Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic single amino acid mutations that cause skeletal and cranial dysplasias, as well as cancer, we also study the effects of these mutations on dimerization. First, we show that the A391E mutation, linked to Crouzon syndrome with acanthosis nigricans and to bladder cancer, significantly enhances FGFR3 dimerization in the absence of ligand and thus induces aberrant receptor interactions. Second, we present results about the effect of three cysteine mutations that cause thanatophoric dysplasia, a lethal phenotype. Such cysteine mutations have been hypothesized previously to cause constitutive dimerization, but we find instead that they have a surprisingly modest effect on dimerization. Most of the studied pathogenic mutations also altered FGFR3 dimer structure, suggesting that both increases in dimerization propensities and changes in dimer structure contribute to the pathological phenotypes. The results acquired with the QI-FRET method further our understanding of the interactions between FGFR3 molecules and RTK molecules in general. Since RTK dimerization regulates RTK signaling, our findings advance our knowledge of RTK activity in health and disease. The utility of the QI-FRET method is not restricted to RTKs, and we thus hope that in the future the QI-FRET method will be applied to other classes of membrane proteins, such as channels and G protein-coupled receptors.

A novel FGFR2 mutation in tyrosine kinase II domain, L617F, in Crouzon syndrome.

The purposes of this study were to find a novel mutation of FGFR2 in Korean Crouzon syndrome patients and to identify the functional consequences of this mutation. The samples consisted of 16 Crouzon patients. Peripheral venous blood was collected from the patients. FGFR2 mutation screening was performed by direct PCR sequencing of all exons and part of the introns. Restriction fragment length polymorphism (RFLP) analysis was performed to confirm the novel mutation. For functional studies, we performed luciferase assay for Runx2 transcriptional activity, real-time PCR for the bone markers (osteocalcin and alkaline phosphatase), and Western blot for phosphorylated FGFR2 and ERK1/2-MAPK protein. Among 16 patients, 10 showed FGFR2 mutations that had already been reported elsewhere. A novel FGFR2 mutation associated with tyrosine kinase II (TK-II) domain, L617F, was found in one Crouzon syndrome patient by direct PCR sequencing. Presence of this mutation was confirmed using RFLP analysis. Runx2 transcriptional activity and expression of osteocalcin and alkaline phosphatase significantly increased in L617F-transfected cells compared to wild-type cells. FGFR2 autophosphorylation in L617F-transfected cells increased in 1% serum, but ERK1/2-MAPK protein was not activated. The FGFR2-L617F mutation associated with the TK domain is potentially related to premature suture closure in Crouzon syndrome patient.

Síndrome de Crouzon: presentación de un caso / Crouzon syndrome: case report

Es una displasia ósea no letal caracterizada por cierre prematuro de las suturas coronales, hipoplasia de la parte media de la cara y proptosis ocular. Son los defectos congénitos del cráneo más frecuentes en humanos. El síndrome de Crouzon tiene una prevalencia de 15 en 1 x 106 nacimientos. Existen varias teorías que intentan explicar su patogenia. Hipótesis más actuales sugieren que el defecto se produce por una mutación del gen que codifica el receptor del factor del crecimiento fibroblástico tipo 2 (FGFR2). Los hallazgos ecográficos incluyen braquicefalia, oxicefalia, hipertelorismo e hipoplasia de la zona media de la cara. La sensibilidad del ultrasonido para detectar anomalías craneofaciales depende mucho del operador. La ecografía 3D no incrementa la tasa de detección de la 2D. Recientes estudios radiográficos revelaron frecuentes malformaciones intrauterinas no diagnosticadas. El diagnóstico prenatal se puede realizar por técnicas moleculares. La evolución posnatal es variable.(AU)

Síndrome de Crouzon: presentación de un caso / Crouzon syndrome: case report

Es una displasia ósea no letal caracterizada por cierre prematuro de las suturas coronales, hipoplasia de la parte media de la cara y proptosis ocular. Son los defectos congénitos del cráneo más frecuentes en humanos. El síndrome de Crouzon tiene una prevalencia de 15 en 1 x 106 nacimientos. Existen varias teorías que intentan explicar su patogenia. Hipótesis más actuales sugieren que el defecto se produce por una mutación del gen que codifica el receptor del factor del crecimiento fibroblástico tipo 2 (FGFR2). Los hallazgos ecográficos incluyen braquicefalia, oxicefalia, hipertelorismo e hipoplasia de la zona media de la cara. La sensibilidad del ultrasonido para detectar anomalías craneofaciales depende mucho del operador. La ecografía 3D no incrementa la tasa de detección de la 2D. Recientes estudios radiográficos revelaron frecuentes malformaciones intrauterinas no diagnosticadas. El diagnóstico prenatal se puede realizar por técnicas moleculares. La evolución posnatal es variable.

Amniotic bands as a cause of congenital anterior staphyloma.

BACKGROUND: Congenital anterior staphyloma is a rare, complex malformation syndrome of the anterior segment. Only a few reports on associated systemic malformations have been published. We herein present a rare manifestation of congenital anterior staphyloma (CAS) combined with amniotic band disruption syndrome (ABS). PATIENT AND METHODS: Shortly after birth, a massive enlargement of the left eye was observed in a female child. Furthermore, an extensive bilateral congenital cleft lip and cleft alveolar ridge with oblique facial cleft extending into the left medial canthal region, coloboma(s) of the left eyelids, extensive adhesions between lids and eye bulb, as well as circumferential grooves, clubfeet, and terminal transverse defects in both hands and feet were present. Due to severe progression of eye bulb protrusion with thinning of the sclera, enucleation of the left eye was performed at the age of 3 years in order to prevent complications including perforation of the globe and with the aim of improving cosmetic aspects. RESULTS: Histopathological examination of the enucleated eye disclosed findings typical of congenital anterior staphyloma, including massive corneal staphylomatic deformation with superficial vascularization and elapsed corneoscleral margin, destruction of Bowman's layer, absence of Descemet's layer, corneal endothelium, and angle structures. The lens was only partially formed, and had mainly dissolved. The neural retina appeared normal. The optic nerve disc revealed a pronounced excavation. Facial clefts, lid colobomas, congenital constriction bands, and amputation of distal limbs match ABS. This malformation complex develops in early pregnancy, probably prior to 35 days post conception. CONCLUSION: This is the first report on an association of these two rare complex congenital malformations, congenital anterior staphyloma and amniotic band syndrome. The anterior staphyloma was unilateral, and related to facial clefts and lid coloboma in the area adjacent to the anterior staphyloma. Furthermore, the systemic deformities are clearly due to the amniotic bands, and the timing of the development of both complex malformations seems to be similar. All findings suggest that the presence of amniotic bands is a causative factor for all observed abnormalities including anterior staphyloma.

[Biomechanical study of internal midface distraction after different types of maxillary osteotomy in patients with cleft lip and palate].

OBJECTIVE: To investigate the biomechanical changes of internal midface distraction after different types of maxillary osteotomy in patients with cleft lip and palate (CLP). METHODS: 3-D finite element (FEM) analysis was used. 3-D models of Le Fort I, II, III osteotomy and soft tissue were established. Based on the new pattern of internal midface distractor, the distraction of maxillary complex was simulated to advance 10 mm anteriorly. The mechanical change was studied. RESULTS: The maxillary complex in CLP were advanced after distraction. Constriction of alveolar crest and palate occurred in Le Fort I osteotomy, but not in Le Fort II and III osteotomy. The maxillary complex was moved anteriorly en bloc after Le Fort III osteotomy, but some degree of rotation of maxillary complex was observed during the distraction after Le Fort I and II osteotomy. In vertical direction, the maxillary complex had more counterclockwise rotation after Le Fort II osteotomy. CONCLUSION: 3-D FEM analysis can be used for the study of internal distraction. It can reflect the maxillary movement and provide the theory basis for preoperative design.

Síndrome de Crouzon: reporte de un caso / Crouzon syndrome: case report

Con oportunidad de presentar este caso clínico, el objetivo es realizar una revisión bibliográfica y actualizar conocimientos sobre esta enfermedad, que se caracteriza por un desorden genético que cursa con anormalidades en cráneo, cara y cerebro causado por un cierre prematuro de las suturas. Las deformidades faciales están a menudo presentes desde el nacimiento y progresan en el tiempo. Puede desarrollarse en algunos casos, disturbios de la visión y sordera. Con el tratamiento la presión del cráneo puede ser aliviada y los síntomas mayores pueden mejorar.

[Advances in the understanding of the etiopathogenesis of non syndromic craniostenoses and therapeutic approaches]. / Avancées dans la compréhension de l'étiopathogénie des craniosténoses non syndromiques et applications thérapeutiques.

The treatment of craniostenoses has not been revolutionized over the last decade, but the methods of investigation, particularly 3D CT scan, histological studies, and refinement of techniques, have allowed substantial progress in the understanding of the aetiopathogenesis and treatment of craniostenoses. The deformities of the components of the base of the skull can now be more precisely analysed. The authors present these elements for each of the various types of craniostenosis. The role of soft tissues has been more clearly defined; the dura mater, corresponding to an internal periosteum, has an osteogenic, but also directional role. The concept of the brain as the essential determinant of cranial morphology has led to the proposal of treatments adapted to each deformity. Specific craniectomies have been completed by mobilization of flaps, and segmentation of the vault allowing almost ideal reconstruction of the skull. In most treated cases, directed osteogenesis results in good consolidation after seven to eight months; partial non-ossification remains frequent in children treated after the age of eighteen months.

Critical analysis of results of craniofacial surgery for nonsyndromic bicoronal synostosis.

We retrospectively assessed the intermediate and long-term results of craniofacial surgery in 22 consecutive patients with nonsyndromic bicoronal synostosis to determine the outcome of corrective surgery. The study population consisted of 13 males and 9 females whose ages ranged from 6 weeks to 24 months (mean, 5.6 months) at the time of initial surgery. All patients had been assigned a diagnosis of nonsyndromic bicoronal synostosis. Each patient underwent resection of both coronal sutures and frontal orbital advancement with cranial vault remodeling using a floating forehead technique. Age at initial operation was 5 months or less in 13 patients and 6 months or more in 9. Complications occurred in 5 patients (23%), and 1 patient with an associated metabolic disorder died from respiratory arrest postoperatively. Follow-up ranged from 6 to 168 months (mean, 53.2 months). Results were graded according to the need for and extent of reoperation. Residual aesthetic deformities were documented in 12 patients (55%). Three patients (14%) required calvarial recontouring or cranioplasty to achieve satisfactory forehead contour or bony continuity. Total reoperation for recurrent deformity was required at a mean age of 29.7 months in 8 patients (36%) and is pending in another (4%). Four patients (18%) required a third operation (two total reoperations and two cranioplasties) to achieve satisfactory results. Eight of 13 patients (62%) operated on at 5 months of age or younger required total reoperation compared with 1 of 9 (11%) operated at 6 months of age or older. When analyzed alone, age of operation was a statistically significant determinant of the need for reoperation (p < 0.03). However, when subjected to multivariate analysis, neither age at operation nor the presence of an associated anomaly or positive family history had a significant effect on outcome.

Crouzon syndrome: cephalometric analysis and evaluation of pathogenesis.

Crouzon syndrome is a craniofaciostenosis characterized by brachycephaly, ocular proptosis, and maxillary retrusion. The hypothesis has been forwarded that an alteration in anterior cranial base synchondrosis activity is responsible for the skeleton abnormalities which are associated with this disorder. The present work was aimed at assessing this pathogenetic hypothesis. Cephalometry was used as the analysis method and care was taken in determining the three-dimensional measurements of some functional spaces (e.g., orbit, rhinopharynx, and nasal cavity). The results indicate that in Crouzon syndrome the craniofacial alterations depend not only on reduced synchondrosis activity of the anterior cranial base, but also of the posterior cranial base.

9p-Syndrome.

A 2 1/2 month old male child was admitted with loose motions and mild dehydration. He was full term normal delivery, born of a non-consanguinous marriage. On examination, he had trigonocephaly; anteverted nostrils, long philtrum and hypoplastic supraorbital ridges. X-ray showed sutural separation. Karyotyping confirmed deletion of short arm of chromosome 9 distal to band p22.

[The Silver-Russell syndrome]. / Sindrome di Silver-Russell.

The authors describe a case of Silver-Russell Syndrome with severe deficit growth. They display the major and minor features of the syndrome, and discuss about the possible pathogenetic causes.

Síndrome de Rubinstein Taybi. Enfoque rehabilitador, a propósito de un caso clínico / Rubinstein Taybi syndrome. Rehabilitor focuse, a case report

Presentamos el caso de un paciente pediátrico, con manifestaciones de Retardo mental y características físicas singulares, correspondientes al denominado síndrome de Rubinstein-Taybi, el cual está incluido en un programa integral de Rehabilitación para potenciar las áreas Psicofísicas sobre las cuales presenta deficiencias.

Enfermedad de Crouzon en tres de una familia de cinco miembros en el departamento de Cajamarca / Crouzon's disease in three members of a family of five members in Cajamarca city

En este trabajo se describen 3 casos de enfermedad de Crouzon en una familia de cinco miembros procedentes de la localidad de Saullo Grande en Cajamarca

[Deformities, caused by premature ossification of the cranial sutures, and their treatment]. / A koponyavarratok korai csontosodása miatti deformitások és azok kezelése.

The establishment of the Hungarian cranio-facial surgical team is reported on and the activity sphere of this special field is described. The consequences of early ossification of the cranial sutures as well as the therapeutical possibilities are dealt with. Brief review is given on the development of cranio-facial surgery. The age, diagnosis and interventions performed with the patients in 1987 are summarized in a table.