A novel interaction between ATOH8 and PPP3CB.

ATOH8 is a bHLH transcription factor playing roles in a variety of developmental processes such as neurogenesis, differentiation of pancreatic precursor cells, development of kidney and muscle, and differentiation of endothelial cells. PPP3CB belongs to the catalytic subunit of the serine/threonine phosphatase, calcineurin, which can dephosphorylate its substrate proteins to regulate their physiological activities. In our study, we demonstrated that ATOH8 interacts with PPP3CB in vitro with different approaches. We show that the conserved catalytic domain of PPP3CB interacts with both the N-terminus and the bHLH domain of ATOH8. Although the interaction domain of PPP3CB is conserved among all isoforms of calcineurin A, ATOH8 selectively interacts with PPP3CB instead of PPP3CA, probably due to the unique proline-rich region present in the N-terminus of PPP3CB, which controls the specificity of its interaction partners. Furthermore, we show that inhibition of the interaction with calcineurin inhibitor, cyclosporin A (CsA), leads to the retention of ATOH8 to the cytoplasm, suggesting that the interaction renders nuclear localization of ATOH8 which may be critical to control its activity as transcription factor.

Induction of neoplasia after deep anterior lamellar keratoplasty in a CXL-treated cornea.

PURPOSE: Corneal collagen crosslinking (CXL) with ultraviolet-A energy plus riboflavin has become a ubiquitous treatment in early keratoconus, although its long-term safety is unknown. We describe severe sequelae in a CXL-treated patient after he underwent a standard deep anterior lamellar keratoplasty procedure. METHODS: In April 2009, a healthy 49-year-old male patient (R.H.) underwent bilateral CXL according to the Dresden protocol for progressive keratoconus stage 3. The best-corrected visual acuity did not improve over 20/100 within a postoperative period of 2 years, and contact lenses were not tolerated. Consequently, a unilateral deep anterior lamellar keratoplasty was performed, to transplant an 8-mm fully epithelialized button onto an 8-mm bed with a bared Descemet membrane (surgeon: J.H.K.). RESULTS: The postoperative healing course was unusually disturbed. Sutures pulled through the recipient tissue, which required suture replacement. Portions of the epithelium sloughed off repeatedly, and bulky regrowth displayed no attachment to the Bowman membrane. Within the first weeks, the transplant became cloudy. Two biopsies were removed from the limbus area and submitted to independent histopathological laboratories, both of which diagnosed the condition as epithelial neoplasia. Pathology tests indicated conjunctival intraepithelial neoplasia, the preliminary stage of invasive squamous cell carcinoma, in the keratocyte-voided bed of the recipient. CONCLUSIONS: This case suggests that CXL might hamper the ocular healing process and, combined with subsequent corneal surgery, could potentially initiate neoplasia. Further investigation is warranted to determine the safety of the combination of ultraviolet-A/riboflavin treatment and subsequent corneal tissue transplantation.

A thymosin beta15-like peptide promotes intersegmental myotome extension in the chicken embryo.

Beta-thymosins constitute a group of small actin-sequestering peptides. These highly conserved peptides are involved in cytoskeleton dynamics and can influence different cell properties such as motility, substrate adhesion, shape and chemotaxis. As a marker for tumour metastasis, the mammalian thymosin beta15 is believed to have an important diagnostic relevance in cancer prognosis, although little is known about its physiological function. In order to study the role of thymosin beta15(avian) in embryogenesis, we cloned the chicken and quail orthologues of thymosin beta15 and used the chicken as a model for vertebrate development. Avian thymosin beta15, the first known non-mammalian thymosin beta15-like gene, encodes a peptide that possesses a cysteine at position one after the methionine which is a significant difference compared to its mammalian counterparts. Thymosin beta15(avian) expression starts at an early stage of development. The expression pattern changes rapidly with development and differs from that of the related thymosin beta4 gene. The most prominent expression domain is seen in developing muscles of limbs and trunk. Gain-of-function experiments revealed that thymosin beta15(avian) has a function in normal myotome development. Ectopic over-expression of thymosin beta15(avian) leads to premature elongation of myotome cells trespassing segment borders. We conclude that thymosin beta15(avian) has a still undescribed function in promoting myocyte elongation.

Four subunits (αßγδ) of the epithelial sodium channel (ENaC) are expressed in the human eye in various locations.

PURPOSE: The epithelial sodium channel (ENaC) is typically expressed in sodium-absorbing epithelia. Several reports suggest that ENaC is also expressed in ocular tissues and may play a role in aqueous humor secretion and glaucoma. However, the precise localization of ENaC in the human eye is still unclear. Here, the authors studied ENaC expression in 12 normal human donor eyes and in six eyes of patients with glaucoma. METHODS: Quantitative real-time PCR was used to investigate the expression of α-, ß-, γ-, and δ-ENaC transcripts in ocular tissues. In addition, the authors performed immunohistochemical studies using recently generated antibodies against human ß- and γ-ENaC. RESULTS: At the mRNA level, all four ENaC subunits were found to be expressed in a wide range of ocular tissues from normal and glaucomatous human eyes, with the cornea, ciliary body, iris, and retina showing the highest expression levels. At the protein level, ß- and γ-ENaC subunits showed distinct distribution patterns and could be immunolocalized primarily to the cell membranes of epithelial cells of the cornea and to the conjunctiva, iris, ciliary body, lens, and retinal pigment epithelium but also to vascular endothelial cells, smooth muscle cells, stromal cells, and retinal neurons. The authors found no altered mRNA level of any subunit in glaucomatous eyes. CONCLUSIONS: All four ENaC subunits (αßγδ) are expressed in the normal human eye, with distinct localization of subunits possibly reflecting different functional states of the channel. The (patho-)physiological roles of ENaC in the various localizations in the eye remain to be determined.

Diversification and molecular evolution of ATOH8, a gene encoding a bHLH transcription factor.

ATOH8 is a bHLH domain transcription factor implicated in the development of the nervous system, kidney, pancreas, retina and muscle. In the present study, we collected sequence of ATOH8 orthologues from 18 vertebrate species and 24 invertebrate species. The reconstruction of ATOH8 phylogeny and sequence analysis showed that this gene underwent notable divergences during evolution. For those vertebrate species investigated, we analyzed the gene structure and regulatory elements of ATOH8. We found that the bHLH domain of vertebrate ATOH8 was highly conserved. Mammals retained some specific amino acids in contrast to the non-mammalian orthologues. Mammals also developed another potential isoform, verified by a human expressed sequence tag (EST). Comparative genomic analyses of the regulatory elements revealed a replacement of the ancestral TATA box by CpG-islands in the eutherian mammals and an evolutionary tendency for TATA box reduction in vertebrates in general. We furthermore identified the region of the effective promoter of human ATOH8 which could drive the expression of EGFP reporter in the chicken embryo. In the opossum, both the coding region and regulatory elements of ATOH8 have some special features, such as the unique extended C-terminus encoded by the third exon and absence of both CpG islands and TATA elements in the regulatory region. Our gene mapping data showed that in human, ATOH8 was hosted in one chromosome which is a fusion product of two orthologous chromosomes in non-human primates. This unique chromosomal environment of human ATOH8 probably subjects its expression to the regulation at chromosomal level. We deduce that the great interspecific differences found in both ATOH8 gene sequence and its regulatory elements might be significant for the fine regulation of its spatiotemporal expression and roles of ATOH8, thus orchestrating its function in different tissues and organisms.