Gradients in surface nanotopography used to study platelet adhesion and activation.

Gradients in surface nanotopography were prepared by adsorbing gold nanoparticles on smooth gold substrates using diffusion technique. Following a sintering procedure the particle binding chemistry was removed, and integration of the particles into the underlying gold substrate was achieved, leaving a nanostructured surface with uniform surface chemistry. After pre-adsorption of human fibrinogen, the effect of surface nanotopography on platelets was studied. The use of a gradient in nanotopography allowed for platelet adhesion and activation to be studied as a function of nanoparticle coverage on one single substrate. A peak in platelet adhesion was found at 23% nanoparticle surface coverage. The highest number of activated platelets was found on the smooth control part of the surface, and did not coincide with the number of adhered platelets. Activation correlated inversely with particle coverage, hence the lowest fraction of activated platelets was found at high particle coverage. Hydrophobization of the gradient surface lowered the total number of adhering cells, but not the ratio of activated cells. Little or no effect was seen on gradients with 36nm particles, suggesting the existence of a lower limit for sensing of surface nano-roughness in platelets. These results demonstrate that parameters such as ratio between size and inter-particle distance can be more relevant for cell response than wettability on nanostructured surfaces. The minor effect of hydrophobicity, the generally reduced activation on nanostructured surfaces and the presence of a cut-off in activation of human platelets as a function of nanoparticle size could have implications for the design of future blood-contacting biomaterials.

The kidney-expressed winged helix transcription factor FREAC-4 is regulated by Ets-1. A possible role in kidney development.

In this paper we show that the kidney-expressed winged helix transcription factor FREAC-4 is regulated by Ets-1, another kidney-expressed transcription factor. Through transfection experiments three Ets-1 cis-elements are identified within the first 152 nucleotides upstream of the transcription start in the freac-4 promoter. These sites are confirmed in a DNase I in vitro protection assay using recombinant Ets-1 protein. In cotransfection experiments using an Ets-1 expression vector, the induction of freac-4 reporter gene activity is attenuated approximately 6-fold when the three Ets-1 binding sites are mutated. Furthermore, we demonstrate that overexpression of Ets-1 in the human embryonic kidney cell line 293 is sufficient to increase freac-4 mRNA levels. These results are compatible with the hypothesis that Ets-1 acts as an upstream regulator of FREAC-4 expression during kidney development.

The winged helix transcription factor Fkh10 is required for normal development of the inner ear.

Fkhl0 is a member of the forkhead family of winged helix transcriptional regulators. Genes encoding forkhead proteins are instrumental during embryogenesis in mammals, in particular during development of the nervous system. Here we report that mice with a targeted disruption of the Fkh10 locus exhibit circling behaviour, poor swimming ability and abnormal reaching response-all common findings in mice with vestibular dysfunction. These animals also fail to elicit a Preyer reflex in response to a suprathreshold auditory stimulation, as seen in mice with profound hearing impairment. Histological examination of the inner ear reveals a gross structural malformation of the vestibulum as well as the cochlea. These structures have been replaced by a single irregular cavity in which neither proper semicircular ducts nor cochlea can be identified. We also show that at 9.5 days post coitum (dpc), Fkh10 is exclusively expressed in the otic vesicle. These findings implicate Fkh10 as an early regulator necessary for development of both cochlea and vestibulum and identify its human homologue FKHL10 as a previously unknown candidate deafness gene at 5q34.

Chromosome localization, sequence analysis, and expression pattern identify FKHL 18 as a novel human forkhead gene.

The forkhead gene family of transcription factors belongs to the "winged helix" class of DNA-binding proteins. Today over 40 members of this gene family have been identified. Forkhead genes have been shown to be involved in embryonic development, tumorigenesis, and direction of tissue specificity of gene expression. Here we describe a new human forkhead gene called freac-10 (HGMW-approved symbol FKHL 18). A combination of fluorescence in situ hybridization and somatic cell hybrids localizes freac-10 to the chromosomal region of 20q11.1-q11.2. Hybridization to a panel consisting of RNA derived from 50 different tissues shows that freac-10 is transcribed predominantly in the aorta, thus having a unique expression pattern compared with other forkhead genes. Sequence comparison reveals a striking similarity, over the conserved DNA binding region, to a murine forkhead gene-fkh-3. We propose, based on sequence differences in the N- and C-terminal regions of the forkhead domain and a clear difference in expression pattern between freac-10 and fkh-3, that freac-10 represents a novel member of this gene family.

Characterization of the human forkhead gene FREAC-4. Evidence for regulation by Wilms' tumor suppressor gene (WT-1) and p53.

We describe the cloning and sequence analysis of a nearly full-length cDNA as well as a corresponding 5.2-kilobase pair genomic fragment encoding FREAC-4, a member of the forkhead family of transcription factors. The cDNA is collinear with respect to the coding region of the intronless genomic clone. The conceptual translation product predicts a protein of 465 amino acids with a hyperacidic amino-terminal end, a DNA binding forkhead domain and a carboxyl-terminal part that is rich in homopolymeric runs of prolines and alanines. The transcription start is identified using an RNase protection assay. A 2.7-kilobase pair genomic DNA fragment, located immediately upstream of the translation start, was fused to a luciferase reporter gene. Significant levels of luciferase activity were detected when this construct was transfected into two kidney-derived cell lines, 293 and COS-7 cells, whereas only background reporter gene expression was observed in a cell line of nonkidney origin. Cotransfections with plasmids expressing WT-1, WTAR (a mutated form of WT-1), p53, and a mutated form of p53 revealed a complex pattern of regulation with a 3-fold induction with WT-1, a 7-fold induction with mutated p53, and a 4-fold repression with wild-type p53. A 5'-promoter deletion series delimits a DNA fragment necessary for WT-1 inducibility in cotransfection experiments. This fragment is shown to contain at least one cis-element that is capable of interacting with recombinant WT-1.