RESUMO
BACKGROUND: The Kindler syndrome (KS) protein kindlin-1 is a member of a protein complex that links cortical actin to integrins on the surface of basal keratinocytes. Loss of kindlin-1 leads to abnormalities of cell adhesion and motility, and to skin blistering and progressive poikiloderma as clinical symptoms. OBJECTIVES: Here we investigated a severely affected patient, disclosed the mutation that caused the disease and delineated its biological consequences. METHODS: Mutation screening of the kindlin-1 gene, KIND1 (now called FERMT1), was performed with polymerase chain reaction (PCR) amplification of all exons and sequencing. Mutated kindlin-1 was characterized by reverse transcriptase (RT)-PCR and immunoblotting, and genotype-phenotype correlations were analysed using immunohistochemical staining of skin biopsies and keratinocytes from the patient's skin. Cell adhesion and motility were assessed with functional tests. RESULTS: We disclosed a splice site mutation in the first position of intron 13 of the FERMT1 gene, which caused skipping of exon 13. The short transcript partially escaped nonsense-mediated mRNA decay and was translated into a truncated protein. CONCLUSION: A C-terminally truncated kindlin-1 in keratinocytes could not function correctly even if it were expressed.
Assuntos
Adesão Celular/genética , Queratinócitos/citologia , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Dermatopatias Genéticas/patologia , Adulto , Éxons , Mutação da Fase de Leitura , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Reação em Cadeia da Polimerase , Dermatopatias Genéticas/genéticaRESUMO
Kindlin-1 is an epithelium-specific phosphoprotein and focal adhesion adaptor component. Mutations in the corresponding gene (KIND1) cause Kindler syndrome (KS), which is manifested by skin blistering, poikiloderma, photosensitivity and carcinogenesis. Some patients also exhibit gastrointestinal symptoms, but it has remained unclear whether these represent a feature of Kindler syndrome or a coincidence. We examined kindlin-1 in human gastrointestinal epithelia and showed that it is involved in the aetiopathology of Kindler syndrome-associated colitis. Kindlin-1 expression was assessed by indirect immunofluorescence, western blot and RT-PCR. Kindlin-1 is expressed in oral mucosa, colon and rectum. Both the full-length 74 kDa kindlin-1 protein and a 43 kDa isoform were detected in CaCo2 cells, the latter resulting from alternative splicing. In the first months of life, patients (homozygous for null mutations) had severe intestinal involvement with haemorrhagic diarrhoea and showed morphological features of severe ulcerative colitis. Later in childhood, histopathology demonstrated focal detachment of the epithelium in all segments of the colon, chronic inflammation and mucosal atrophy. These findings define an intestinal phenotype for Kindler syndrome as a consequence of a primary epithelial barrier defect. The different clinical intestinal manifestations in Kindler syndrome patients may be explained by partial functional compensation of kindlin-1 deficiency by the intestinal isoform or by the presence of truncated mutant kindlin-1.
Assuntos
Colite Ulcerativa/patologia , Mucosa Intestinal/patologia , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Vesícula/genética , Vesícula/metabolismo , Criança , Doença Crônica , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Colite Ulcerativa/fisiopatologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo/métodos , Humanos , Recém-Nascido , Mucosa Intestinal/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Síndrome de Rothmund-Thomson/genética , Síndrome de Rothmund-Thomson/metabolismo , Dermatopatias Genéticas/genética , Dermatopatias Genéticas/metabolismo , SíndromeRESUMO
PURPOSE: Allograft vascular thrombosis occurs in 5% to 10% of pediatric renal transplants. The hemodynamics of renal allograft immediately after implantation is unclear. High resolution Doppler ultrasound of the renal allograft performed in the operating room after incision closure is an effective and objective method to advance our understanding of baseline renal allograft hemodynamics, and identify unsuspected vascular complications early enough to ensure prompt surgical repair. MATERIALS AND METHODS: Between September 1998 and July 2000 high resolution, color power Doppler ultrasound was prospectively performed on 21 living related renal transplants in the operating room immediately after incision closure. Each ultrasound described allograft anastomotic blood flow, direction of diastolic flow, parenchymal perfusion and resistive indexes. RESULTS: There were 20 (95%) allografts with good power Doppler perfusion that had satisfactory immediate function with no vascular complications at 9 to 26-month followup. Initially, anastomotic turbulence was described in 15 (71%) allografts, and resistive indexes were abnormal in 8 (38%). Turbulence and abnormal resistive index normalized in all allografts by 1-month followup. Ultrasound of 1 allograft identified unsuspected poor perfusion and reversal of diastolic flow in the operating room after incision closure. In another allograft in which a 4-hour post-transplant ultrasound was compared with the baseline study in the operating room an unsuspected thrombosis of the right common iliac vein was confirmed. CONCLUSIONS: Good parenchymal perfusion and forward diastolic flow after renal reperfusion correlated well with immediate graft function. Initial turbulence and abnormal resistive index in the presence of favorable perfusion are misleading and not independent predictors of graft function. Ultrasound performed in the operating room identified 2 unsuspected major vascular complications facilitating prompt surgical correction.
Assuntos
Transplante de Rim/diagnóstico por imagem , Transplante de Rim/fisiologia , Ultrassonografia Doppler em Cores , Adolescente , Anastomose Cirúrgica , Criança , Pré-Escolar , Feminino , Hemodinâmica , Humanos , Transplante de Rim/efeitos adversos , Transplante de Rim/métodos , Doadores Vivos , Masculino , Estudos Prospectivos , Reperfusão , Transplante Homólogo , Trombose Venosa/diagnóstico por imagem , Trombose Venosa/etiologiaRESUMO
The incidence of postoperative hyperamylasemia was evaluated in 131 patients who underwent obstetric and gynecologic procedures. Preoperative and postoperative serum amylase levels were determined in 178 patients who underwent routine surgical procedures. In our sample, we could not document any elevations in serum amylase levels after operations. These findings contradict those of previous reports of a high incidence of postoperative hyperamylasemia after surgical procedures except those performed upon the gastrointestinal tract. Furthermore, in spite of the fact that the female internal genitalia is rich in amylase and that pregnancy is considered a predisposing condition for the development of postoperative pancreatitis, the preoperative and postoperative serum amylase levels were consistently within normal range. We would like to conclude that the manipulation of female internal genitalia, pregnant or not, does not induce hyperamylasemia. Therefore, hyperamylasemia in postoperative gynecologic and obstetric patients should alert the clinician to the possibility of postoperative pancreatitis. We believe that our findings should be confirmed on large samples of patients.
Assuntos
Amilases/sangue , Genitália Feminina/cirurgia , Complicações na Gravidez/cirurgia , Feminino , Humanos , Gravidez , Complicações na Gravidez/enzimologiaRESUMO
Human TK- 143 cells were converted to TK+ phenotype with a plasmid containing the native herpes simplex virus 1 (HSV-1), thymidine kinase, a beta gene, and a chimeric ovalbumin gene consisting of the coding sequences of the ovalbumin gene linked to the promoter-regulatory region of the HSV-1 alpha 4 gene. Comparison of the synthesis of ovalbumin and the alpha 4 gene product in the converted cells infected with ts mutants in alpha 4 gene and incubated at the permissive (33 degrees C) and nonpermissive (39 degrees C) temperatures revealed the following. (i) The synthesis of both ovalbumin and alpha 4 gene product was transiently induced at the permissive temperature but continued at elevated levels for many hours at the nonpermissive temperature. (ii) The synthesis of both ovalbumin and alpha 4 gene products resumed when the infected cells were shifted from permissive to nonpermissive temperature after the shut-off of alpha protein synthesis. (iii) Although both the beta-TK and alpha 4-ovalbumin chimeric genes were covalently linked on the same plasmid, each was regulated independently. We conclude that alpha gene regulation is determined solely by (a) the inducer and (b) the induction sequence contained in the promoter-regulatory region and not by the location or the higher order structure of the immediate environment of the gene.
Assuntos
Óperon , Simplexvirus/genética , Proteínas Virais/genética , DNA Recombinante , Regulação da Expressão Gênica , Ovalbumina/genéticaAssuntos
Vírus da Influenza A/metabolismo , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Transcrição Gênica , Replicação Viral , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Embrião de Galinha , Cricetinae , Citoplasma/metabolismo , Rim , Metilação , Capuzes de RNA/metabolismo , Moldes GenéticosRESUMO
Influenza viral mRNA, i.e., complementary RNA (cRNA), isolated from infected cells , was resolved into six different species by electrophoresis in 2.1% acrylamide gels containing 6 M urea. The cRNA's were grouped into three size classes: L (large), M (medium-size), and S (small). Similarly, when gels were sliced for analysis, the virion RNA (vRNA) also distributed into six peaks because the three largest vRNA segments were closely spaced and were resolved only when the gels were autoradiographed or stained. Because of their attached polyadenylic acid [poly(A)]sequences, the cRNA segments migrated more slowly than did the corresponding vRNA segments during gel electrophoresis. After removal of the poly(A) by RNase H, the cRNA and vRNA segments comigrated, indicating that they were approximately the same size. One of the cRNA segments, S2, was shown by annealing to contain the genetic information in the vRNA segment with which it comigrated, strongly suggesting that each cRNA segment was transcribed from the vRNA segment of the same size. In contrast to the vRNA segments, which when isolated from virions were present in approximately 1:1 molar ratios, the segments of the isolated cRNA were present in unequal amounts, with the segments M2 and S2 predominating, suggesting that different amounts of the cRNA segments were synthesized in the infected cell. The predominant cRNA segments, M2 and S2, and also the S1 segment, were active as mRNA's in wheat germ extracts. The M2 cRNA was the mRNA for the nucleocapsid protein; S1 for the membrane protein; and S2 for the nonstructural protein NS1.