Second, the cremaster muscle is continuous with abdominal wall muscles, but most of its growth occurs in the distal gubernacular tip. Finally, gubernacular eversion at birth brings the cranial proliferative zone to the external distal tip, enabling gubernacular elongation similar to a limb bud.


Subject(s)
Fetus/embryology , Inguinal Canal/growth & development , Ligaments/growth & development , Scrotum/growth & development , Testis/growth & development , Abdominal Muscles/growth & development , Animals , Animals, Newborn , Fetus/anatomy & histology , Inguinal Canal/anatomy & histology , Inguinal Canal/embryology , Ligaments/anatomy & histology , Ligaments/embryology , Male , Rats , Rats, Sprague-Dawley , Scrotum/anatomy & histology , Scrotum/embryology , Testis/anatomy & histology , Testis/embryology
4.
Placenta ; 31(9): 747-55, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20659767

ABSTRACT

Colonisation of the maternal uterine wall by the trophoblast involves a series of alterations in the behaviour and morphology of trophoblast cells. Villous cytotrophoblast cells change from a well-organised coherently layered phenotype to one that is extravillous, acquiring a proliferative, migratory and invasive capacity, to facilitate fetal-maternal interaction. These changes are similar to those of other developmental processes falling under the umbrella of an epithelial-mesenchymal transition (EMT). Modulation of cell adhesion and cell polarity occurs through changes in cell-cell junctional molecules, such as the cadherins. The cadherins, particularly the classical cadherins (e.g. Epithelial-(E)-cadherin), and their link to adaptors called catenins at cell-cell contacts, are important for maintaining cell attachment and the layered phenotype of the villous cytotrophoblast. In contrast, reduced expression and re-organization of cadherins from these cell junctional regions promote a loosened connection between cells, coupled with reduced apico-basal polarity. Certain non-classical cadherins play an active role in cell migration processes. In addition to the classical cadherins, two other cadherins which have been reported in placental tissues are vascular endothelial (VE) cadherin and cadherin-11. Cadherin molecules are well placed to be key regulators of trophoblast cell behaviour, analogous to their role in other developmental EMTs. This review addresses cadherin expression and function in normal and diseased human placental tissues, especially in fetal growth restriction and pre-eclampsia where trophoblast invasion is reduced.


Subject(s)
Cadherins/physiology , Epithelial-Mesenchymal Transition/physiology , Placentation/physiology , Cadherins/biosynthesis , Cell Adhesion , Cell Movement/genetics , Female , Fetal Growth Retardation/physiopathology , Humans , Placenta/metabolism , Pre-Eclampsia/physiopathology , Pregnancy , Trophoblasts/cytology
5.
Neurogastroenterol Motil ; 22(3): 331-e86, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19775251

ABSTRACT

BACKGROUND Most enteric neurones arise from neural crest cells that originate in the post-otic hindbrain, and migrate into and along the developing gastrointestinal tract. There is currently great interest in the possibility of cell therapy to replace diseased or absent enteric neurones in patients with enteric neuropathies, such as Hirschsprung's disease. However, it is unclear whether neural crest stem/progenitor cells will be able to colonize colon (i) in which the mesenchyme has differentiated into distinct layers, (ii) that already contains enteric neurones or (iii) that lacks a gene expressed by the gut mesenchyme, such as endothelin-3 (Et-3). METHODS Co-cultures were used to examine the ability of enteric neural crest-derived cells (ENCCs) from E11.5 mouse gut to colonize a variety of recipient hindguts. KEY RESULTS Enteric neural crest-derived cells migrated and gave rise to neurones in E14.5 and E16.5 aneural colon in which the external muscle layers had differentiated, but they did not migrate as far as in younger colon. There was no evidence of altered ENCC proliferation, cell death or neuronal differentiation in older recipient explants. Enteric neural crest-derived cells failed to enter most recipient E14.5 and E16.5 colon explants already containing enteric neurones, and the few that did showed very limited migration. Finally, ENCCs migrated a shorter distance and a higher proportion expressed the pan-neuronal marker, Hu, in recipient E11.5 Et-3(-/-) colon compared to wild-type recipient colon. CONCLUSIONS & INFERENCES Age and an absence of Et-3 from the recipient gut both significantly reduced but did not prevent ENCC migration, but the presence of neurones almost totally prevented ENCC migration.


Subject(s)
Cell Movement/physiology , Colon/innervation , Endothelin-3/metabolism , Neurogenesis/physiology , Neurons/physiology , Age Factors , Analysis of Variance , Animals , Coculture Techniques , Colon/cytology , Colon/metabolism , ELAV Proteins/metabolism , Endothelin-3/genetics , Immunohistochemistry , Mice , Mice, Knockout , Neural Crest/cytology , Neural Crest/physiology , Neurons/cytology , Stem Cells/cytology , Stem Cells/physiology
6.
Dev Dyn ; 238(12): 3152-5, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19877279

ABSTRACT

Gene expression fields in embryogenesis are spatially precise and often small, so experimental gene expression often requires similar spatial definition. For in ovo electroporation, typically a gene construct is injected into a natural body cavity in the embryo prior to electroporation. Limited control of the size and location of the electroporated field can be obtained by varying electrode placement and geometry, and by altering the miscibility and viscosity of the construct vehicle but it is difficult to tightly constrain electroporation to small regions. Electroporation of different constructs in close proximity has not been possible. We show that loading the construct into an agarose bead, which is then microsurgically implanted, allows for focal electroporation. Different constructs can be electroporated in close proximity by emplacing several agarose beads. This technique is simple, cheap, rapid, and requires no more specialised equipment than that required for conventional in ovo electroporation.


Subject(s)
Electroporation/methods , Gene Transfer Techniques , Ovum , Animals , Animals, Genetically Modified , Embryo, Nonmammalian , Fluorescent Antibody Technique/methods , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Microspheres , Ovum/cytology , Quail/embryology , Transgenes
7.
Endocrinology ; 150(9): 4044-55, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19477940

ABSTRACT

We have recently demonstrated that fibroblast growth factor (FGF)-2 promotes neuroblastoma cell differentiation and overrides their mitogenic response to IGF-I. However, the mechanisms involved are unknown. SK-N-MC cells were cultured with FGF-2 (50 ng/ml) and/or IGF-I (100 ng/ml) up to 48 h. Fluorescence-activated cell sorting analysis indicated that FGF-2 promotes G1/G0 cell cycle phase arrest. Gene expression by RT2-PCR and cellular localization showed up-regulation of p21. We then investigated whether FGF-2-induced differentiation of SK-N-MC cells (by GAP43 and NeuroD-6 expression) involves epithelium-mesenchyme transition interconversion. Real-time PCR (RT2-PCR) showed modulation of genes involved in maintenance of the epithelial phenotype and cell-matrix interactions (E-cadherin, Snail-1, MMPs). Zymography confirmed FGF-2 up-regulated MMP2 and induced MMP9, known to contribute to neuronal differentiation and neurite extension. Id1-3 expression was determined by RT2-PCR. FGF-2 induced Id2, while down-regulating Id1 and Id3. FGF-2 induced nuclear accumulation of ID2 protein, while ID1 and ID3 remained cytoplasmic. RNA interference demonstrated that Id3 regulates differentiation and cell cycle (increased Neuro-D6 and p21 mRNA), while d Id2 modulates epithelium-mesenchyme transition-like events (increased E-cadherin mRNA). In conclusion, we have shown for the first time that FGF-2 induces differentiation of neuroblastoma cells via activation of a complex gene expression program enabling modulation of cell cycle, transcription factors, and suppression of the cancer phenotype. The use of RNA interference indicated that Id-3 is a key regulator of these events, thus pointing to a novel therapeutic target for this devastating childhood cancer.


Subject(s)
Cell Differentiation/genetics , Cyclin-Dependent Kinase Inhibitor p21/physiology , Fibroblast Growth Factor 2/physiology , G1 Phase/drug effects , Inhibitor of Differentiation Protein 1/physiology , Inhibitor of Differentiation Protein 2/physiology , Inhibitor of Differentiation Proteins/physiology , Neoplasm Proteins/physiology , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Line, Tumor , Extracellular Matrix/physiology , Gene Expression Regulation, Neoplastic , Humans , Neuroblastoma/genetics , RNA Interference , RNA, Messenger/metabolism , Signal Transduction
8.
Dev Biol ; 270(2): 455-73, 2004 Jun 15.
Article in English | MEDLINE | ID: mdl-15183726

ABSTRACT

Neural crest-derived cells that form the enteric nervous system undergo an extensive migration from the caudal hindbrain to colonize the entire gastrointestinal tract. Mice in which the expression of GFP is under the control of the Ret promoter were used to visualize neural crest-derived cell migration in the embryonic mouse gut in organ culture. Time-lapse imaging revealed that GFP(+) crest-derived cells formed chains that displayed complicated patterns of migration, with sudden and frequent changes in migratory speed and trajectories. Some of the leading cells and their processes formed a scaffold along which later cells migrated. To examine the effect of population size on migratory behavior, a small number of the most caudal GFP(+) cells were isolated from the remainder of the population. The isolated cells migrated slower than cells in large control populations, suggesting that migratory behavior is influenced by cell number and cell-cell contact. Previous studies have shown that neurons differentiate among the migrating cell population, but it is unclear whether they migrate. The phenotype of migrating cells was examined. Migrating cells expressed the neural crest cell marker, Sox10, but not neuronal markers, indicating that the majority of migratory cells observed did not have a neuronal phenotype.


Subject(s)
Cell Movement/physiology , Gastrointestinal Tract/cytology , Mice/embryology , Neural Crest/embryology , Animals , Cell Count , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Female , Gastrointestinal Tract/physiology , Green Fluorescent Proteins , High Mobility Group Proteins/metabolism , Immunohistochemistry , Luminescent Proteins/metabolism , Mice/metabolism , Mice, Transgenic , Microscopy, Confocal , Pregnancy , Proto-Oncogene Proteins c-ret , Receptor Protein-Tyrosine Kinases/metabolism , SOXE Transcription Factors , Time Factors , Transcription Factors
9.
J Cell Physiol ; 199(3): 371-80, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15095284

ABSTRACT

The insulin-like growth factor (IGF) system is a key regulator of cell growth, survival and differentiation, and these functions are co-modulated by other growth factors including fibroblast growth factor-2 (FGF-2). To investigate IGF/FGF interactions in neuronal cells, we employed neuroblastoma cells (SK-N-MC). In serum free conditions proliferation of the SK-N-MC cells was promoted by IGF-I (25 ng/ml), but blunted by FGF-2 (50 ng/ml). IGF-I-induced proliferation was abolished in the presence of FGF-2 even when IGF-I was used at 100 ng/ml. In addition to our previously described FGF-2 induced proteolytic cleavage of IGFBP-2, we found that FGF-2 increased IGFBP-6 levels in conditioned medium (CM) without affecting IGFBP-6 mRNA abundance. Modulation of IGFBP-2 and -6 levels were not significant mechanisms involved in the blockade of IGF-I action since the potent IGF-I analogues [QAYL]IGF-I and des(1-3)IGF-I (minimal IGFBP affinity) were unable to overcome FGF-2 inhibition of cell proliferation. FGF-2 treated cells showed morphological differentiation expressing the TUJ1 neuronal marker while cells treated with IGF-I alone showed no morphological change. When IGF-I was combined with FGF-2, however, cell morphology was indistinguishable from that seen with FGF-2 alone. FGF-2 inhibited proliferation and enhanced differentiation was also associated with a 70% increase in cell death. Although IGF-I alone was potently anti-apoptotic (60% decreased), IGF-I was unable to prevent apoptosis when administrated in combination with FGF-2. Gene-array analysis confirmed FGF-2 activation of the intrinsic and extrinsic apoptotic pathways and blockade of IGF anti-apoptotic signaling. FGF-2, directly and indirectly, overcomes the proliferative and anti-apoptotic activity of IGF-I by complex mechanisms, including enhancement of differentiation and apoptotic pathways, and inhibition of IGF-I induced anti-apoptotic signalling. Modulation of IGF binding protein abundance by FGF-2 does not play a significant role in inhibition of IGF-I induced mitogenesis.


Subject(s)
Fibroblast Growth Factor 2/pharmacology , Insulin-Like Growth Factor I/pharmacology , Neurons/drug effects , Apoptosis/drug effects , Apoptosis/physiology , Blotting, Northern , Blotting, Western , Cell Differentiation , Cell Division/drug effects , Cell Line, Tumor , Electrophoresis, Polyacrylamide Gel , Gene Expression/drug effects , Humans , Immunohistochemistry , Insulin-Like Growth Factor Binding Protein 2/drug effects , Insulin-Like Growth Factor Binding Protein 6/drug effects , Neuroblastoma , Neurons/metabolism , Oligonucleotide Array Sequence Analysis
10.
BJU Int ; 92(3): 297-305, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12887488

ABSTRACT

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.


Subject(s)
Connexins/metabolism , Electric Conductivity , Urinary Bladder Diseases/metabolism , Urinary Bladder Diseases/physiopathology , Blotting, Northern , Electric Impedance , Extracellular Space/physiology , Humans , Immunohistochemistry , Sarcoplasmic Reticulum/physiology
11.
Dev Dyn ; 227(3): 395-401, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12815625

ABSTRACT

The ability of glial cell line-derived neurotrophic factor (GDNF), neurturin, and artemin to induce neurite outgrowth from dorsal root, superior cervical, and lumbar sympathetic ganglia from mice at a variety of development stages between embryonic day (E) 11.5 and postnatal day (P) 7 was examined by explanting ganglia onto collagen gels and growing them in the presence of agarose beads impregnated with the different GDNF family ligands. Artemin, GDNF, and neurturin were all capable of influencing neurite outgrowth from dorsal root and sympathetic ganglia, but the responses of each neuron type to the different ligands varied during development. Neurites from dorsal root ganglia responded to artemin at P0 and P7, to GDNF at E15.5 and P0, and to neurturin at E15.5, P0, and P6/7; thus, artemin, GDNF, and neurturin are all capable of influencing neurite outgrowth from dorsal root ganglion neurons. Neurites from superior cervical sympathetic ganglia responded significantly to artemin at E15.5, to GDNF at E15.5 and P0, and to neurturin at E15.5. Neurites from lumbar sympathetic ganglia responded to artemin at all stages from E11.5 to P7, to GDNF at P0 and P7 and to neurturin at E11.5 to P6/7. Combined with the data from previous studies that have examined the expression of GDNF family members, our data suggest that artemin plays a role in inducing neurite outgrowth from young sympathetic neurons in the early stages of sympathetic axon pathfinding, whereas GDNF and neurturin are likely to be important at later stages of sympathetic neuron development in inducing axons to enter particular target tissues once they are in the vicinity or to induce branching within target tissues. Superior cervical and lumbar sympathetic ganglia showed temporal differences in their responsiveness to artemin, GDNF, and neurturin, which probably partly reflects the rostrocaudal development of sympathetic ganglia and the tissues they innervate.


Subject(s)
Ganglia, Spinal/embryology , Ganglia, Sympathetic/embryology , Gene Expression Regulation, Developmental , Nerve Growth Factors/metabolism , Nerve Tissue Proteins/metabolism , Neurons/physiology , Animals , Axons , Cell Movement , Collagen/metabolism , Epidermal Growth Factor/metabolism , Ganglia, Sympathetic/metabolism , Glial Cell Line-Derived Neurotrophic Factor , Lumbosacral Region , Mice , Microscopy, Confocal , Nerve Growth Factors/physiology , Nerve Tissue Proteins/physiology , Neurons/metabolism , Neurturin , Time Factors
12.
Bull Math Biol ; 65(2): 235-62, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12675331

ABSTRACT

During the development of vertebrate embryos, cell migrations occur on an underlying tissue domain in response to some factor, such as nutrient. Over the time scale of days in which this cell migration occurs, the underlying tissue is itself growing. Consequently cell migration and colonization is strongly affected by the tissue domain growth. Numerical solutions for a mathematical model of chemotactic migration with no domain growth can lead to travelling waves of cells with constant velocity; the addition of domain growth can lead to travelling waves with nonconstant velocity. These observations suggest a mathematical approximation to the full system equations, allowing the method of characteristics to be applied to a simplified chemotactic migration model. The evolution of the leading front of the migrating cell wave is analysed. Linear, exponential and logistic uniform domain growths are considered. Successful colonization of a growing domain depends on the competition between cell migration velocity and the velocity and form of the domain growth, as well as the initial penetration distance of the cells. In some instances the cells will never successfully colonize the growing domain. These models provide an insight into cell migration during embryonic growth, and its dependence upon the form and timing of the domain growth.


Subject(s)
Chemotaxis/physiology , Growth/physiology , Models, Biological , Animals , Cell Count/statistics & numerical data , Cell Division/physiology , Diffusion , Numerical Analysis, Computer-Assisted , Time Factors
13.
J Urol ; 168(3): 1235-9, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12187274

ABSTRACT

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.


Subject(s)
Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/pharmacology , Muscle Contraction/drug effects , Urinary Bladder Neck Obstruction/physiopathology , Urinary Bladder, Neurogenic/physiopathology , Urinary Bladder/drug effects , Adenosine Triphosphate/analogs & derivatives , Animals , Dose-Response Relationship, Drug , Female , Guinea Pigs , Humans , In Vitro Techniques , Male , Middle Aged , Muscle, Smooth/drug effects , Muscle, Smooth/enzymology , Muscle, Smooth/physiology , Urinary Bladder/enzymology , Urinary Bladder/physiology , Urinary Bladder Neck Obstruction/enzymology , Urinary Bladder, Neurogenic/enzymology
14.
Plast Reconstr Surg ; 108(3): 597-604, 2001 Sep 01.
Article in English | MEDLINE | ID: mdl-11698829

ABSTRACT

The close spatial relationship between peripheral nerves and blood vessels in the adult is well known. However, evidence supporting the congruent development of these structures in embryos remains anecdotal. Neurovascular relationships also have been shown to be conserved in other vertebrates. This homology suggests that either peripheral nerves or blood vessels, or both, might have fundamental morphogenetic roles during embryologic development. Both peripheral nerves and blood vessels have been independently implicated as etiologic agents in the pathogenesis of congenital disabilities, and several congenital anomalies fit their distribution patterns. This article presents a technique for the simultaneous visualization of peripheral nerves and blood vessels at different stages in the developing embryo. The forelimbs of 310 quail embryos were dissected over a 1-year period. Peripheral nerves were labeled with the neural crest and axon antibody, HNK-1, followed by fluorescein-conjugated secondary antibodies. Blood vessels were labeled by a perfusion technique using the fluorescent dye, dioctadecyl-tetramethylindocarbocyanine. Specimens were processed and imaged in whole-mount with confocal microscopy, and images were reconstructed using three-dimensional modeling software. Both nerves and blood vessels seem to undergo a highly stereotypic sequence of development in the embryonic quail forelimb. Furthermore, the existence of a close spatial relationship between nerves and blood vessels suggests either a high degree of developmental interdependence or shared patterning mechanisms. This technique permits further evaluation of the possible role peripheral nerves and blood vessels might play in the pathogenesis of congenital disabilities and provides a starting point for further studies aimed at elucidating the means by which peripheral nerves and blood vessels are patterned in the forelimb of the avian embryo.


Subject(s)
Blood Vessels/embryology , Fluorescent Dyes , Microscopy, Confocal/methods , Peripheral Nerves/embryology , Animals , Histological Techniques , Immunohistochemistry , Quail
15.
Genes Cells ; 6(7): 619-29, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11473580

ABSTRACT

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.


Subject(s)
Face/embryology , Facial Bones/embryology , Fibroblasts/cytology , Nuclear Proteins/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , Cell Nucleus/metabolism , Conserved Sequence , Evolution, Molecular , Gene Expression/physiology , Humans , In Situ Hybridization, Fluorescence , Mice , Molecular Sequence Data , Morphogenesis/genetics , Morphogenesis/physiology , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Quail/embryology , Quail/genetics , Transfection
16.
Dev Biol ; 229(2): 503-16, 2001 Jan 15.
Article in English | MEDLINE | ID: mdl-11150245

ABSTRACT

In situ hybridization revealed that GDNF mRNA in the mid- and hindgut mesenchyme of embryonic mice was minimal at E10.5 but was rapidly elevated at all gut regions after E11, but with a slight delay (0.5 days) in the hindgut. GDNF mRNA expression was minimal in the mesentery and in the pharyngeal and pelvic mesenchyme adjacent to the gut. To examine the effect of GDNF on enteric neural crest-derived cells, segments of E11.5 mouse hindgut containing crest-derived cells only at the rostral ends were attached to filter paper supports and grown in catenary organ culture. With GDNF (100 ng/ml) in the culture medium, threefold fewer neurons developed in the gut explants and fivefold more neurons were present on the filter paper outside the gut explants, compared to controls. Thus, in controls, crest-derived cells colonized the entire explant and differentiated into neurons, whereas in the presence of exogenous GDNF, most crest-derived cells migrated out of the gut explant. This is consistent with GDNF acting as a chemoattractant. To test this idea, explants of esophagus, midgut, superior cervical ganglia, paravertebral sympathetic chain ganglia, or dorsal root ganglia from E11.5-E12.5 mice were grown on collagen gels with a GDNF-impregnated agarose bead on one side and a control bead on the opposite side. Migrating neural cells and neurites from the esophagus and midgut accumulated around the GDNF-impregnated beads, but neural cells in other tissues showed little or no chemotactic response to GDNF, although all showed GDNF-receptor (Ret and GFRalpha1) immunoreactivity. We conclude that GDNF may promote the migration of crest cells throughout the gastrointestinal tract, prevent them from straying out of the gut (into the mesentery and pharyngeal and pelvic tissues), and promote directed axon outgrowth.


Subject(s)
Enteric Nervous System/embryology , Gene Expression Regulation, Developmental , Intestines/embryology , Nerve Growth Factors/genetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/pharmacology , Neurons/physiology , Animals , Cecum/embryology , Cecum/innervation , Cell Division/drug effects , Embryonic and Fetal Development , Glial Cell Line-Derived Neurotrophic Factor , Intestines/innervation , Mice , Nerve Growth Factors/pharmacology , Nerve Growth Factors/physiology , Nerve Tissue Proteins/physiology , Neurons/cytology , Neurons/drug effects , Organ Culture Techniques , RNA, Messenger/genetics , Transcription, Genetic
20.
Nat Genet ; 25(4): 414-8, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10932185

ABSTRACT

Secondary palate formation is a complex process that is frequently disturbed in mammals, resulting in the birth defect cleft palate. Gene targeting has identified components of cytokine/growth factor signalling systems such as Tgf-alpha/Egfr, Eph receptors B2 and B3 (Ephb2 and Ephb3, respectively), Tgf-beta2, Tgf-beta3 and activin-betaA (ref. 3) as regulators of secondary palate development. Here we demonstrate that the mouse orphan receptor 'related to tyrosine kinases' (Ryk) is essential for normal development and morphogenesis of craniofacial structures including the secondary palate. Ryk belongs to a subclass of catalytically inactive, but otherwise distantly related, receptor protein tyrosine kinases (RTKs). Mice homozygous for a null allele of Ryk have a distinctive craniofacial appearance, shortened limbs and postnatal mortality due to feeding and respiratory complications associated with a complete cleft of the secondary palate. Consistent with cleft palate phenocopy in Ephb2/Ephb3-deficient mice and the role of a Drosophila melanogaster Ryk orthologue, Derailed, in the transduction of repulsive axon pathfinding cues, our biochemical data implicate Ryk in signalling mediated by Eph receptors and the cell-junction-associated Af-6 (also known as Afadin). Our findings highlight the importance of signal crosstalk between members of different RTK subfamilies.


Subject(s)
Craniofacial Abnormalities/genetics , Receptor Cross-Talk/physiology , Receptor Protein-Tyrosine Kinases/metabolism , Animals , Animals, Newborn , Craniofacial Abnormalities/embryology , Embryonic and Fetal Development/genetics , Female , Genotype , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Mutation , Phenotype , Receptor Protein-Tyrosine Kinases/deficiency , Receptor Protein-Tyrosine Kinases/genetics , Receptor, EphB2 , Signal Transduction
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