RESUMEN
OBJECTIVES: To review the short-term outcome of endoscopic resection of superficial upper gastro-intestinal lesions in Hong Kong. DESIGN: Historical cohort study. SETTING: All Hospital Authority hospitals in Hong Kong. PATIENTS: This was a multicentre retrospective study of all patients who underwent endoscopic resection of superficial upper gastro-intestinal lesions between January 2010 and June 2013 in all government-funded hospitals in Hong Kong. MAIN OUTCOME MEASURES: Indication of the procedures, peri-procedural and procedural parameters, oncological outcomes, morbidity, and mortality. RESULTS: During the study period, 187 lesions in 168 patients were resected. Endoscopic mucosal resection was performed in 34 (18.2%) lesions and endoscopic submucosal dissection in 153 (81.8%) lesions. The mean size of the lesions was 2.6 (standard deviation, 1.8) cm. The 30-day morbidity rate was 14.4%, and perforations and severe bleeding occurred in 4.3% and 3.2% of the patients, respectively. Among patients who had dysplasia or carcinoma, R0 resection was achieved in 78% and the piecemeal resection rate was 11.8%. Lateral margin involvement was 14% and vertical margin involvement was 8%. Local recurrence occurred in 9% of patients and 15% had residual disease. The 2-year overall survival rate and disease-specific survival rate was 90.6% and 100%, respectively. CONCLUSION: Endoscopic mucosal resection and endoscopic submucosal dissection were introduced in low-to-moderate-volume hospitals with acceptable morbidity rates. The short-term survival was excellent. However, other oncological outcomes were higher than those observed in high-volume centres and more secondary procedures were required.
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Adenoma/cirugía , Carcinoma/cirugía , Neoplasias Duodenales/cirugía , Neoplasias Esofágicas/cirugía , Perforación Intestinal/etiología , Hemorragia Posoperatoria/etiología , Neoplasias Gástricas/cirugía , Adenoma/patología , Anciano , Pérdida de Sangre Quirúrgica , Carcinoma/patología , Disección/efectos adversos , Neoplasias Duodenales/patología , Endoscopía Gastrointestinal , Neoplasias Esofágicas/patología , Femenino , Mucosa Gástrica/cirugía , Hong Kong , Humanos , Mucosa Intestinal/cirugía , Masculino , Auditoría Médica , Persona de Mediana Edad , Neoplasia Residual , Estudios Retrospectivos , Neoplasias Gástricas/patología , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
We present an all-fiber passively mode-locked fiber laser incorporating three-dimensional (3D) graphene as a saturable absorber (SA) for the first time to the best of our knowledge. The 3D graphene is synthesized by template-directed chemical vapor deposition (CVD). The SA is then simply formed by sandwiching the freestanding 3D graphene between two conventional fiber connectors without any deposition process. It is demonstrated that such 3D graphene based SA is capable to produce high quality mode-locked pulses. A passively mode-locked fiber laser is constructed and stable output pulses with a fundamental repetition rate of ~9.9 MHz and a pulse width of ~1 ps are generated from the fiber laser. The average output power of the laser is ~10.5 mW while the output pulse is operating at single pulse region. The results imply that the freestanding 3D graphene can be applied as an effective saturable absorption material for passively mode-locked lasers.
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Carcinoma de Células Escamosas/etiología , Neoplasias Esofágicas/etiología , Infecciones por Papillomavirus/complicaciones , Estudios de Casos y Controles , Estudios Transversales , ADN Viral/análisis , Femenino , Genes p53 , Humanos , Masculino , Persona de Mediana Edad , Fumar/efectos adversosRESUMEN
The retinal cyclic guanosine 3',5'-monophosphate (cGMP) phosphodiesterase (PDE) is a key regulator of phototransduction in the vertebrate visual system. PDE consists of a catalytic core of alpha and beta subunits associated with two inhibitory gamma subunits. A gene-targeting approach was used to disrupt the mouse PDEgamma gene. This mutation resulted in a rapid retinal degeneration resembling human retinitis pigmentosa. In homozygous mutant mice, reduced rather than increased PDE activity was apparent; the PDEalphabeta dimer was formed but lacked hydrolytic activity. Thus, the inhibitory gamma subunit appears to be necessary for integrity of the photoreceptors and expression of PDE activity in vivo.
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3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , GMP Cíclico/metabolismo , Retina/patología , Degeneración Retiniana/enzimología , Células Fotorreceptoras Retinianas Bastones/enzimología , 3',5'-GMP Cíclico Fosfodiesterasas/deficiencia , 3',5'-GMP Cíclico Fosfodiesterasas/genética , Animales , Secuencia de Bases , Quimera , Cruzamientos Genéticos , Electrorretinografía , Activación Enzimática , Femenino , Marcación de Gen , Humanos , Luz , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutación , Fenotipo , Retina/metabolismo , Retina/fisiopatología , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Retinitis Pigmentosa/patologíaRESUMEN
Heterotrimeric guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) are deactivated by hydrolysis of the GTP that they bind when activated by transmembrane receptors. Transducin, the G protein that relays visual excitation from rhodopsin to the cyclic guanosine 3',5'-monophosphate phosphodiesterase (PDE) in retinal photoreceptors, must be deactivated for the light response to recover. A point mutation in the gamma subunit of PDE impaired transducin-PDE interactions and slowed the recovery rate of the flash response in transgenic mouse rods. These results indicate that the normal deactivation of transducin in vivo requires the G protein to interact with its target enzyme.
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3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Transducina/metabolismo , Visión Ocular , 3',5'-GMP Cíclico Fosfodiesterasas/genética , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , Electrorretinografía , Activación Enzimática , Femenino , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Trifosfato/metabolismo , Hidrólisis , Luz , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación Puntual , Retina/citología , Retina/fisiología , Degeneración Retiniana , TransgenesRESUMEN
PURPOSE: To determine whether local immunosuppression with Cyclosporin A can influence the survival of human fetal retinal pigment epithelium (RPE) xenografts in the rabbit's subretinal space. METHODS: Cultured human fetal RPE cells were transduced with the gene for green fluorescent protein (GFP) using a lentiviral vector. The RPE was transplanted into the subretinal space of rabbits that received intravitreal cyclosporine either by weekly injections (0. 25-0.5 mg) or by slow release (approximately 2 microg/d) from a capsule sutured into the vitreal cavity after prior cryopexy. The transplanted RPE was followed by GFP fluorescence scanning laser ophthalmoscopy and by histology of the transplant site. RESULTS: RPE xenografts in eyes receiving intravitreal cyclosporine survived longer (several months) than they did in control eyes without cyclosporine. Survival was as long with slow release capsules as it was with weekly intravitreal injections at much higher concentrations of cyclosporine. CONCLUSIONS: Local immunosuppression of the eye with cyclosporine prolongs the survival of RPE xenografts in the subretinal space of rabbits, implying that rejection involves activated T lymphocytes. Local immunosuppression with slow release capsules is as effective as weekly injections at much higher concentrations.
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Ciclosporina/farmacología , Trasplante de Tejido Fetal , Supervivencia de Injerto , Inmunosupresores/farmacología , Epitelio Pigmentado Ocular/trasplante , Retina/cirugía , Animales , Células Cultivadas , Fluorofotometría , Técnicas de Transferencia de Gen , Vectores Genéticos , Proteínas Fluorescentes Verdes , Humanos , Terapia de Inmunosupresión , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Oftalmoscopía , Epitelio Pigmentado Ocular/metabolismo , Epitelio Pigmentado Ocular/patología , Conejos , Retina/metabolismo , Retina/patología , Retroviridae/genética , Trasplante HeterólogoRESUMEN
PURPOSE: To examine the corneal electroretinogram (ERG) of transgenic mice (W70A mice) carrying a point mutation (W70A) in the gene encoding for the gamma-subunit of rod cGMP phosphodiesterase (PDEgamma). METHODS: The ERG of W70A mice was compared with that of normal mice. Cone responses were separated from rod responses by light adaptation, whereas rod sensitivity was assessed by threshold stimulation with dim light. Spectral sensitivity curves of the ERG were obtained using a constant response criterion. RESULTS: The ERG of the W70A mouse has a desensitized, delayed rod b-wave at threshold, and a prolonged rod b-wave at higher flash intensities. The a-wave is absent even at maximal stimulation. The cone ERG of the W70A mouse is indistinguishable from that of normal mice. The spectral sensitivity of the W70A mouse is maximal in the UV spectrum, in contrast to the normal mouse, which is most sensitive in the green region of the spectrum. This supports the interpretation of the results as normal cone and abnormal rod function in the W70A mouse. CONCLUSIONS: The W70A mouse represents new model of stationary nyctalopia that can be recognized by its unusual ERG features.
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3',5'-GMP Cíclico Fosfodiesterasas/genética , Ceguera Nocturna/genética , Mutación Puntual , Degeneración Retiniana/genética , Segmento Externo de la Célula en Bastón/enzimología , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , Adaptación a la Oscuridad , Electrorretinografía , Ratones , Ratones Transgénicos , Ceguera Nocturna/enzimología , Ceguera Nocturna/fisiopatología , Estimulación Luminosa , Células Fotorreceptoras Retinianas Conos/fisiología , Degeneración Retiniana/enzimología , Degeneración Retiniana/fisiopatología , Segmento Externo de la Célula en Bastón/fisiopatología , Umbral SensorialRESUMEN
PURPOSE: Mice (Pdegtm1/Pdegtm1) homozygous for a mutant allele of the gamma subunit of retinal cyclic guanosine monophosphate phosphodiesterase (PDE gamma) suffer a severe photoreceptor degeneration. To determine whether the antiapoptotic BCL2 gene is effective in delaying the cell death pathway in this new strain of mutant mice, a transgene encoding the BCL2 gene product was introduced by mating into the mutant background, and the resulting mice were examined for possible rescue of the retinal degeneration. METHODS: Electroretinograms (ERGs) of the Pdegtm1/Pdegtm1 mice carrying BCL2 were taken to monitor the responses to light. Light and electron microscopy of sections were used to examine degeneration at different times after birth. RESULTS: The ERGs of the mutants with the transgene were larger than those without the transgene at 2 and 3 weeks after birth. The maximum differences occurred at 2 weeks postpartum. At 4 weeks after birth, no ERG could be detected in either strain. Histologic analysis showed a greater preservation of photoreceptor nuclei in the Pdegtm1/Pdegtm1 mice containing the BCL2 transgene, which paralleled the electroretinography. CONCLUSIONS: The introduction of an antiapoptotic transgene BCL2 can delay temporarily and partially the degeneration of photoreceptors in a new autosomal-recessive murine model of retinal degeneration.
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3',5'-GMP Cíclico Fosfodiesterasas/genética , Apoptosis/genética , Genes Supresores , Genes bcl-2/genética , Células Fotorreceptoras/ultraestructura , Degeneración Retiniana/genética , Degeneración Retiniana/prevención & control , Animales , Supervivencia Celular , Electroforesis en Gel de Agar , Electrorretinografía , Femenino , Expresión Génica , Genotipo , Masculino , Ratones , Ratones Mutantes , Ratones Transgénicos/genética , Células Fotorreceptoras/enzimología , Células Fotorreceptoras/fisiopatología , Reacción en Cadena de la Polimerasa , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatologíaRESUMEN
PURPOSE: To determine whether human retinal pigment epithelium (RPE) can be modified by retroviral-mediated gene transfer and to monitor the human RPE cells in the subretinal space of living rabbits with scanning laser ophthalmoscopy (SLO). METHODS: Cultured human fetal retinal pigment epithelium (HFRPE) was exposed to green fluorescent protein (GFP)-transducing retroviral vectors, Moloney murine leukemia virus, and lentivirus. The cultured cells were followed by fluorescence microscopy. Suspensions of GFP-expressing HFRPE were transplanted into the subretinal space of pigmented rabbits, and the transplant sites were examined by SLO for fluorescence, including fluorescein and indocyanine green angiography. The rabbits were euthanatized at different times after transplantation, and the retinas were studied histologically. RESULTS: Retroviral gene transfer can introduce a foreign gene such as GFP into cultured HFRPE. Gene expression is maintained in cultured RPE for at least 3 months. The lentiviral vector transduced both nondividing and dividing cells; the Moloney vector only transduced the latter. GFP-expressing cells can be followed in the living retina. Their changes reflect the rejection response followed histologically. CONCLUSIONS: Cultured HFRPE could be transduced to express GFP for long periods of time by retroviral gene transfer. GFP allowed retinal transplants and gene expression to be monitored in vivo. These results provide a model for potential ex vivo gene therapy in the subretinal space.
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Trasplante de Tejido Fetal , Técnicas de Transferencia de Gen , Proteínas Luminiscentes/metabolismo , Epitelio Pigmentado Ocular/trasplante , Retina/cirugía , Retroviridae/genética , Animales , Células Cultivadas , Virus Defectuosos , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Oftalmoscopía , Epitelio Pigmentado Ocular/metabolismo , Epitelio Pigmentado Ocular/virología , Conejos , Retina/citología , Retina/metabolismo , Retroviridae/metabolismoRESUMEN
The vasorelaxant actions of adenosine 5'-triphosphate (ATP)-dependent K+ channel openers and sodium nitroprusside in isolated thoracic aorta and pulmonary artery of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats (14-18 weeks old) were investigated. Cumulative addition of sodium nitroprusside and different ATP-dependent K+ channel openers (pinacidil, cromakalim, nicorandil, 2-(2"(1",3"-dioxolone)-2-methyl-4-(2'-oxo-1'-pyrrolidinyl)-6-nitro -2H-1-benzopyren (KR-30450) and aprikalim) to these preparations caused a concentration-dependent relaxation of noradrenaline-pre-contracted aorta and pulmonary artery from both strains. The relative order of relaxation potency, estimated by comparing the IC50, was sodium nitroprusside > KR-30450 > aprikalim > or = cromakalim > pinacidil > nicorandil in pulmonary artery and aorta from both strains. At high concentrations (> or =1 microM), cromakalim, aprikalim and KR-30450 produced a greater percentage relaxation in SHR aorta than in WKY aorta. However, there was no apparent difference between SHR and WKY in the relaxation response to all drugs tested on the pulmonary artery. The effects of cromakalim, aprikalim, pinacidil and KR-30450 observed in aorta and pulmonary artery were significantly attenuated by 3 microM glibenclamide. 6-Anilino-5,8-quinolinequinone (LY 83583, 1 microM), a soluble guanylate cyclase inhibitor, abolished the vasorelaxant effects of nicorandil and sodium nitroprusside. In conclusion, sodium nitroprusside and ATP-dependent K+ channel openers cause relaxation of noradrenaline-pre-contracted aorta and pulmonary artery from both strains. However, all the drugs tested failed to cause selective relaxation of the pulmonary artery relative to the thoracic aorta.
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Aorta Torácica/efectos de los fármacos , Hipertensión/fisiopatología , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Canales de Potasio/agonistas , Arteria Pulmonar/efectos de los fármacos , Vasodilatadores/farmacología , Adenosina Trifosfato/farmacología , Aminoquinolinas/farmacología , Animales , Apamina/farmacología , Benzopiranos/farmacología , Caribdotoxina/farmacología , Cromakalim/farmacología , Relación Dosis-Respuesta a Droga , Gliburida/farmacología , Técnicas In Vitro , Masculino , NG-Nitroarginina Metil Éster/farmacología , Nicorandil/farmacología , Nitroprusiato/farmacología , Norepinefrina/farmacología , Picolinas/farmacología , Pinacidilo/farmacología , Potasio/farmacología , Piranos/farmacología , Pirrolidinonas/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
This paper demonstrates lentiviral transduction of the humanized form of the Aequoria victoria gene for green fluorescent protein (GFP) into human fetal retinal pigment epithelium (RPE) in vitro and rabbit RPE in vivo. In vitro GFP expression of cultured human fetal RPE begins within two to three days after 12-16 h of maintained exposure to the virus at titers of 10(8)-10(9) infectious units (IU)/ml. Both stationary and dividing cells are transduced using a lenti viral vector with a cytomegalovirus (CMV) promoter. Expression remains stable for at least three to four months without evidence of toxicity and continues through cell division. In vivo expression is followed non-invasively in rabbit eye using a scanning laser ophthalmoscope (SLO), which can detect single fluorescing retinal cells. In vivo expression begins within a few days after a viral solution is introduced into the subretinal space. A solution of 10(9) IU/ml produces fluorescence within three to four days. Less concentrated solutions lead to slower and less expression. No expression is detectable at concentrations of 10(6) IU/ml. Within one to two weeks after introduction of the viral solution, there is evidence of rejection seen by SLO as a loss of GFP fluorescence and disruption of the RPE. Histology shows damage to the RPE layer and monocytic cell infiltrates in the choroid and subretinal space within the area receiving the viral solution. Strong GFP expression leads to rejection within two weeks. With less expression, rejection is delayed and in some cases undetectable for at least six months. If the GFP gene is not included in the viral vector or if the viral concentration is insufficient to produce detectable GFP expression, rejection is not seen. Using a rhodopsin promoter or injecting the virus intra rather than subretinally produces weak expression and no rejection. Lentivirus can induce expression of a foreign gene in the RPE. Viral induced transduction and GFP expression have no effect on the viability of the RPE in vitro. Continued expression of GFP after cell division implies chromosomal integration of the gene. In vivo expression of GFP in RPE encounters rejection. Rejection may not occur with low GFP expression. The latter occurs with low viral titers, a rhodopsin promoter or intra-retinal injection of viral solution. The results are relevant to gene therapy in retina when gene transduction leads to the expression of foreign proteins.
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Vectores Genéticos/administración & dosificación , Lentivirus/genética , Proteínas Luminiscentes/genética , Epitelio Pigmentado Ocular/metabolismo , Transducción Genética/métodos , Animales , División Celular , Técnicas de Cultivo , Expresión Génica , Rechazo de Injerto , Proteínas Fluorescentes Verdes , Humanos , Inyecciones , Proteínas Luminiscentes/inmunología , Microscopía Confocal , Epitelio Pigmentado Ocular/embriología , Epitelio Pigmentado Ocular/inmunología , Conejos , Factores de TiempoRESUMEN
PURPOSE: To evaluate the use of surgical treatment with amniotic membrane for long-term atopic keratoconjunctivitis. Damaged corneas were repaired with various techniques: amniotic membrane transplantations, amniotic membrane coverings, amniotic membrane fillings (AMFs), and amniotic membrane inlay fillings, the latter of which were combined with glycerol-preserved corneal transplants. METHODS: This retrospective study was conducted on 37 eyes belonging to 37 patients with atopic keratoconjunctivitis. Thirty-two patients were classified into four groups according to surgical technique. Five patients undergoing medical management served as controls. Surgical outcome was measured by recovery time and long-term visual improvement. RESULTS: In all surgical eyes, integrity of ocular tissues was effectively restored and symptoms were reduced at 24.4 ± 13 days post recovery. Mean best-corrected visual acuity improved from 0.6 ± 0.2 to 0.198 ± 0.16 logarithm of the minimum angle of resolution (P<0.001). There were no intraoperative or postoperative complications, with the exception of two recurring cases, both controlled by medication. Recovery time of the control groups lasted 52 ± 16 days. In controls, mean best-corrected visual acuity improved from 0.74 ± 0.15 to 0.54 ± 0.29 logarithm of the minimum angle of resolution (P ≤ 0.05). The vision improvement has significant difference for surgical treatment vs medical. (Mann-Whitney U-test, U = 119, P < 0.05, one tailed).Vision improvements remained stable during a mean follow-up period of 21.7 ± 3.8 months. CONCLUSION: Patients suffering from severe chronic atopic keratoconjunctivitis and its complications can benefit from suitable surgical treatments: transplants, covers, fillings, or corneal graft surgeries supplemented with AMFs.
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Amnios/trasplante , Queratoconjuntivitis/cirugía , Procedimientos Quirúrgicos Oftalmológicos , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Preescolar , Enfermedad Crónica , Trasplante de Córnea/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Agudeza Visual , Adulto JovenRESUMEN
Formation of nanocrystals with preferred orientation within the amorphous carbon matrix has attracted lots of theoretical and experimental attentions recently. Interesting properties of this films, easy fabrication methods and practical problems associated with the growth of other carbon nanomaterials such as carbon nanotubes (CNTs) and graphene gives this new class of carbon nanostructure a potential to be considered as a replacement for some applications such as thermal management at nanoscale and interconnects. In this short review paper, the fabrication techniques and associated formation mechanisms of these nanostructured films have been discussed. Besides, electrical and thermal properties of these nanostructured films have been compared with CNTs and graphene.
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Carbono/química , Nanotecnología/métodos , Electricidad , Grafito/química , TemperaturaRESUMEN
AIM: To characterise new clinical features in a family with enhanced S-cone syndrome (ESCS) and investigate the pathogenesis of these clinical features in the homozygous Nr2e3(rd7) (rd7) mutant mice. METHODS: Four patients from an affected family were included for genotypic and phenotypic study. Eye tissues from rd7 mice were used to detect a possible relationship between macrophages and autofluorescent material by immunohistochemistry (IHC) staining. RESULTS: Homozygous mutation in R311Q in NR2E3 was detected in this family. Colour photographs revealed that white dots do not correlate to hyperautofluorescent spots seen in autofluorescence imaging of the macula. OCT showed rosette-like lesions similar to those found in rd7 mice histology sections. From IHC analysis, we observed that F4/80 (a pan macrophage marker) and autofluorescence were colocalised to the same cells within the retina rosettes. CONCLUSIONS: The retinal structure of a young ESCS patient with homozygous R311Q mutation in the NR2E3 gene is similar to that seen in the rd7 mice. The macrophages were found to contain autofluorescent materials in the retinal rosettes of rd7 mice. These data are consistent with macrophage infiltration contributing to the hyperautofluorescent spots found in our patients.
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Regulación del Desarrollo de la Expresión Génica/genética , Mutación/genética , Receptores Citoplasmáticos y Nucleares/genética , Células Fotorreceptoras Retinianas Conos/fisiología , Degeneración Retiniana/genética , Factores de Transcripción/genética , Animales , Niño , Angiografía con Fluoresceína , Fondo de Ojo , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Mutantes , Receptores Nucleares Huérfanos , Linaje , Degeneración Retiniana/fisiopatologíaRESUMEN
The phosphodiesterase 6 gamma (PDE6 gamma) inhibitory subunit of the rod PDE6 effector enzyme plays a central role in the turning on and off of the visual transduction cascade, since binding of PDE6 gamma to the transducin alpha subunit (T alpha) initiates the hydrolysis of the second messenger cGMP, and PDE6 gamma in association with RGS9-1 and the other GAP complex proteins (G beta 5, R9AP) accelerates the conversion of T alpha GTP to T alpha GDP, the rate-limiting step in the decay of the rod light response. Several studies have shown that PDE6 gamma can be phosphorylated at two threonines, T22 and T35, and have proposed that phosphorylation plays some role in the physiology of the rod. We have examined this possibility by constructing mice in which T22 and/or T35 were replaced with alanines. Our results show that T35A rod responses rise and decay more slowly and are less sensitive to light than wild-type (WT). T22A responses show no significant difference in initial time course with WT but decay more rapidly, especially at dimmer intensities. When the T22A mutation is added to T35A, double mutant rods no longer showed the prolonged decay of T35A rods but remained slower than WT in initial time course. Our experiments suggest that the polycationic domain of PDE6 gamma containing these two phosphorylation sites can influence the rate of PDE6 activation and deactivation and raise the possibility that phosphorylation or dephosphorylation of PDE6 gamma could modify the time course of transduction, thereby influencing the wave form of the light response.
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Luz , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , Electrofisiología , Humanos , Masculino , Ratones , Ratones Mutantes , Ratones Transgénicos , Mutación/genética , Fosforilación , Fotones , Transducción de Señal/fisiologíaRESUMEN
The nuclear magnetic resonance spin-lattice (T1) and spin-spin (T2) relaxation times are closely related to the molecular motions of the molecules in a liquid sample. T1 and T2 of human epidermal cells were measured at 300 MHz as functions of harvesting methods (i.e., scraping vs trypsinization) and age in culture. It was found that T1 and T2 values have smaller variances when the cell is harvested by trypsinization rather than scraping. The correlation coefficients for both T1 and T2, obtained from cells harvested by scraping. More importantly, this is the first report to monitor in vitro aging through relaxation times measurement. There is a significant increase in the values of T1 and T2 from the third to seventh passages. Human keratinocytes slowed down and even ceased to grow the seventh passage. Therefore, the cellular water molecules of human keratinocytes have higher mobility in a more differentiated state. The factors contributing to the change in relaxation times as cells progress toward senescence are discussed.
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Queratinocitos/citología , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Diploidia , Humanos , Espectroscopía de Resonancia Magnética/métodos , Protones , Tripsina , AguaRESUMEN
Basic fibroblast growth factor (FGF2) is a wide-spectrum mitogenic, angiogenic, and neurotrophic factor that is expressed at low levels in many tissues and cell types and reaches high concentrations in brain and pituitary. FGF2 has been implicated in a multitude of physiological and pathological processes, including limb development, angiogenesis, wound healing, and tumor growth, but its physiological role is still unclear. To determine the function of FGF2 in vivo, we have generated FGF2 knockout mice, lacking all three FGF2 isoforms, by homologous recombination in embryonic stem cells. FGF2(-/-) mice are viable, fertile and phenotypically indistinguishable from FGF2(+/+) littermates by gross examination. However, abnormalities in the cytoarchitecture of the neocortex, most pronounced in the frontal motor-sensory area, can be detected by histological and immunohistochemical methods. A significant reduction in neuronal density is observed in most layers of the motor cortex in the FGF2(-/-) mice, with layer V being the most affected. Cell density is normal in other regions of the brain such as the striatum and the hippocampus. In addition, the healing of excisional skin wounds is delayed in mice lacking FGF2. These results indicate that FGF2, although not essential for embryonic development, plays a specific role in cortical neurogenesis and skin wound healing in mice, which, in spite of the apparent redundancy of FGF signaling, cannot be carried out by other FGF family members.
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Factor 2 de Crecimiento de Fibroblastos/fisiología , Neuronas/fisiología , Cicatrización de Heridas , Animales , Recuento de Células , Factor 2 de Crecimiento de Fibroblastos/deficiencia , Factor 2 de Crecimiento de Fibroblastos/genética , Ratones , Ratones Noqueados , Neocórtex/citología , Neocórtex/crecimiento & desarrollo , Piel/lesiones , Cicatrización de Heridas/genéticaRESUMEN
The inhibitory rod cGMP phosphodiesterase gamma subunit (PDEgamma) is a major component of the photoresponse and is required to support rod integrity. Pdeg(tm1)/Pdeg(tm1) mice (which lack PDEgamma owing to a targeted disruption of the Pdeg gene) suffer from a very rapid and severe photoreceptor degeneration. The Y84G (Tyr(84)-->Gly) allele of PDEgamma has previously been shown in experiments carried out in vitro to reduce the regulatory control of the PDE catalytic core (PDEalphabeta) exerted by the wild-type gamma subunit. To determine the effects of this mutation on in vivo function, the murine opsin promoter was used to direct expression to the photoreceptors of +/Pdeg(tm1) mice of a mutant Y84G and a wild-type PDEgamma control transgene. The transgenic mice were crossed with Pdeg(tm1)/Pdeg(tm1) mice to generate animals able to synthesize only the transgenic PDEgamma. Our results showed that wild-type PDEgamma and Y84G transgenes could complement the Pdeg(tm1)/Pdeg(tm1) mutant for photoreceptor survival. The mutation caused a significant biochemical defect in PDE activation by transducin. However, the Y84G mutation did not fully eliminate the control of PDEgamma on the PDE catalytic core in vivo; the expression of the mutant subunit was associated with only a 10-fold reduction in the amplitude of the a-wave and a 1.5-fold decrease in the b-wave of the corneal electroretinogram. Unexpectedly, the mutation caused a much 'milder' phenotype in vivo than was predicted from the biochemical assays in vitro.