Rho GTPase Cdc42 is essential for human T-cell development.

BACKGROUND: Rho GTPases are involved in the regulation of many cell functions, including some related to the actin cytoskeleton. Different Rho GTPases have been shown to be important for T-cell development in mice. However, their role in human T-cell development has not yet been explored. DESIGN AND METHODS: We examined the expression and activation of Rho GTPases along different stages of T-cell development in the human thymus. Early stage human thymocytes were transduced with constitutively active and dominant negative mutants of different Rho GTPases to explore their role in human T-cell development, as analyzed in fetal thymus organ cultures. The use of these mutants as well as Rho GTPase-specific inhibitors allowed us to explore the role of GTPases in thymocyte migration. RESULTS: We found that the expression of several Rho GTPases is differently regulated during successive stages of T-cell development in man, suggesting a specific role in human thymopoiesis. In chimeric fetal thymus organ culture, T-cell development was not or only mildly affected by expression of dominant negative Rac1 and Rac2, but was severely impaired in the presence of dominant negative Cdc42, associated with enhanced apoptosis and reduced proliferation. Kinetic analysis revealed that Cdc42 is necessary in human T-cell development both before and after expression of the pre-T-cell receptor. Using inhibitors and retrovirally transferred mutants of the aforementioned Rho GTPases, we showed that only Rac1 is necessary for migration of different thymocyte subsets, including the early CD34(+) fraction, towards stromal cell-derived factor-1 alpha. Constitutively active mutants of Rac1, Rac2 and Cdc42 all impaired migration towards stromal cell-derived factor-1 alpha and T-cell development to different degrees. CONCLUSIONS: This is the first report on Rho GTPases in human T-cell development, showing the essential role of Cdc42. Our data suggest that enhanced apoptotic death and reduced proliferation rather than disturbed migration explains the decreased thymopoiesis induced by dominant negative Cdc42.

An interlaboratory comparison of ITS2-PCR for the identification of yeasts, using the ABI Prism 310 and CEQ8000 capillary electrophoresis systems.

BACKGROUND: Currently, most laboratories identify yeasts routinely on the basis of morphology and biochemical reactivity. This approach has quite often limited discriminatory power and may require long incubation periods. Due to the increase of fungal infections and due to specific antifungal resistence patterns for different species, accurate and rapid identification has become more important. Several molecular techniques have been described for fast and reliable identification of yeast isolates, but interlaboratory exchangeability of identification schemes of molecular techniques has hardly been studied. Here, we compared amplified ITS2 fragment length determination by an ABI Prism 310 (Applied Biosystems, Foster City, Ca.) capillary electrophoresis system with that obtained by a CEQ8000 (Beckman Coulter, Fullerton, Ca.) capillary electrophoresis system. RESULTS: Although ITS2 size estimations on both systems differed and separate libraries had to be constructed for each system, both approaches had the same discriminatory power with regard to the 44 reference strains, identical identifications were obtained for 39/ 40 clinical isolates in both laboratories and strains from 51 samples were correctly identified using CEQ8000, when compared to phenotypic identification. CONCLUSION: Identification of yeasts with ITS2-PCR followed by fragment analysis can be carried out on different capillary electrophoresis systems with comparable discriminatory power.

Mutations in the calcium-binding motifs of CDH23 and the 35delG mutation in GJB2 cause hearing loss in one family.

We have ascertained a multi-generation family with apparent autosomal recessive non-syndromic childhood hearing loss (DFNB). Failure to demonstrate linkage in a genome-wide scan with 300 polymorphic markers has suggested genetic heterogeneity for the hearing loss in this family. This heterogeneity could be demonstrated by analysis of candidate loci and genes for DFNB. Patients in one branch of the family (branch C) are homozygous for the 35delG mutation in the GJB2 gene (DFNB1). Patients in two other branches (A and B) carry two new mutations in the cadherin 23 ( CDH23) gene (DFNB12). A homozygous CDH23 c.6442G-->A (D2148N) mutation is present in branch A. Patients in branch B are compound heterozygous for this mutation and the c.4021G-->A (D1341N) mutation. The substituted aspartic acid residues are highly conserved and are part of the calcium-binding sites of the extracellular cadherin (EC) domains. Molecular modeling of the mutated EC domains of CDH23 based on the structure of E-cadherin indicates that calcium-binding is impaired. In addition, other aspartic and glutamic acid residue substitutions in the highly conserved calcium-binding sites reported to cause DFNB12 are also likely to result in a decreased affinity for calcium. Since calcium provides rigidity to the elongated structure of cadherin molecules enabling homophilic lateral interaction, these mutations are likely to impair interactions of CDH23 molecules either with CDH23 or with other proteins. DFNB12 is the first human disorder that can be attributed to inherited missense mutations in the highly conserved residues of the extracellular calcium-binding domain of a cadherin.