RESUMO
BACKGROUND: The difference in diagnostic yield between surgical lung biopsy and transbronchial lung cryobiopsy (TBLC) in diffuse parenchymal lung diseases (DPLD) has been reported to be due to differences in the rate of interpathologist agreement, specimen size, and specimen adequacy. In TBLC, the specimens containing large airway components are generally believed as inadequate specimens for histological evaluation, but the detailed characteristics of TBLC specimens including the large airway and the impact on histological diagnostic rates of DPLD have not been investigated. METHODS: We retrospectively reviewed the specimen characteristics of patients with DPLD who underwent TBLC. RESULTS: Between February 2018 and January 2020, 74 patients and 177 specimens were included. There were 85 (48.0%) large airway specimens (LAS) that contained bronchial gland or bronchial cartilage. The ideal specimen ratio was significantly lower in the LAS-positive group than that in the LAS-negative group (5.8% vs. 45.6%), and the proportion of bronchioles, alveoli, and perilobular area were similarly lower in the LAS-positive group. The presence of traction bronchiectasis and diaphragm overlap sign on high-resolution computed tomography (HRCT) were also significantly higher in the LAS-positive group than those in the LAS-negative group. We observed a statistically significant trend in histological diagnostic yield (40.7% in LAS positive group; 60.8% in LAS positive and negative group; 91.6% in LAS negative group) (Cochran-Armitage trend test). CONCLUSION: LAS is a specimen often collected in TBLC and contains a low percentage of bronchioles, alveoli, and perilobular area. Since the histological diagnostic yield tends to be higher in cases that do not contain LAS, it may be important to determine the biopsy site that reduces the frequency of LAS collection by referring to the HRCT findings in TBLC.
Assuntos
Broncoscopia , Doenças Pulmonares Intersticiais , Humanos , Broncoscopia/métodos , Estudos Retrospectivos , Doenças Pulmonares Intersticiais/diagnóstico , Doenças Pulmonares Intersticiais/patologia , Pulmão/diagnóstico por imagem , Pulmão/patologia , Biópsia/métodosRESUMO
BACKGROUND: Pulmonary alveolar proteinosis (PAP) is characterized by the accumulation of surfactant proteins within the alveolar spaces. Autoimmune PAP (APAP) caused by elevated levels of GM-CSF autoantibodies (GM-Ab) is very rarely associated with systemic autoimmune disease. Here we report a case of APAP manifested during immunosuppressive treatment for polymyositis with interstitial lung disease. CASE PRESENTATION: A 52-year-old woman treated at our hospital because of polymyositis with interstitial pneumonia had maintained remission by immunosuppressive treatment for 15 years. She had progressive dyspnea subsequently over several months with her chest CT showing ground-glass opacities (GGO) in bilateral geographic distribution. Her bronchoalveolar lavage fluid with cloudy appearance revealed medium-sized foamy macrophages and PAS-positive amorphous eosinophilic materials by cytological examination. We diagnosed her as APAP due to an increased serum GM-CSF autoantibody level. Attenuating immunosuppression failed to lead GGO improvement, but whole lung lavage (WLL) was effective in her condition. CONCLUSIONS: PAP should be considered as one of the differential diseases when the newly interstitial shadow was observed during immunosuppressive treatment. WLL should be regarded as the treatment option for APAP concurred in connective tissue disease (CTD).
Assuntos
Autoanticorpos/sangue , Doenças Autoimunes/diagnóstico , Doenças Pulmonares Intersticiais/complicações , Polimiosite/complicações , Proteinose Alveolar Pulmonar/diagnóstico , Doenças Autoimunes/imunologia , Doenças Autoimunes/fisiopatologia , Líquido da Lavagem Broncoalveolar/citologia , Dispneia/etiologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Imunossupressores/efeitos adversos , Pulmão/fisiopatologia , Doenças Pulmonares Intersticiais/tratamento farmacológico , Pessoa de Meia-Idade , Polimiosite/tratamento farmacológico , Proteinose Alveolar Pulmonar/imunologia , Proteinose Alveolar Pulmonar/fisiopatologia , Proteinose Alveolar Pulmonar/terapia , Tomografia Computadorizada por Raios XRESUMO
The microscopic mechanisms of Bi electrodeposition on Au(111) and Au(100) electrodes in the overpotential regime were studied by in situ scanning tunneling microscopy with high spatial and temporal resolution. Atomic resolution images of the needle-like Bi(110) deposits formed on Au(111) reveal the central influence of covalent Bi-Bi bonds on the deposit morphology. In the straight steps along the needle edges the Bi atoms are interlinked by these bonds, whereas at the needle tip and at kinks along the needle edges dangling bonds exist, explaining the rapid structural fluctuations at these sites. For ultrathin Bi deposits on Au(100) a more open atomic arrangement was found within the surface plane, which was tentatively assigned to an epitaxially stabilised Bi(111) film. Furthermore, well-defined nanowires, consisting of zigzag chains of Bi surface atoms, were observed on this surface.
RESUMO
Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment.
Assuntos
Fatores de Crescimento Neural/farmacologia , Neurônios/citologia , Receptores de Superfície Celular/fisiologia , Células Tumorais Cultivadas/citologia , Animais , Autorradiografia , Diferenciação Celular , Linhagem Celular , DNA de Neoplasias/genética , Citometria de Fluxo , Humanos , Melanoma , Fatores de Crescimento Neural/metabolismo , Neuroblastoma , Neurônios/efeitos dos fármacos , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Receptores de Superfície Celular/genética , Receptores de Fator de Crescimento Neural , Transdução de Sinais , Timidina/metabolismo , Transfecção , Trítio , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
The human trkA cDNA was transfected into a malignant human neuroblastoma (NB) cell line (HTLA230) to investigate its role in NB growth and differentiation. This cell line lacks expression of both endogenous trkA and gp75NGFR genes. Transfectants expressing the trkA mRNA and surface-bound receptors transcriptionally activate immediate-early genes (c-fos, c-jun, and jun-B) following nerve growth factor (NGF) stimulation. NGF treatment induces growth arrest as well as down-regulation of the amplified N-myc oncogene. Genes selectively expressed in mature neurons (SCG-10, ret proto-oncogene, GAP-43, etc.) are transcriptionally activated, and neurite outgrowth further demonstrates differentiation of transfectants following NGF stimulation. trkA-expressing NB cells remain tumorigenic in nude mice; however, subcutaneous treatment of tumor-bearing mice with NGF induces Schwannian and neuronal cell differentiation similar to the induction seen in human ganglioneuroblastomas. Thus, trkA expression in HTLA230 cells is sufficient to generate a functional NGF receptor complex that leads to growth-arrested and differentiated NB cells in vitro and in vivo in the presence of NGF. Hence, NGF may play a crucial role in NB cell differentiation and regression in vivo.
Assuntos
DNA Complementar/genética , DNA de Neoplasias/genética , Neuroblastoma/genética , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator de Crescimento Neural/genética , Animais , Diferenciação Celular/genética , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fatores de Crescimento Neural/farmacologia , Neuroblastoma/patologia , Proto-Oncogene Mas , Receptor trkA , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/patologiaRESUMO
BACKGROUND: Stimulation of transient receptor potential ankyrin 1 (TRPA1), which abundantly expressed in enterochromaffin cells (ECC), has been reported to exert apparently contradictory results in in vitro contractility and in vivo gastrointestinal (GI) transit evaluations. The pharmaceutical-grade Japanese traditional medicine daikenchuto (TU-100) has been reported to be beneficial for postoperative ileus (POI) and accelerate GI transit in animals and humans. TU-100 was recently shown to increase intestinal blood flow via stimulation of TRPA1 in the epithelial cells of the small intestine (SI). METHODS: The effects of various TRPA1 agonists on motility were examined in a manipulation-induced murine POI model, in vitro culture of SI segments and an ECC model cell line, RIN-14B. KEY RESULTS: Orally administered TRPA1 agonists, aryl isothiocyanate (AITC) and cinnamaldehyde (CA), TU-100 ingredients, [6]-shogaol (6S) and γ-sanshool (GS), improved SI transit in a POI model. The effects of AITC, 6S and GS but not CA were abrogated in TRPA1-deficient mice. SI segments show periodic peristaltic motor activity whose periodicity disappeared in TRPA1-deficient mice. TU-100 augmented the motility. AITC, CA and 6S increased 5-HT release from isolated SI segments and the effects of all these compounds except for CA were lost in TRPA1-deficient mice. 6S and GS induced a release of 5-HT from RIN-14B cells in a dose- and TRPA1-dependent manner. CONCLUSIONS & INFERENCES: Intraluminal TRPA1 stimulation is a potential therapeutic strategy for GI motility disorders. Further investigation is required to determine whether 5-HT and/or ECC are involved in the effect of TRPA1 on motility.
Assuntos
Modelos Animais de Doenças , Trânsito Gastrointestinal/efeitos dos fármacos , Trânsito Gastrointestinal/fisiologia , Íleus/tratamento farmacológico , Canal de Cátion TRPA1/agonistas , Canal de Cátion TRPA1/fisiologia , Acroleína/análogos & derivados , Acroleína/farmacologia , Acroleína/uso terapêutico , Amidas/farmacologia , Amidas/uso terapêutico , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Íleus/fisiopatologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Naftoquinonas/farmacologia , Naftoquinonas/uso terapêutico , Técnicas de Cultura de ÓrgãosRESUMO
The effect of constitutively expressed N-myc gene on nerve growth factor (NGF) induced neuronal differentiation was investigated. B104, a rat central nervous system-derived cell line and its N-myc gene expressing derivative lines (C6, C7) (Bernards et al., 1986), were stably transfected with the trkA proto-oncogene and independent clones for each cell line were analysed. NGF induced phosphorylation of the trkA receptor, activated a cascade of cellular intermediaries such as phospholipase C gamma 1 and ERK proteins, and stimulated c-fos gene transcription in all trkA-expressing clones. NGF-mediated neuronal differentiation was observed solely in trkA-expressing B104-derived clones and was characterized by reduced cell growth, activation of NGF-regulated genes, and downregulation of the endogenous low-affinity NGF receptor gene (gp75NGFR). No such phenotypical changes occurred in trkA-expressing C6 or C7-derived clones following NGF treatment. These results are consistent with the hypothesis that constitutive expression of N-myc inhibits exit from cell cycle and blocks neuronal cell differentiation.
Assuntos
Genes myc/fisiologia , Fatores de Crescimento Neural/farmacologia , Neurônios/citologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fos/fisiologia , Glicoproteínas de Membrana/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptor de Fator de Crescimento Neural , Receptor trkA , Receptores de Fator de Crescimento Neural/genéticaRESUMO
Functional nerve growth factor (NGF) responsiveness was investigated in human neuroblastoma (NB) cell lines in vitro and retinoic acid (RA) was found to transcriptionally enhance expression of the trkA, but not the trkB gene in GOTO cells, while the reverse was found in HTLA230 NB cells. Ciliary neurotrophic factor (CNTF) specifically induced trkA gene transcription in both cell lines. Transcriptional activation of the trkA gene increased total trkA protein and surface bound receptor, which was tyrosine phosphorylated upon NGF stimulation inducing immediate early response gene transcription (i.e. c-fos, Egr-1). Newly synthesized trkA protein had a molecular weight of 110 kDa and was post-translationally modified. Rapid down regulation of the receptor protein occurred upon NGF stimulation, despite the presence of high levels of trkA mRNA, due to an increased rate of receptor degradation. Transient DNA synthesis and cell proliferation upon NGF treatment occurred in GOTO cells with elevated trkA expression. In contrast, NGF induced neuronal differentiation in HTLA230 cells expressing the endogenous trkA receptor gene, despite the lack of p75 expression. Hence, transcriptional activation of trkA gene expression can be achieved by different signal pathways in human NB cells, but NGF can act either as mitogen or inducer of cell differentiation, depending on the tumor from which cells are derived.
Assuntos
Proteínas Imediatamente Precoces , Fatores de Crescimento Neural/farmacologia , Neuroblastoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Tretinoína/farmacologia , Fator Neurotrófico Ciliar , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas do Tecido Nervoso , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptor do Fator Neutrófico Ciliar , Receptor de Fator de Crescimento Neural , Receptor trkA , Receptores de Fator de Crescimento Neural/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
Ultrasound high-resolution B-mode imaging was used to assess the carotid arteries in 105 patients with insulin-dependent diabetes mellitus (IDDM), 4-25 years of age, with duration of diabetes ranging from 0.5-17 years, 529 patients with non-insulin-dependent diabetes (NIDDM), 31-86 years of age, with duration of diabetes ranging from 0.5-49 years, and 104 nondiabetic healthy subjects, 7-76 years of age, to determine the intimal plus medial thickness (IMT) of the arterial wall. The IMT values for IDDM patients 10-19 years of age (0.525 +/- 0.123 mm, n = 68) or 20-25 years of age (0.696 +/- 0.124 mm, n = 14) were significantly greater than those in age-matched nondiabetic subjects (0.444 +/- 0.057 mm, n = 12, P = 0.01169; 0.538 +/- 0.098 mm, n = 34, P < 0.00006). NIDDM patients showed IMT values equivalent to those in normal adults > or = 20 years of age. Multiple regression analysis showed that IMT in IDDM patients was positively related to the duration of diabetes (P = 0.00061) as well as to age (P = 0.00046). No other possible risk factors, such as serum total cholesterol level, serum high-density lipoprotein (HDL)-cholesterol level, serum low-density lipoprotein-cholesterol level, serum triglycerides, serum lipoprotein(a) level, or systolic or diastolic blood pressure, have shown significant correlations with IMT in IDDM patients. However, non-HDL-cholesterol, smoking, and systolic hypertension were independently responsible for increases in IMT values of NIDDM patients as well as age and duration of diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Arteriosclerose/diagnóstico por imagem , Artérias Carótidas/diagnóstico por imagem , Diabetes Mellitus Tipo 1/diagnóstico por imagem , Diabetes Mellitus Tipo 2/diagnóstico por imagem , Angiopatias Diabéticas/diagnóstico por imagem , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Arteriosclerose/sangue , Pressão Sanguínea , Criança , Pré-Escolar , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/sangue , Feminino , Hemoglobinas Glicadas/análise , Humanos , Masculino , Pessoa de Meia-Idade , UltrassonografiaRESUMO
OBJECTIVE: To quantitatively assess atherosclerosis of the carotid artery in subjects with and without diabetes. RESEARCH DESIGN AND METHODS: Ultrasound high resolution B-mode imaging of carotid arteries was conducted on 71 nondiabetic subjects without hyperlipidemia or hypertension and 295 diabetic patients to determine IMT of the arterial wall. RESULTS: IMT was linearly related with age in nondiabetic (IMT = [0.0087 x age] + 0.3318) and diabetic subjects (IMT = [0.0155 x age] + 0.32450). The regression coefficient for age was significantly greater in diabetic than nondiabetic subjects. IMT (mean +/- SD) of diabetic subjects aged 20-29 was significantly greater than that of nondiabetic subjects aged 20-29 (0.73 +/- 0.27 vs. 0.52 +/- 0.07 mm, P less than 0.01). Multivariate regression analysis of 275 NIDDM patients indicated smoking, hyperlipidemia, duration of diabetes, hypertension, and age were factors determining thickness of the carotid arterial wall. CONCLUSIONS: Diabetes, along with age, hyperlipidemia, smoking, and hypertension, aggravates carotid atherosclerosis.
Assuntos
Arteriosclerose/diagnóstico por imagem , Arteriosclerose/epidemiologia , Artérias Carótidas/diagnóstico por imagem , Diabetes Mellitus/diagnóstico por imagem , Angiopatias Diabéticas/diagnóstico por imagem , Arteriosclerose/sangue , Pressão Sanguínea , Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Diabetes Mellitus/sangue , Diabetes Mellitus/fisiopatologia , Angiopatias Diabéticas/sangue , Angiopatias Diabéticas/fisiopatologia , Feminino , Hemoglobinas Glicadas/análise , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Triglicerídeos/sangue , UltrassonografiaRESUMO
We have previously demonstrated that PLC-delta was abnormally accumulated in autopsied brains with Alzheimer's disease (AD). As nonneuronal tissue involvement in AD is also suggested and PLC activity is reduced in AD platelets, we examined the changes of the protein level of PLC-delta and its enzyme activity in platelets taken from patients with AD and age-matched controls. PLC-delta in human platelets was detected as a 72 kDa protein using a specific antibody against PLC-delta. Western blots revealed that the protein level of PLC-delta was significantly higher in the cytosolic fraction prepared from AD platelets compared to controls. We investigated the activity of PLC-delta which hydrolyzes phosphatidylinositol and found that the PLC-delta activity in the cytosolic fraction from AD platelets was significantly reduced compared to the control. This finding that the enzyme activity per PLC-delta molecule is reduced in AD platelets is consistent with the study using Alzheimer brains. These results suggest that aberrant phosphoinositide metabolism is present in nonneuronal tissues as well as the brains of patients with AD.
Assuntos
Doença de Alzheimer/sangue , Plaquetas/enzimologia , Fosfolipases Tipo C/metabolismo , Adulto , Distribuição por Idade , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Alzheimer's disease (AD) has been suggested to be a systemic disease, and signal transduction abnormalities have been reported in non-neuronal AD cells. We have previously quantified the protein kinase C (PKC) subtypes in AD and control brains using a two-site enzyme immunoassay (EIA), and have shown that type II PKC levels were significantly reduced in the temporal cortex of AD patients. In this study, we used this EIA to assess the platelet levels of type II PKC in age-matched groups of AD patients and normal controls. The cytosolic level of type II PKC was significantly higher in AD platelets than in control platelets but was unchanged in the membranous fraction. Platelet proteins showed no differences between the AD and control groups. Therefore, the type II PKC content of the cytosolic fraction was increased in AD platelets. These results suggest that type II PKC may be altered in both the brain and platelets of AD patients and support the hypothesis that AD is a systemic disease.
Assuntos
Doença de Alzheimer/enzimologia , Plaquetas/enzimologia , Proteína Quinase C/sangue , Idoso , Idoso de 80 Anos ou mais , Encéfalo/enzimologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Análise por Pareamento , Proteína Quinase C/classificaçãoRESUMO
Using a two-site enzyme immunoassay, we determined the content of type II protein kinase C (PKC) in human platelets from 24 donors of various ages without any neurological, hematological or malignant disorders. The content of type II PKC in the membranous fraction was positively correlated with aging. The content of PKC in the cytosolic fraction tended to decline with aging, but the correlation was not significant. The total amount of PKC also had no significant correlation with aging. Age-related changes in platelet protein content were not observed. These results suggested that the subcellular distribution of type II PKC in human platelets is altered with normal aging.
Assuntos
Envelhecimento/sangue , Plaquetas/enzimologia , Proteína Quinase C/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Preservação de Sangue , Criopreservação , Citosol/enzimologia , Feminino , Humanos , Masculino , Membranas/enzimologia , Pessoa de Meia-IdadeRESUMO
We assessed the amount of protein kinase C (PKC) in samples from postmortem normal human and Alzheimer's disease (AD) brains by a two-site enzyme immunoassay that quantitatively identified types alpha, beta, and gamma isozymes. In the normal human brain matter, type beta was the main type present, the majority of each isozyme of PKC being present in the membranous fraction of the brain tissues. In AD brains, the amount of type beta PKC was significantly reduced in the membranous fraction of the temporal cortical tissues. The amounts of types alpha and gamma in the membranous fraction and types alpha, beta, and gamma in the cytosolic fraction in AD brains were lower than in the control brains, but the difference was not significant. There was also a significant decrease in the levels of PKC in the membranous fraction of AD brains, as measured by radioactive phorbol ester binding. These results suggest that the type beta PKC isozyme is mainly present in the human temporal cortex and that reduced levels of type beta PKC in the membranous fraction may reflect a biochemical deficit related specifically to the pathogenesis of AD.
Assuntos
Doença de Alzheimer/enzimologia , Encéfalo/enzimologia , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Feminino , Humanos , Técnicas Imunoenzimáticas , Cinética , Masculino , Dibutirato de 12,13-Forbol , Ensaio RadioliganteRESUMO
PURPOSE: To examine the intracellular and extracellular expression of myocilin in the human and primate trabecular meshwork (TM) in the presence and absence of glucocorticoids. METHODS: Myocilin expression was examined in cultured human TM cells by Northern blot analysis and myocilin antibody-mediated immunoprecipitation. Myocilin expression was quantified using high-resolution two-dimensional polyacrylamide gel electrophoresis of radiolabeled proteins from human TM cells, TM tissue explants, and perfused human anterior segments cultured with and without dexamethasone (DEX) for 14 to 21 days, as well as TM tissue from pigtailed monkeys treated orally for 1 year with cortisone acetate. Immunofluorescence with anti-myocilin antibodies was used to localize cellular and extracellular expression of myocilin in cultured human TM cells. RESULTS: Glucocorticoid treatment caused a significant induction of myocilin mRNA, a tetrad of cell-associated proteins, and 8 to 20 secreted proteins (molecular mass [M(r)] 56 and 59 kDa and isoelectric point [pI] 5.2 and 5.3) in some, but not all the cultured human TM cells and explanted tissues. Western immunoblot analysis using anti-myocilin peptide antibodies identified these proteins as encoded by the MYOC gene. There was significant induction of the myocilin proteins in three perfusion-cultured human eyes, in which DEX-induced elevated intraocular pressure developed. Monkeys treated 1 year with cortisol acetate showed steroid glaucoma-like morphologic changes in the TM that correlated with the induction of myocilin in the TM. Immunofluorescence analysis of cultured TM cells localized myocilin intracellularly in discrete perinuclear and cytoplasmic vesicular deposits as well as extracellularly on the cell surface associated with the extracellular matrix. In several DEX-treated TM cell lines, there were significant levels of myocilin secreted into the media. Enzymatic deglycosylation of proteins in the TM media converted the higher molecular weight isoforms of myocilin (approximately 57 kDa) to the lower molecular weight isoforms ( approximately 55 kDa). CONCLUSIONS: Although the function of myocilin is unknown, induction of these TM proteins was found in eyes in which glucocorticoid-induced ocular hypertension developed. Therefore, myocilin may play an important pathogenic role in ocular hypertension in addition to its role in certain forms of POAG.
Assuntos
Proteínas do Olho/biossíntese , Glucocorticoides/farmacologia , Glicoproteínas/biossíntese , Hipertensão Ocular/induzido quimicamente , Malha Trabecular/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Animais , Northern Blotting , Western Blotting , Células Cultivadas , Cortisona/análogos & derivados , Cortisona/farmacologia , Proteínas do Citoesqueleto , Dexametasona/farmacologia , Eletroforese em Gel Bidimensional , Proteínas do Olho/genética , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica/efeitos dos fármacos , Glicoproteínas/genética , Humanos , Pressão Intraocular/efeitos dos fármacos , Macaca nemestrina , Pessoa de Meia-Idade , Hipertensão Ocular/metabolismo , Hipertensão Ocular/patologia , RNA Mensageiro/biossíntese , Malha Trabecular/metabolismo , Malha Trabecular/ultraestruturaRESUMO
Single-photon emission computed tomography (SPECT) using thallium-201 was compared with 12-lead electrocardiography (ECG) in patients with Duchenne (29), facioscapulohumeral (7), limb-girdle (6) and myotonic (5) dystrophies, by dividing the left ventricular (LV) wall into 5 segments. SPECT showed thallium defects (37 patients, mostly in the posteroapical wall), malrotation (23), apical aneurysm (5) and dilatation (7). ECG showed abnormal QRS (36 patients), particularly as a posterolateral pattern (13). Both methods of assessment were normal in only 7 patients. The Duchenne type frequently showed both a thallium defect (particularly in the posteroapical wall) and an abnormal QRS (predominantly in the posterolateral wall); the 3 other types showed abnormalities over the 5 LV wall segments in both tests. The percent of agreement between the 2 tests was 64, 66, 70, 72 and 72 for the lateral, apical, anteroseptal, posterior and inferior walls, respectively. The 2 tests were discordant in 31% of the LV wall, with SPECT (+) but ECG (-) in 21% (mostly in the apicoinferior wall) and SPECT (-) but ECG (+) in 10% (mostly in the lateral wall). Some patients showed large SPECT hypoperfusion despite minimal electrocardiographic changes. ECG thus appeared to underestimate LV fibrosis and to reflect posteroapical rather than posterolateral dystrophy in its posterolateral QRS pattern. In this disease, extensive SPECT hypoperfusion was also shown, irrespective of clinical subtype and skeletal involvement.
Assuntos
Cardiomiopatias/etiologia , Eletrocardiografia , Coração/diagnóstico por imagem , Distrofias Musculares/complicações , Radioisótopos de Tálio , Tomografia Computadorizada de Emissão , Cardiomiopatias/diagnóstico , Feminino , Coração/fisiopatologia , Humanos , MasculinoRESUMO
Several naturally occurring cyclin-dependent kinase (CDK) inhibitors have been isolated from different lower organisms. In this report, we examined the effect of one of the CDK inhibitors, butyrolactone I (BL), on the expression of cyclins D2, A and B1 in three human prostatic cancer cell lines (DU145, PC-3, LNCaP) using two colored flow cytometric analysis. The percentage of DU145 cells in the 4C phase of the cell cycle were increased significantly at both 70 microM and 100 microM BL. Furthermore, an additional 8C peak was observed which had double the DNA content of the 4C phase at these concentrations of BL. The appearance of the 8C peak increased gradually and was more evident in DU145 and PC-3 than LNCaP. Cells in the 8C peak had either two nuclei or abnormal nuclei as observed by Papanicolaou stain. BL also increased the amount of cyclin B1 positive cells in the 4C phase. This increase was apparent on day 1 and returned to normal by day 3. Since BL selectively inhibits cyclin-dependent kinase, cyclin B1 might accumulate without being degraded. Other cyclins were not significantly changed by BL. The data demonstrate that BL inhibited Cdc2 of unsynchronized cultured prostate cancer cells, and interrupted the cell cycle progression toward cell division. The BL inhibition of Cdc2 led to the accumulation of cells in the 4C phase without mitosis resulting in an accumulation of cyclin B1. The appearance of cells in the 8C phase may be due to the progression of cells in the 4C phase through the cell cycle skipping mitosis. Cyclin B1 decreased in correlation with the progression through a new cell cycle. These results suggest that BL does not cause a complete arrest of the cell cycle in G2/M but that BL occasionally allows for the skipping of mitosis and subsequent progression through the cell cycle to occur.
Assuntos
4-Butirolactona/análogos & derivados , Ciclina B/biossíntese , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fase G2/efeitos dos fármacos , Mitose/efeitos dos fármacos , Neoplasias da Próstata/patologia , 4-Butirolactona/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Ciclina B1 , DNA de Neoplasias/análise , Humanos , Masculino , Células Tumorais CultivadasRESUMO
Two sublines of B16 melanoma cells, F10 and BL6, are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. We found a new member of the GTPase superfamily, namely TIB929, which displayed an induction of expression in BL6 cells. It conserved three consensus sequences for GTP-binding site motifs and showed a significant homology to the yeast Gtr2 gene throughout the coding sequence. TIB929 was expressed ubiquitously in human tumor cells, with a marked expression in highly metastatic cells. TIB929 was mapped on mouse chromosome 4D, syntenic to human chromosome 1p. The results suggested an involvement of TIB929 in malignant progression.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma Experimental/enzimologia , Melanoma Experimental/secundário , Proteínas de Neoplasias , Neoplasias Cutâneas/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/genética , Masculino , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP , Família Multigênica , Transplante de Neoplasias , RNA Mensageiro/biossíntese , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Distribuição Tecidual , Regulação para CimaRESUMO
Numerous human Cytochrome P450 enzymes (CYPs) associated with 'phase I' drug metabolism have been identified. Among them, CYP2D6 is thought to be the major target autoantigen to anti-liver kidney microsome (LKM)-1 autoantibody, a characteristic feature of autoimmune hepatitis (AIH) type II. In this study, we were able to clone CYP2D6 cDNA from a human liver cDNA library and express the CYP2D6 recombinant protein, and also to prepare four other representative human CYP proteins (CYP1A2, 2C9, 2E1, and 3A4). These preparations were used to assay the immunoreactivity of patients with AIH type I (n=35) and type II (n=9). As comparison groups, sera from patients with chronic hepatitis B (n=15), chronic hepatitis C (n=55; 24 anti-LKM-1-positive, 31 anti-LKM-1-negative), and from normal controls (n=30) were included. The five CYP proteins did not react with sera from normal controls nor from patients with chronic hepatitis B. CYP2D6 reacted with sera from 100% (9/9) of AIH type II patients, 79% (19/24) of patients with anti-LKM-1-positive chronic hepatitis C, and 6.5% (2/31) of patients with anti-LKM-1-negative chronic hepatitis C. In contrast, CYP1A2 reacted with serum from one patient with AIH type I, CYP2E1 reacted with sera from two patients with AIH type I, one patient with anti-LKM-1-positive chronic hepatitis C, and two patients with anti-LKM-1-negative chronic hepatitis C, and CYP3A4 reacted with sera from one patient with AIH type II and one patient with anti-LKM-1-positive chronic hepatitis C. CYP2C9 did not react with any of the sera included in this study. From these results, it is suggested that CYPs other than CYP2D6 can function as immunotargets in certain disease conditions.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Citocromo P-450 CYP2D6/imunologia , Sistema Enzimático do Citocromo P-450/imunologia , Hepatite B Crônica/enzimologia , Hepatite C Crônica/enzimologia , Hepatite Autoimune/enzimologia , Esteroide 16-alfa-Hidroxilase , Adulto , Animais , Citocromo P-450 CYP1A2/imunologia , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2E1/imunologia , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/sangue , Sistema Enzimático do Citocromo P-450/genética , Citocromos , Eletroforese em Gel de Poliacrilamida , Feminino , Imunofluorescência , Hepatite B Crônica/sangue , Hepatite B Crônica/imunologia , Hepatite C Crônica/sangue , Hepatite C Crônica/imunologia , Hepatite Autoimune/sangue , Hepatite Autoimune/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Oxigenases de Função Mista/imunologia , Ratos , Dodecilsulfato de Sódio , Esteroide Hidroxilases/imunologia , Células Tumorais Cultivadas , beta-Galactosidase/imunologiaRESUMO
Mutations of p53 tumor suppressor gene occur in a subset of aggressive prostatic carcinomas and are detectable by immunohistochemistry. However, it is uncertain whether p53 overexpression really reflects p53 gene mutation or loss of p53 function. p21WAF1, an inhibitor of cyclin-dependent kinases, is activated by wild-type p53 protein, not by mutant type. Therefore, it is possible that combined analysis of p21WAF1 and p53 proteins aids in determining the functional status of p53 immunostaining. Routinely processed prostatic tissues from 60 patients with prostatic adenocarcinomas were examined by immunohistochemistry for p21WAF1 and p53 expression. As for tissue distribution, p21WAF1 protein was expressed mostly in the luminal layers, in contrast, p53 protein was restricted to the basal layers of benign prostatic glands. In prostatic adenocarcinomas, p21WAF1 protein was more likely to be expressed in well-differentiated areas; in contrast, p53 protein was more likely in poorly differentiated areas in the tumors. The percentage of positive nuclear areas for p21WAF1 and p53 proteins in prostatic adenocarcinomas, assessed by CAS200 computerized image analyzer, were 8.6+/-10% and 16+/-14% (mean+/-SE), respectively. The survival study showed that the p53+/ p21- phenotype showed poorer prognosis than p53+/p21+. Multivariate analysis showed that p21WAF1 expression, clinical stage, and Gleason score were independent prognosticators. In conclusion, p21WAF1 immunohistochemistry is a useful method for interpretation of p53 immunohistochemical results. Combined analysis by p21WAF1 and p53 immunostaining would predict the patient survival more accurately than p53 immunostaining alone.