RESUMEN
We have developed a reliable, cost-effective and non-toxic fixative to meet the needs of contemporary molecular pathobiology research, particularly in respect of RNA and DNA integrity. The effects of 25 different fixative recipes on the fixed quality of tissues from C57BL/6 mice were investigated. Results from IHC, PCR, RT-PCR, RNA Agilent Bioanalyser and Real-Time PCR showed that a novel zinc-based fixative (Z7) containing zinc trifluoroacetate, zinc chloride and calcium acetate was significantly better than the standard zinc-based fixative (Z2) and neutral buffered formalin (NBF) for DNA, RNA and protein preservation. DNA sequences up to 2.4 kb in length and RNA fragments up to 361 bp in length were successfully amplified from Z7 fixed tissues, as demonstrated by PCR, RT-PCR and Real-Time PCR. Total protein analysis was achieved using 2-D gel electrophoresis. In addition, nucleic acids and proteins were very stable over a 6-14-month period. This improved, non-toxic and economical tissue fixative could be applied for routine use in pathology laboratories to permit subsequent genomic/proteomic studies.
Asunto(s)
Acetatos/química , Cloruros/química , ADN/normas , Fijadores/química , Proteínas/normas , ARN/normas , Ácido Trifluoroacético/química , Compuestos de Zinc/química , Zinc/química , Animales , Compuestos de Calcio/química , ADN/análisis , Electroforesis en Gel Bidimensional , Femenino , Formaldehído , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Adhesión en Parafina , Reacción en Cadena de la Polimerasa , Proteínas/análisis , Proteómica , ARN/análisis , Fijación del Tejido/métodosRESUMEN
Tumor cells proliferate rapidly and thus are frequently subjected to replication stress and the risk of incomplete duplication of the genome. Fragile sites are replicated late, making them more vulnerable to damage when DNA replication fails to complete. Therefore, genomic alterations at fragile sites are commonly observed in tumors. FRA16D is one of the most common fragile sites in lung cancer, however, the nature of the tumor suppressor genes affected by FRA16D alterations has been controversial. Here, we show that the ATMIN gene, which encodes a cofactor required for activation of ATM kinase by replication stress, is located close to FRA16D and is commonly lost in lung adenocarcinoma. Low ATMIN expression was frequently observed in human lung adenocarcinoma tumors and was associated with reduced patient survival, suggesting that ATMIN functions as a tumor suppressor in lung adenocarcinoma. Heterozygous Atmin deletion significantly increased tumor cell proliferation, tumor burden, and tumor grade in the LSL-KRasG12D; Trp53 F/F (KP) mouse model of lung adenocarcinoma, identifying ATMIN as a haploinsufficient tumor suppressor. ATMIN-deficient KP lung tumor cells showed increased survival in response to replication stress and consequently accumulated DNA damage. Thus, our data identify ATMIN as a key gene affected by genomic deletions at FRA16D in lung adenocarcinoma. SIGNIFICANCE: These findings identify ATMIN as a tumor suppressor in LUAD; fragility at chr16q23 correlates with loss of ATMIN in human LUAD and deletion of Atmin increases tumor burden in a LUAD mouse model.
Asunto(s)
Adenocarcinoma/genética , Sitios Frágiles del Cromosoma/genética , Cromosomas Humanos Par 16/genética , Genes Supresores de Tumor , Neoplasias Pulmonares/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Animales , Células Cultivadas , Cromosomas Humanos Par 16/ultraestructura , Daño del ADN , Regulación Neoplásica de la Expresión Génica , Genotipo , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Ratones , Clasificación del Tumor , Factores de Transcripción/deficiencia , Factores de Transcripción/fisiología , Carga Tumoral/genética , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Segmental mediolytic arteriopathy (SMA) is a rare condition. It was first defined in 1976 and has been well described in the literature, although to date the aetiology of the condition is unknown. In most case reports SMA is diagnosed retrospectively once tissue has undergone histological examination. We present the first known case of SMA of the colic, mid-jejunal, common hepatic, intrahepatic and gastric arteries to be diagnosed at angiography after multiple episodes of undiagnosed intraperitoneal bleeding, and, perhaps related to this, one of the few reported patients with SMA involving multiple intra-abdominal arteries to have survived.
Asunto(s)
Hemoperitoneo/etiología , Enfermedades Vasculares/complicaciones , Aneurisma Roto/diagnóstico por imagen , Angiografía , Colon/irrigación sanguínea , Embolización Terapéutica , Hemoperitoneo/diagnóstico por imagen , Hemoperitoneo/terapia , Humanos , Yeyuno/irrigación sanguínea , Masculino , Arterias Mesentéricas/diagnóstico por imagen , Persona de Mediana Edad , Reoperación , Enfermedades Vasculares/diagnóstico por imagen , Enfermedades Vasculares/terapiaRESUMEN
Detection, characterization, and staging constitute the fundamental elements in the endoscopic diagnosis of gastrointestinal diseases, but histology still remains the diagnostic gold standard. New developments in endoscopic techniques may challenge histopathology in the near future. An ideal endoscopic technique should combine a wide-field, "red flag" screening technique with an optical contrast or microscopy method for characterization and staging, all simultaneously available during the procedure. In theory, biophotonic advances have the potential to unite these elements to allow in vivo "optical biopsy." These techniques may ultimately offer the potential to increase the rates of detection of high risk lesions and the ability to target biopsies and resections, and so reduce the need for biopsy, costs, and uncertainty for patients. However, their utility and sensitivity in clinical practice must be evaluated against those of conventional histopathology. This review describes some of the most recent applications of biophotonics in endoscopic optical imaging and metrology, along with their fundamental principles and the clinical experience that has been acquired in their deployment as tools for the endoscopist. Particular emphasis has been placed on translational label-free optical techniques, such as fluorescence spectroscopy, fluorescence lifetime imaging microscopy (FLIM), two-photon and multi-photon microscopy, second harmonic generation (SHG) and third harmonic generation (THG) imaging, optical coherence tomography (OCT), diffuse reflectance, Raman spectroscopy, and molecular imaging.
RESUMEN
Androgen independence is the major cause of endocrine therapy failure in advanced prostate cancer (PC). To examine the effects of human androgen receptor (AR) expression on growth of human PC cells, transfection of full-length AR cDNA in an androgen-insensitive human prostatic adenocarcinoma cell line (DU145) was performed. Transcriptional activity of AR was confirmed by the MMTV luciferase assay and AR expression was assessed by reverse transcriptase polymerase chain reaction, Western blotting, and immunocytochemistry. Two stable transfectant cell lines expressing functional AR were established and passaged over 60 times. Under standard culture conditions, AR expression in transfected cells was predominantly cytoplasmic. Exposure to dihydrotestosterone (DHT; 60 pM-10 nM) resulted in a rapid (maximal at 30 minutes) translocation of AR to the nucleus. Treatment with DHT (5 nM) caused a significant reduction in cell-cell adhesion and aggregation accompanied by a decrease in E-cadherin expression. This was associated with up to 40% inhibition of proliferation and approximately two-fold increase in apoptosis. These results suggest that gene transfer-mediated AR expression in DU145 cells confers sensitivity to DHT, modulates cell-cell adhesion through E-cadherin, and suppresses cell growth by inhibiting proliferation and promoting apoptosis. This provides amodelfor studies ofAR-regulated cell signalling and identification of novel androgen-regulated genes in PC.
Asunto(s)
Apoptosis , Cadherinas/metabolismo , Receptores Androgénicos/biosíntesis , Transcripción Genética , Transporte Activo de Núcleo Celular , Western Blotting , Adhesión Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Separación Celular , Colorantes/farmacología , Citoplasma/metabolismo , ADN Complementario/metabolismo , Densitometría , Dihidrotestosterona/farmacología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Genes Reporteros , Humanos , Inmunohistoquímica , Ligandos , Luciferasas/metabolismo , Microscopía Fluorescente , Plásmidos/metabolismo , Pruebas de Precipitina , ARN/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Sales de Tetrazolio/farmacología , Tiazoles/farmacología , Factores de Tiempo , Transfección , Azul de Tripano/farmacologíaRESUMEN
This study examines the coexpression of MUC1 mucin and trefoil factor 1 (TFF1) and their relationship to progression of renal cell carcinoma (RCC). Immunohistochemistry was performed on tumor and adjacent normal tissue from clear-cell RCC (n = 60) and tissues from normal controls (n = 5) using a set of well-characterized monoclonal antibodies recognizing different epitopes of MUC1 and TFF1. Results of immunohistochemistry were compared with clinical parameters, including tumor grade, tumor size, presence of metastasis, and progression-free survival of patients after surgery. In normal tissue, MUC1 and TFF1 were absent from the normal proximal tubular epithelium but were identified in distal and collecting tubular epithelium. In RCC, increased MUC1 expression positively correlated to tumor progression. MUC1 recognized by HMFG1 was associated with large tumor size (P < .05), distant metastasis (P < .05), and invasion of large veins (P < .05). Expression of the under-glycosylated form of MUC1 recognized by SM3 was found to correlate to time to progression (recurrence, metastasis, or death of patient; P < .001). Expression of TFF1 did not significantly correlate with any prognostic parameters. However, there was a significant correlation (P < .01) between TFF1 and MUC1 expression (HMFG2 epitope) in RCCs. These results are consistent with the following conclusions: (1) MUC1 may be an independent prognostic marker in RCC; (2) TFF1 is frequently coexpressed with MUC1 and may act synergistically; and (3) RCC may originate from distal tubular epithelium.
Asunto(s)
Carcinoma de Células Renales/metabolismo , Proteínas de Unión al ADN/biosíntesis , Neoplasias Renales/metabolismo , Mucina-1/biosíntesis , Proteínas Nucleares/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/secundario , Recuento de Células , Epítopos , Femenino , Humanos , Inmunohistoquímica , Corteza Renal/anatomía & histología , Corteza Renal/metabolismo , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Mucina-1/clasificación , Pronóstico , Factores de TranscripciónRESUMEN
Trefoil factor family (TFF) domain peptides, products of mucin-secreting epithelial cells, are thought to influence mucosal integrity. Molecular studies revealed that mammalian TFFs lack transmembrane domains. Using immunocytochemistry and FACS analysis we demonstrated the association of TFF1 with the cell membrane in MCF-7 (a breast adenocarcinoma cell line), and tested the hypothesis that glycosylphosphatidylinositol (GPI) linkage is the mechanism for this association. Cleavage of GPI anchorage using phospholipase C did not affect TFF1 binding to the cell membrane. Our results demonstrate for the first time that TFF1 is associated with the cell membrane of MCF-7 cells and is not linked via a GPI anchor.
Asunto(s)
Membrana Celular/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Proteínas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Neoplasias de la Mama/metabolismo , Femenino , Citometría de Flujo , Humanos , Factor Trefoil-1 , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
The acute phase response is strictly connected with modulation of gene expression. Transcriptional control of many genes is mediated by binding of diverse transcription factors to cis-acting DNA motifs in the respective promoter sequence. We now describe such regulatory elements for the TFF3 gene coding for a peptide involved in response to gut irritation. TNF-alpha stimulation, which induces NF-kappaB activation, evoked up to 10-fold reduction of TFF3 expression in the colon tumour cell line HT-29. Several consensus binding sites for members of the NF-kappaB transcription factor subunits are located in the 5'-flanking region of TFF3. Mutating these sites was aimed at discovering which one is responsible for NF-kappaB binding and thus regulation of TFF3 expression.
Asunto(s)
Mucinas/genética , Proteínas Musculares/genética , FN-kappa B/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Activación Transcripcional/efectos de los fármacos , Proteínas Potenciadoras de Unión a CCAAT/genética , Ensayo de Cambio de Movilidad Electroforética , Células HT29 , Humanos , Mutación/genética , Péptidos , Factor de Transcripción ReIA , Factor Trefoil-3 , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
We have recently demonstrated that human TFF2 inhibits apoptosis in the non-TFF2 expressing breast adenocarcinoma cell line MCF-7. In this study we examined the impact of TFF2 and an anti-TFF2 antibody (hSP3) on the survival of other human adenocarcinoma cell lines; TFF2-positive (LS174T and SW480) and TFF2-negative (MCF-7 and T47D). Addition of TFF2 protected the (TFF2-) lines but had no effect on those constitutively expressing TFF2. Blocking with hSP3 significantly increased apoptosis in the (TFF2+) cell lines with minimal effect on the (TFF2-) cells. Our results show that the cytoprotective effect of TFF2 seen in MCF-7 cells is not cell line-specific and can be abrogated by inhibition of its expression.
Asunto(s)
Anticuerpos/metabolismo , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias Colorrectales/metabolismo , Mucinas/farmacología , Proteínas Musculares/farmacología , Péptidos/farmacología , Humanos , Proteínas Recombinantes/farmacología , Factor Trefoil-2 , Células Tumorales CultivadasRESUMEN
We present the first detailed study using multispectral multiphoton fluorescence lifetime imaging to differentiate basal cell carcinoma cells (BCCs) from normal keratinocytes. Images were acquired from 19 freshly excised BCCs and 27 samples of normal skin (in & ex vivo). Features from fluorescence lifetime images were used to discriminate BCCs with a sensitivity/specificity of 79%/93% respectively. A mosaic of BCC fluorescence lifetime images covering >1 mm(2) is also presented, demonstrating the potential for tumour margin delineation. Using 10,462 manually segmented cells from the image data, we quantify the cellular morphology and spectroscopic differences between BCCs and normal skin for the first time. Statistically significant increases were found in the fluorescence lifetimes of cells from BCCs in all spectral channels, ranging from 19.9% (425-515 nm spectral emission) to 39.8% (620-655 nm emission). A discriminant analysis based diagnostic algorithm allowed the fraction of cells classified as malignant to be calculated for each patient. This yielded a receiver operator characteristic area under the curve for the detection of BCC of 0.83. We have used both morphological and spectroscopic parameters to discriminate BCC from normal skin, and provide a comprehensive base for how this technique could be used for BCC assessment in clinical practice.
Asunto(s)
Carcinoma Basocelular/diagnóstico , Fotones , Neoplasias Cutáneas/diagnóstico , Tomografía/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Carcinoma Basocelular/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Cutáneas/patología , Espectrometría de Fluorescencia , Factores de Tiempo , Adulto JovenRESUMEN
OBJECTIVE: To characterize possible endometrial defects in infertile women with isolated PCO morphology. DESIGN: Prospective study. SETTING: An academic hospital with an IVF unit. PATIENT(S): Women with primary unexplained infertility and isolated PCO, fertile women, and women with infertility secondary to male factor. INTERVENTION(S): Thirty-one women (fertile and with male factor infertility) had endometrial sampling across the menstrual cycle. Nine fertile women and 10 infertile women with isolated PCO had sampling on day LH +7, adjusted for histologic dating. MAIN OUTCOME MEASURE(S): Expression of alphavbeta3 and its ligand, osteopontin, were determined using real-time quantitative polymerase chain reaction and immunohistochemistry. In vitro regulation of osteopontin was assessed using the Ishikawa cell line. RESULT(S): Cyclic variations revealed a fall in integrin alphavbeta3 mRNA during the secretory phase with concomitant up-regulation of osteopontin mRNA. Immunohistochemistry on day LH +7 demonstrated a significant reduction in expression of osteopontin in the isolated PCO group with no difference in expression of alphavbeta3. In vitro studies confirmed up-regulation of osteopontin by estrogen with no apparent effect of androgen. CONCLUSION(S): These results demonstrate an apparent reduction of osteopontin expression, important in cell-cell adhesion, during the window of implantation in infertile women with isolated PCO morphology.
Asunto(s)
Implantación del Embrión , Endometrio/metabolismo , Infertilidad Femenina/metabolismo , Integrina alfaVbeta3/metabolismo , Osteopontina/metabolismo , Síndrome del Ovario Poliquístico/metabolismo , Adulto , Línea Celular , Regulación hacia Abajo , Estradiol/metabolismo , Femenino , Humanos , Inmunohistoquímica , Infertilidad Femenina/etiología , Infertilidad Femenina/fisiopatología , Integrina alfaVbeta3/genética , Masculino , Metribolona/farmacología , Osteopontina/genética , Síndrome del Ovario Poliquístico/complicaciones , Síndrome del Ovario Poliquístico/fisiopatología , Progesterona/metabolismo , Estudios Prospectivos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Congéneres de la Testosterona/farmacología , Factores de TiempoRESUMEN
The kisspeptins are KiSS-1 gene-derived peptides that signal through the G protein-coupled receptor-54 (GPR54) and have recently been shown to be critical regulators of reproduction. Acute intracerebroventricular or peripheral administration of kisspeptin stimulates the hypothalamic-pituitary-gonadal (HPG) axis. This effect is thought to be mediated via the hypothalamic gonadotropin-releasing hormone (GnRH) system. Chronic administration of GnRH agonists paradoxically suppresses the HPG axis after an initial agonistic stimulation. We investigated the effects of continuous peripheral kisspeptin administration in male rats by use of Alzet minipumps. Initially we compared the effects of acute subcutaneous administration of kisspeptin-10, -14, and -54 on the HPG axis. Kisspeptin-54 produced the greatest increase in plasma LH and total testosterone at 60 min postinjection and was used in the subsequent continuous administration experiments. Chronic subcutaneous long-term administration of 50 nmol kisspeptin-54/day for 13 days decreased testicular weight. Histological examination showed degeneration of the seminiferous tubules associated with a significant decrease in the circulating levels of the testes-derived hormone, inhibin B. Plasma free and total testosterone were also lower, although these changes did not reach statistical significance. Further studies examined the effects of shorter periods of continuous kisspeptin administration. Subcutaneous administration of 50 nmol kisspeptin-54 for 1 day increased plasma LH and testosterone. This effect was lost after 2 days of administration, suggesting a downregulation of the HPG axis response to kisspeptin following continuous administration. These findings indicate that kisspeptin may provide a novel tool for the manipulation of the HPG axis and spermatogenesis.
Asunto(s)
Endocrinología/métodos , Testículo/efectos de los fármacos , Testículo/patología , Proteínas Supresoras de Tumor/farmacología , Factores de Edad , Animales , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Bombas de Infusión Implantables , Inyecciones Subcutáneas , Kisspeptinas , Hormona Luteinizante/sangre , Masculino , Oligopéptidos/farmacología , Tamaño de los Órganos/efectos de los fármacos , Hipófisis/efectos de los fármacos , Ratas , Ratas Wistar , Testosterona/sangreRESUMEN
BACKGROUND AND AIMS: The gastrointestinal trefoil factor family (TFF1, TFF2, TFF3) peptides are considered to play an important role in maintaining the integrity of the mucosa. The physiological role of TFF2 in the protection of the GI tract was investigated in TFF2 deficiency. METHODS: TFF2-/- mice were generated and differential expression of various genes was assessed by using a mouse expression microarray, quantitative real time PCR, Northern blots or immunohistochemistry. RESULTS: On an mRNA level we found 128 differentially expressed genes. We observed modulation of a number of crucial genes involved in innate and adaptive immunity in the TFF2-/- mice. Expression of proteasomal subunits genes (LMP2, LMP7 and PSMB5) involved in the MHC class I presentation pathway were modulated indicating the formation of immunoproteasomes improving antigen presentation. Expression of one subunit of a transporter (TAP1) responsible for importing degraded antigens into ER was increased, similarly to the BAG2 gene that modulates chaperone activity in ER helping proper loading on MHC class I molecules. Several mouse defensin (cryptdin) genes coding important intestinal microbicidal proteins were up-regulated as a consequence of TFF2 deficiency. Normally moderate expression of TFF3 was highly increased in stomach.
Asunto(s)
Sistema Digestivo/inmunología , Sistema Digestivo/metabolismo , Mucinas/deficiencia , Proteínas Musculares/deficiencia , Péptidos/deficiencia , Animales , Presentación de Antígeno , Secuencia de Bases , Cartilla de ADN/genética , Perfilación de la Expresión Génica , Marcación de Gen , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mucinas/genética , Mucinas/inmunología , Proteínas Musculares/genética , Proteínas Musculares/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Péptidos/genética , Péptidos/inmunología , Complejo de la Endopetidasa Proteasomal/genética , Precursores de Proteínas/genética , Factor Trefoil-2RESUMEN
Fluorescence lifetime imaging (FLIM) depends on the fluorescence decay differences between tissues to generate image contrast. In the present study FLIM has been applied to fixed (but unstained) breast cancer tissues to demonstrate the feasibility of this approach for histopathological assessment. As the FLIM method relies on natural autofluorescence, it may be possible to circumvent tissue processing altogether and so FLIM has the potential to be a powerful new method of in vivo tissue imaging via an endoscopic or per-operative approach in a variety of organs, as well as a research tool for in vivo animal models of disease. Unstained, alcohol-fixed tissue samples from 13 patients were stimulated by laser pulses at 415 nm. The temporal decay of the autofluorescence was imaged over a period of 2 ns after cessation of the pulse. The decay rate at each image pixel was calculated as the 'lifetime' factor tau. A tissue classification scheme was used to define regions in each image. The average lifetimes of different tissue regions were compared. A total of 167 tissue regions were measured. Within individual fields, stroma had a larger tau (slower decay) than epithelium (p < 0.001). Within individual patients (taking the mean tau of a given tissue type across all fields from each patient), there was a statistically significant difference between benign and malignancy-associated stroma (p < 0.05). Also, benign collagen had a longer tau than benign epithelium (p < 0.05). Multivariate analysis showed a significant difference between benign stroma, malignancy-associated stroma, blood vessels, and malignant epithelium (p < 0.05). Statistically significant differences between benign and malignancy-associated stroma were obtained even with small patient numbers, indicating that lifetime-based instruments can be developed for real-time diagnostic imaging with microscopic resolution.
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Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/metabolismo , Colágeno/metabolismo , Diagnóstico Diferencial , Estudios de Factibilidad , Femenino , Fluorescencia , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Rayos Láser , Microscopía Fluorescente/métodosRESUMEN
To address the hypothesis that certain disease-associated mutants of the breast-ovarian cancer susceptibility gene BRCA1 have biological activity in vivo, we have expressed a truncated Brca1 protein (trBrca1) in cell-lines and in the mammary gland of transgenic mice. Immunofluorescent analysis of transfected cell-lines indicates that trBRCA1 is a stable protein and that it is localized in the cell cytoplasm. Functional analysis of these cell-lines indicates that expression of trBRCA1 confers an increased radiosensitivity phenotype on mammary epithelial cells, consistent with abrogation of the BRCA1 pathway. MMTV-trBrca1 transgenic mice from two independent lines displayed a delay in lactational mammary gland development, as demonstrated by altered histological profiles of lobuloalveolar structures. Cellular and molecular analyses indicate that this phenotype results from a defect in differentiation, rather than altered rates of proliferation or apoptosis. The results presented in this paper are consistent with trBrca1 possessing dominant-negative activity and playing an important role in regulating normal mammary development. They may also have implications for germline carriers of BRCA1 mutations.
Asunto(s)
Proteína BRCA1/genética , Células Epiteliales/metabolismo , Lactancia/fisiología , Glándulas Mamarias Animales/fisiología , Preñez/metabolismo , Animales , Proteína BRCA1/metabolismo , Biomarcadores/análisis , Western Blotting , Cartilla de ADN/química , Células Epiteliales/efectos de la radiación , Femenino , Técnica del Anticuerpo Fluorescente , Eliminación de Gen , Expresión Génica , Vectores Genéticos , Etiquetado Corte-Fin in Situ , Masculino , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/efectos de la radiación , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Reacción en Cadena de la Polimerasa , Embarazo , Tolerancia a Radiación , TransfecciónRESUMEN
We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro, including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance.