Clemens Stockklausner

A sequence motif responsible for ER export and surface expression of Kir2.0 inward rectifier K(+) channels.

Integral membrane proteins are sorted via the secretory pathway. It was proposed that this pathway is non‐selective provided that the cargo protein is properly assembled and lacks an endoplasmic reticulum (ER) retention signal. However, recent experimental evidence suggests that efficient export of proteins from the ER to the Golgi complex is not simply a default pathway. Here we demonstrate a novel sequence motif (FxYENEV) in the cytoplasmic C‐terminus of mammalian inward rectifier potassium (Kir) channels which determines ER export. This motif is found to be both necessary and sufficient for efficient export from the ER that eventually leads to efficient surface expression of Kir2.1 channels.

The uORF-containing thrombopoietin mRNA escapes nonsense-mediated decay (NMD)

Platelet production is induced by the cytokine thrombopoietin (TPO). It is physiologically critical that TPO expression is tightly regulated, because lack of TPO causes life-threatening thrombocytopenia while an excess of TPO results in thrombocytosis. The plasma concentration of TPO is controlled by a negative feedback loop involving receptor-mediated uptake of TPO by platelets. Furthermore, TPO biosynthesis is limited by upstream open reading frames (uORFs) that curtail the translation of the TPO mRNA. uORFs are suggested to activate RNA degradation by nonsense-mediated decay (NMD) in a number of physiological transcripts. Here, we determine whether NMD affects TPO expression. We show that reporter mRNAs bearing the seventh TPO uORF escape NMD. Importantly, endogenously expressed TPO mRNA from HuH7 cells is unaffected by abrogation of NMD by RNAi. Thus, regulation of TPO expression is independent of NMD, implying that mRNAs bearing uORFs cannot generally be considered to represent NMD targets.

Interaction of PSD-95 with potassium channels visualized by fluorescence lifetime-based resonance energy transfer imaging

Resonance energy transfer (RET) has been extensively used to estimate the distance between two different fluorophores. This study demonstrates how protein-protein interactions can be visualized and quantified in living cells by time-correlated single-photon counting (TCSPC) imaging techniques that exploit the RET between appropriate fluorescent labels. We used this method to investigate the association of the potassium inward rectifier channel Kir2.1 and the neuronal PDZ protein PSD-95, which has been implicated in subcellular targeting and clustering of ion channels. Our data show that the two proteins not only colocalize within clusters but also interact with each other. Moreover, the data allow a spatially resolved quantification of this protein-protein interaction with respect to the relative number and the proximity between interacting molecules. Depending on the subcellular localization, a fraction of 20 to 60% of PSD-95 molecules interacted with Kir2.1 channels, approximating their fluorescent labels by less than 5 nm.

Significance of image-defined risk factors for surgical complications in patients with abdominal neuroblastoma.

INTRODUCTION Neuroblastoma (NB) is one of the most common malignant tumors in infancy. The commonly used International Neuroblastoma Staging System is not suitable for determining the surgical risks. To address this, we aimed to evaluate the correlation between so-called image-defined risk factors (IDRFs) and the surgical risks in abdominal neuroblastoma. MATERIAL AND METHODS We evaluated 60 cases who underwent surgical intervention and examined the pre-surgical radiological imaging to look for IDRFs and surgical complications in children with abdominal neuroblastoma. RESULTS The MRI- and CT-scans showed a total of 122 IDRFs in 39 cases. Complete resection was carried out in 50%, partial excision in 32%, and biopsy in 18% of cases. Total resection was possible in 100% of cases with no IDRF. Where IDRFs were present, total resection was only possible in 26% of cases (p<0.0001). We found a highly significant, negative correlation between the number of IDRFs and the possibility of performing complete resection of NB (p<0.0001). 7 (11.6%) complications were detected, all in patients who showed at least one IDRF previously. CONCLUSION Our findings indicate that IDRFs are useful indicators for predicting surgical risk and surgical outcome and thus should be taken into account when planning surgery.

13-cis retinoic acid treatment of a patient with chemotherapy refractory nephroblastomatosis

A 9-month-old girl presented with massive bilateral diffuse nephroblastomatosis. After response to actinomycin D and vincristine over a period of 1 year, the nephroblastomatosis continuously progressed under this treatment. As retinoic acid signaling is critical for normal renal development and nephroblastomatosis seems histologically as undifferentiated embryonal tissue, we added 13-cis retinoic acid to the chemotherapy regimen. Three months thereafter, kidney volumes declined significantly over a period of 1 year. Interestingly, nephroblastomatosis-associated acquired von Willebrand disease also resolved. Retinoic acid maybe a novel nontoxic treatment option for nephroblastomatosis requiring further systematic evaluation.

Long-term remission of children with relapsed and secondary anaplastic large cell non-Hodgkin lymphoma (ALCL) following treatment with pulsed dexamethasone and low dose etoposide.

Anaplastic large cell lymphoma (ALCL) accounts for approximately 15% of childhood NHL. Relapsed ALCL represents a formidable challenge because outcome is poor despite the use of high‐dose chemotherapy regimens. We report two patients with relapsed T‐type and 0‐type ALCL who achieved long‐term 3rd and 4th remissions with 4‐weekly oral dexamethasone (DEX) and etoposide pulses for 2 years. This regimen also induced and maintained remission in a third patient with Nijmegen breakage syndrome (NBS) with secondary T‐type ALCL. These patients demonstrate that low‐intensity oral chemotherapy can induce long‐term remissions and offer a curative perspective in refractory, relapsed and secondary ALCL. Pediatr Blood Cancer 2008;50:126–129.

The thrombopoietin receptor P106L mutation functionally separates receptor signaling activity from thrombopoietin homeostasis

The interaction between thrombopoietin (THPO) and its receptor c-Mpl regulates downstream cytokine signaling and platelet homeostasis. Hereditary mutations of c-Mpl can either result in loss-of-function and thrombocytopenia or in gain-of-function and thrombocythemia (HT), and are important models to analyze the mechanism of c-Mpl activity. We have analyzed the effect of the c-Mpl P106L gain-of-function and the nearby loss-of-function R102P and F104S mutations, which cause HT or thrombocytopenia, respectively, on posttranslational processing, intracellular trafficking, cell surface expression, and cell proliferation. In contrast to R102P and F104S, the P106L mutant confers cytokine-independent growth and stimulates downstream signaling after THPO treatment in Ba/F3 cells. Despite their opposite function, R102P and P106L, both lead to abnormal subcellular receptor distribution, lack of membrane localization, impaired glycosylation, and elevated THPO serum levels in effected patients. These findings indicate that the activation of downstream signaling by c-Mpl P106L does not require correct processing, trafficking, and cell surface expression of c-Mpl, whereas the negative feedback loop controlling THPO serum levels requires cell surface expression of the receptor. Thus, we propose that the P106L mutation functionally separates the activity of c-Mpl in downstream signaling from that in maintaining platelet homeostasis.

Hereditary thrombocythemia caused by a thrombopoietin (THPO) gain-of-function mutation associated with multiple myeloma and congenital limb defects

Hereditary thrombocythemia (HT) has been described as a rare benign disorder caused by mutations in the thrombopoietin (THPO) or the c-Mpl receptor genes. Here we report two families with HT resulting from a THPO c.13+1 G>C mutation in the splice donor of intron 3. In one family there were coexisting distal limb defects, whereas in the other one member developed early-onset multiple myeloma. These observations, together with previously reported patients, suggest that THPO gain of function may dysregulate the hemangioblast and disturb vasculogenesis and hematopoietic development. Overstimulation of the THPO pathway might therefore predispose to clonal hematopoietic disease and to congenital abnormalities.

DNA methylation in PRDM8 is indicative for dyskeratosis congenita.

Dyskeratosis congenita (DKC) is associated with impaired telomere maintenance and with clinical features of premature aging. In this study, we analysed global DNA methylation (DNAm) profiles of DKC patients. Age-associated DNAm changes were not generally accelerated in DKC, but there were significant differences to DNAm patterns of healthy controls, particularly in CpG sites related to an internal promoter region of PR domain containing 8 (PRDM8). Notably, the same genomic region was also hypermethylated in aplastic anemia (AA) – another bone marrow failure syndrome. Site-specific analysis of DNAm level in PRDM8 with pyrosequencing and MassARRAY validated aberrant hypermethylation in 11 DKC patients and 27 AA patients. Telomere length, measured by flow-FISH, did not directly correlate with DNAm in PRDM8. Therefore the two methods may be complementary to also identify patients with still normal telomere length. In conclusion, blood of DKC patients reveals aberrant DNAm patterns, albeit age-associated DNAm patterns are not generally accelerated. Aberrant hypermethylation is particularly observed in PRDM8 and this may support identification and classification of bone marrow failure syndromes.

Pediatric Case Report on Magnetic Resonance Imaging/Transrectal Ultrasound-fusion Biopsy of Rhabdomyosarcoma of the Bladder/Prostate: A New Tool To Reduce Therapy-associated Morbidity?

Rhabdomyosarcomas are the most common soft tissue sarcomas in children. Here we present management of an 18-month-old boy with metastatic rhabdomyosarcoma of the bladder/prostate. After radiochemotherapy, high-spatial-resolution 3-Tesla multiparametric magnetic resonance imaging (MRI) showed regressive systemic disease but a residual mass at the right seminal vesicle. For histologic re-evaluation, 3-dimensional-controlled stereotactic MRI/transrectal ultrasound (TRUS)-fusion biopsy specimens were taken. Because histologic analysis showed nonvital tissue, a decision could be made against adjuvant radical cystoprostatectomy. Advanced 3-Tesla imaging and MRI/TRUS-fusion biopsies in children are feasible and represent an effective tool to examine suspicious pelvic lesions. Depending on histology, this can lead to a significant reduction of therapy-associated morbidity.