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The diagnostics and treatment of pediatric urology patients in the clinical routine can be extremely challenging. In contrast to adult patients, the main concerns in the diagnostics of congenital diseases are time consuming examinations and limited options in addition to the expectations of the parents. The exact knowledge of the diagnostic possibilities in association with the correct interpretation of the indications is essential. Simple processes can be much more time consuming because of a lack of compliance, especially in very young children. Sonography is considered the standard for imaging in pediatric urology. Profound knowledge of the embryonal development and also physiological processes throughout childhood contribute to making the correct diagnosis. This article deals with the possibilities of nuclear medicine diagnostics, advanced diagnostics in bladder voiding disorders and finally imaging diagnostics in the pediatric urological operating room.Diagnostik und Therapie von kinderurologischen Patienten und Patientinnen können im klinischen Alltag äußerst herausfordernd sein. Im Vergleich zu erwachsenen Patienten stehen bei der Diagnostik kongenitaler Erkrankungen neben einer Erwartungshaltung der Eltern des Kindes anspruchsvollere Untersuchungen, erhöhter Zeitaufwand und limitierte Optionen im Vordergrund. Die genaue Kenntnis der diagnostischen Möglichkeiten in Verbindung mit der richtigen Indikationsstellung ist hier essenziell. Einfache Prozesse können aufgrund der fehlenden Compliance besonders kleiner Kinder wesentlich zeitaufwendiger sein. Die Sonographie gilt als der Standard in der bildgebenden Kinderurologie. Ein fundiertes Wissen über die embryonale Entwicklung wie auch über physiologische Prozesse im Laufe der Kindheit trägt zur korrekten Diagnosestellung bei. Diese Arbeit befasst sich mit den Möglichkeiten der nuklearmedizinischen Diagnostik, der weiterführenden Diagnostik bei Blasenentleerungsstörungen sowie zuletzt der bildgebenden Diagnostik im kinderurologischen Operationssaal.

Aim: Stratification of hepatoblastoma (HB) patients is based on clinical and imaging characteristics obtained at the time of diagnosis. We aim to integrate biomarkers into a tool that accurately predicts survival of HB patients. Methods: We retrospectively analysed 174 HB patients for the presence of four biomarkers and explored their prognostic potential by correlating with overall survival (OS) and event-free survival (EFS). Results: Mutations of CTNNB1, NFE2L2 and TERT were found in 135 (78%), 10 (6%) and 10 (6%) patients, respectively, and the adverse C2 subtype of the 16-gene signature in 63 (36%) patients. C2-patients had more frequent metastatic disease, higher alpha-fetoprotein levels, non-fetal histology and significantly worse 3-year OS (68% versus 95%) and EFS (63% versus 87%) than C1-patients. Patients carrying a NFE2L2 mutation had a significantly worse 3-year OS (57% versus 88%) than NFE2L2 wild-type patients and were more likely to have vessel invasive growth and non-fetal histology. TERT mutations were almost exclusively found in older patients, whereas CTNNB1 mutations showed no association with any clinical feature or outcome. In a multivariable analysis, the C2 subtype remained a significant predictor of poor outcome with hazard ratios of 6.202 and 3.611 for OS and EFS, respectively. When added to the Children's Hepatic tumors International Collaboration risk stratification, the presence of the C2 subtype identified a group of high-risk patients with a very poor outcome. Conclusion: We propose a new stratification system based on the combination of clinical factors and the 16-gene signature, which may facilitate a risk-adapted management of HB patients. (C) 2020 The Author(s). Published by Elsevier Ltd.

The diagnostics and treatment of pediatric urology patients in the clinical routine can be extremely challenging. In comparison to adult patients, congenital diseases, more time consuming examinations and limited options in addition to the parents' expectations must be taken into account in the diagnostic work up. In this first of two parts we will delve into ultrasound diagnostics as the cornerstone in the diagnostic pathway of children with hydronephrosis ans take a closer look on contrast enhanced ultrasound (CEUS). Conventional voiding cystourethrography still plays a major role in the diagnostic pathway of vesicoureteric reflux and will be treated in this article. Computed tomography should only be considered in pediatric patients in rare cases, always taking radiation into critical account. Magnetic resonance imaging provides an excellent anatomical overview without exposing the child to unnecessary radiation. This article provides an overview on the diagnostic imaging studies in pediatric urology and brings tips for the diagnostic evaluation.Die Diagnostik und die Therapie von kinderurologischen Patienten und Patientinnen können im klinischen Alltag äußerst herausfordernd sein. Im Vergleich zu erwachsenen Patienten stehen bei der Diagnostik kongenitaler Erkrankungen anspruchsvollere Untersuchungen, erhöhter Zeitaufwand und limitierte Optionen neben einer Erwartungshaltung der Eltern des Kindes im Vordergrund. Die genaue Kenntnis der diagnostischen Möglichkeiten in Verbindung mit der richtigen Indikationsstellung ist hier essenziell. Dieser erste von zwei Beitragsteilen widmet sich zum Einen der Sonographie als primärer Standarduntersuchungsmethode in der Kinderurologie und geht im speziellen auf das contrast enhanced ultrasound (CEUS) ein. Das konventionelle Röntgenunterstützte Miktionszystourethrographie ist weiterhin fester Bestandteil des Diagnosealgorithmus des vesikoureterorenalen Refluxes und wird in dieser Arbeit eingängig behandelt. Der Einsatz der CT beschränkt sich aufgrund der Strahlenexposition in der Kinderurologie auf die Abklärung des schweren Polytraumas, und in Ausnahmefällen auf die Tumor- bzw. Steindiagnostik. Die Magnetresonanztomographie ermöglicht eine hervorragende anatomische Beurteilung des gesamten Urogenitaltraktes, ohne das Kind einer unnötigen Strahlenexposition auszusetzen. Diese Fortbildungseinheit gibt Ihnen eine aktuelle Übersicht und bietet einige Tipps und Tricks in der kinderurologischen Diagnostik.

Background Ethiopia is a rapidly developing country in Eastern Africa. 43.2% of the population is younger than 15. In contrast, until a few years ago, pediatric surgery was only available in Addis Ababa. Now, Ethiopia is making great efforts to improve the care of surgical ill children. JimmaChild was established to set up a pediatric surgery in Jimma. Methods JimmaChild developed from a scientific collaboration between Jimma University (JU) and Ludwig-Maximilians-University. The project was developed and realized by Ethiopian and German colleagues. A curriculum was written for this purpose. The pediatric surgical training of the fellows was carried out on-site by German pediatric surgeons. Results A new pediatric surgery was established at JU with its own operating room, ward, and staff. After two and a half years, two fellows completed their final examinations as pediatric surgeons. Among others, 850 elective surgeries were performed, 82% assisted by the German colleagues. The German colleagues rated the preparation for the trip, the on-site support, and the professional progress of the fellows mostly as good to very good. Reported problems in the program flow were also recognized and solved in part. Conclusions Best possible integration of the project into existing structures was achieved by close cooperation of Ethiopian and German colleagues during the project development. Problems were identified and addressed early on by external monitoring. As the project responsibility was mainly with the Ethiopian colleagues, a department was created that now exists independent of external funding and trains its own fellows.

Hepatoblastoma (HB) is the most common liver tumor in children under the age of 3 years worldwide. While many patients achieve good outcomes with surgical resection and conventional chemotherapy, there is still a high‑risk population that exhibits a poor treatment response and unfavorable prognosis, which warrants the search for novel treatment options. In recent years, it has become clear that genetic events alone are not sufficient to explain the aggressive phenotype of this embryonal malignancy. Instead, epigenetic modifications and aberrant gene expression seem to be key drivers of HB. In the present study, expression analyses such as reverse transcription‑quantitative polymerase chain reaction revealed that the oncogene, MYCN proto‑oncogene basic‑helix‑loop‑helix transcription factor (MYCN) was upregulated in HB and other pediatric liver tumors, due to the transcriptional activity of its antisense transcript MYCN opposite strand (MYCNOS). Pyrosequencing demonstrated the hypomethylated regions in the promoter of MYCN and MYCNOS, suggesting that an epigenetic mechanism may underlie the induction of aberrant expression. Transient MYCN knockdown in HB cells resulted in growth inhibition over time. In addition, treating HB cells with the MYCN inhibitors JQ1 and MLN8237 led to the significant downregulation of MYCN either at the mRNA or protein levels, respectively. The underlying mechanism of action of the two inhibitors was revealed to be associated with the induction of dose‑dependent growth arrest, by arresting cells at either the G1/G0 or G2 phase. Furthermore, MLN8237 and JQ1 were able to cause spindle disturbances and/or apoptosis in HB cells. The present results suggest that MYCN may be a promising biomarker for HB and a potential therapeutic target in patients with tumors overexpressing MYCN.

Despite tremendous progress in therapy, about 30 % of patients with hepatoblastoma still succumb to the disease. Thus, the development of improved therapies as well as the identification of prognostic factors are urgently needed. In the present study, expression and promoter methylation of the N-myc downstream-regulated gene (NDRG2), a tumor suppressor gene contributing to the regulation of the Wnt signalling pathway, was analysed in 38 hepatoblastoma samples by real-time reverse transcription-PCR and pyrosequencing, respectively. The NDRG2 gene was highly expressed in normal pediatric liver tissue, but was significantly downregulated in heptoblastoma primary tumors. Detailed methylation analysis of CpG sites in the NDRG2 promoter region revealed a general high degree of DNA methylation in hepatoblastoma, which correlated with the suppression of NDRG2. By analyzing clinicopathological features we could demonstrate a strong association between low NDRG2 expression and tumor metastasis. Importantly, the overall survival analysis by Kaplan–Meier revealed that high NDRG2 expression was correlated with a higher survival rate in hepatoblastoma patients. Our data show that downregulation of NDRG2 may play an important role in advanced hepatoblastomas.

The option of nephron sparing surgery for unilateral Wilms tumor has been debated in the recent literature. This procedure is being used increasingly to preserve kidney tissue and function. However, nephron sparing surgery is feasible only for selected cases, and a higher local relapse rate has been observed. Moreover, a significant reduction of nephrons is associated with development of renal hypertension and progressive renal failure. We analyzed outcomes after bilateral partial nephrectomy and unilateral partial plus contralateral total nephrectomy in patients with bilateral Wilms tumor.We analyzed data from the Society of Pediatric Oncology and Hematology database on 22 patients with bilateral Wilms tumor. Kidney size was measured using volumetric analysis of magnetic resonance imaging. Patients were matched with children who had undergone magnetic resonance imaging of the abdomen for other malignancies.Mean kidney volumes after unilateral partial plus total contralateral nephrectomy (66.9 cm(3)) were significantly greater than the reference kidneys (p = 0.028), whereas controls were equal to the bilateral partial nephrectomy group (49.7 cm(3), p = 0.959). Total kidney volume was significantly larger after bilateral partial nephrectomy (102.1 cm(3)) vs unilateral partial plus total contralateral nephrectomy (66.9 cm(3), p = 0.0338). Eight patients (66.7%) had renal hypertension after unilateral partial plus total contralateral nephrectomy but only 2 (20%) after bilateral partial nephrectomy (p = 0.043). Overall survival and relapse rates were equal between the groups and did not correlate with unfavorable histology.Our findings suggest that patients with bilateral Wilms tumor benefit from bilateral nephron sparing surgery. Hypertension is less common after bilateral partial nephrectomy, and rates of local relapse or disease associated death are distributed equally between the groups.

Background Complete resection of hepatoblastoma (HB) is a demanding procedure in advanced tumors. Perioperative complications are still common. The influence of complication rates on course of disease and survival of patients with HB has not been analyzed yet. Procedures Patients with high risk (HR) HB and standard-risk (SR) HB registered from 1999 to 2008 to the German prospective multicenter study HB99 were evaluated regarding perioperative complications, reasons (e.g., tumor size and vessel involvement) and impact on further treatment and overall survival (OS). Results Surgical data from 126 patients were available (47 HR–HB, 79 SR–HB). Postoperative complications occurred in 26 (21%) patients consisting of biliary leakage (n = 9), cholestasis (n = 5), deficit of liver perfusion (n = 5) and others (n = 7). Twenty of these 26 patients (77%) required a second operation. The rate of postoperative complications was higher in the HR-group (26%) compared to the SR-group (17%). Patients with vessel involvement had significantly more complications (17% vs. 54%, P = 0.01). Patients with PRETEXT I/II-tumors had the same rate of postoperative complications (19% vs. 20%) as patients with PRETEXT III/IV. Patients of HR-group with postoperative complications showed delayed start in adjuvant chemotherapy (>21d) (75% vs. 25%, n.s.) combined with significant lower 5-year-OS (75% vs. 50%, P = 0.02). In multivariate analysis postoperative complications were an independent negative prognostic factor for HR-patients (HR 3.1, P = 0.04). Conclusions Postoperative complications after HB resection are frequent and associated with worsened OS of patients with HR–HB. One possible reason is delay in postoperative chemotherapy. The approach to precarious liver resection should be carefully planned and executed by specialists. Pediatr Blood Cancer 2015;62:24–28. © 2014 Wiley Periodicals, Inc.

The glycine receptor-deficient mutant mouse spastic carries a full-length long interspersed nuclear element (LINE1) retrotransposon in intron 6 of the glycine receptor β subunit gene, Glrbspa. The mutation arose in the C57BL/6J strain and is associated with skipping of exon 6 or a combination of the exons 5 and 6, thus resulting in a translational frameshift within the coding regions of the GlyR β subunit. The effect of the Glrbspa LINE1 insertion on pre-mRNA splicing was studied using a minigene approach. Sequence comparison as well as motif prediction and mutational analysis revealed that in addition to the LINE1 insertion the inactivation of an exonic splicing enhancer (ESE) within exon 6 is required for skipping of exon 6. Reconstitution of the ESE by substitution of a single residue was sufficient to prevent exon skipping. In addition to the ESE, two regions within the 5′ and 3′ UTR of the LINE1 were shown to be critical determinants for exon skipping, indicating that LINE1 acts as efficient modifier of subtle endogenous splicing phenotypes. Thus, the spastic allele of the murine glycine receptor β subunit gene is a two-hit mutation, where the hypomorphic alteration in an ESE is amplified by the insertion of a LINE1 element in the adjacent intron. Conversely, the LINE1 effect on splicing may be modulated by individual polymorphisms, depending on the insertional environment within the host genome.

Efferent olivocochlear feedback innervation modulates the stream of auditory information from cochlea to brainstem by regulating auditory nerve activity and controlling the contribution of cochlear outer hair cells to basilar membrane motion. In our previous work, we gave a first description of glycine receptors (GlyRs) in the rat cochlea indicating a possible localization at efferent cochlear synapses. Here, we analyze the developmental regulation of GlyR transcripts and protein within the developing murine organ of Corti (postnatal days P0–P21). Using quantitative RT-PCR, GlyRα1 and α2 were identified as the predominant GlyRα subunit transcripts before the onset of hearing (

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated ion channel that mediates fast synaptic inhibition in the vertebrate central nervous system. As a member of the family of Cys-loop receptors, it assembles from five homologous subunits (GlyRalpha1-4 and -beta). Each subunit contains an extracellular ligand binding domain, four transmembrane domains (TM), and an intracellular domain, formed by the loop connecting TM3 and TM4 (TM3-4 loop). The TM3-4 loops of the subunits GlyRalpha1 and -alpha3 harbor a conserved basic motif, which is part of a potential nuclear localization signal. When tested for functionality by live cell imaging of green fluorescent protein and beta-galactosidase-tagged domain constructs, the TM3-4 loops of GlyRalpha1 and -alpha3, but not of GlyRalpha2 and -beta, exhibited nuclear sorting activity. Subunit specificity may be attributed to slight amino acid alterations in the basic motif. In yeast two-hybrid screening and GST pulldown assays, karyopherin alpha3 and alpha4 were found to interact with the TM3-4 loop, providing a molecular mechanism for the observed intracellular trafficking. These results indicate that the multifunctional basic motif of the TM3-4 loop is capable of mediating a karyopherin-dependent intracellular sorting of full-length GlyRs.

The cochlear efferent feedback system exerts direct impact on cochlear nerve activity and balances interaural sensitivity. So far, acetylcholine, GABA and dopamine are known to be transmitters of the inhibitory efferent system. Despite the wealth of information about glycinergic neurotransmission in the central auditory system, the inhibitory glycine receptor (GlyR) has not yet been regarded as a target molecule of efferent transmission in the cochlea. Using RT-PCR, in situ hybridization and immunohistochemistry, we show that GlyRalpha3, GlyRbeta and gephyrin are expressed in the organ of Corti and spiral ganglion neurons. Furthermore, two alternative splice variants of GlyRalpha3, corresponding to the long (alpha3_L) and short (alpha3_K) human isoforms, could be distinguished. The localization of glycine receptors below inner hair cells and in outer hair cells of the adult cochlea suggests that these inhibitory receptors may serve as target molecules of the efferent olivocochlear bundle.

Mutations in the GLRA1 gene, which encodes the alpha1-subunit of the inhibitory glycine receptor (GlyR), are the underlying causes in the majority of cases of hereditary startle disease (OMIM no. 149400). GlyRs are modulated by alcohols and volatile anesthetics, where a specific amino acid at position 267 has been implicated in receptor modulation. We describe a hyperekplexia family carrying the novel dominant missense allele GLRA1(S267N), that affects agonist responses and ethanol modulation of the mutant receptor. This study implies that a disease-related receptor allele carries the potential to alter drug responses in affected patients.

Objectives: Cerebral ischemia has been proposed as a contributing mechanism to secondary neuronal injury after intracranial hemorrhage (ICH). The search for surrogate parameters that allow treatment stratification for spontaneous ICH continues. We aimed to examine perihemorrhagic ischemic changes with an animal experimental MRI study. Methods: A high field MRI compatible setup for male Wistar rats was established using a double injection model. ICH was stereotactically placed into the right basal ganglia of 29 Wistar rats. Coronal T2-WI, T2*-WI and DWI were generated with a 2.35 T animal MRI scanner 15 min, 60 min and 210 min after ICH. Clot signal characteristics, clot volumes and normalized ADC values were analyzed in four hematoma regions (core, periphery, outer rim, healthy ipsilateral tissue) in all sequences. Results: T2*-WI and DWI reliably demonstrated ICH in 100% with only small deviation from the applied volume (−20% to +26%) whereas T2-WI failed to conspicuously show ICH. There were no perihemorrhagic ADC decreases consistent with ischemic cytotoxic edema but a mild vasogenic edema surrounding the ICH could be observed. Conclusion: T2*-WI and DWI are accurate for the diagnosis of hyperacute ICH. According to serial and crossectional ADC analysis, there is no hint towards the existence of a perihemorrhagic ischemic area that might be saved by early intervention. Future studies should focus on perfusion and metabolic/neurotoxic studies of this particular area and neurotoxic properties of the surrounding edema.

Background and Purpose— Cerebral ischemia has been proposed as a contributing mechanism to secondary neuronal injury after intracerebral hemorrhage (ICH). The search for surrogate parameters that allow treatment stratification for spontaneous ICH continues. We sought to assess the presence and prognostic effect of perihemorrhagic ischemic changes and hypoperfusion in a prospective stroke MRI study. Methods— We performed stroke MRI in 32 patients with hyperacute ICH (mean, 16.9±17.2 mL) within 6 hours after symptom onset (mean, 3.1±1.3 hours). Clinical data at baseline (National Institutes of Health Stroke Scale) and on day 90 (Barthel Index, modified Rankin Scale) were assessed. Perihemorrhagic perfusion- and diffusion-weighted imaging changes were assessed in a 1-cm-wide area around the clot. Results— Despite a mild perihemorrhagic mean transit time prolongation of 0.7±1.1 second, there were no significant perihemorrhagic apparent diffusion coefficient or mean transit time changes indicating irreversible ischemia or hypoperfusion. ICH size, time to imaging, or clinical severity at baseline or outcome were not reflected by changes of relative apparent diffusion coefficient or perfusion-weighted imaging. ICH size correlated with baseline clinical severity ( r =0.51, P =0.005). There was a significant association ( P =0.0494) and a significant negative correlation ( r =−0.468, P =0.0103) of perihemorrhagic perfusion change with time from symptom onset not associated with ICH size. Conclusions— Perihemorrhagic hypoperfusion probably is a consequence of reduced metabolic demand (diaschisis) rather than a sign of ischemia. We found no evidence for a perihemorrhagic and potentially salvageable ischemic penumbra in hyperacute ICH. Further studies should address metabolic, toxic, apoptotic, and microvascular aspects.

The effects of a deficiency of glycinergic inhibition deriving from mutations of the glycine-receptor gene Glra1 on the breathing pattern of oscillator mice were studied. We compared the development of breathing frequency, tidal volume and minute ventilation from control mice (wild type- and heterozygous oscillator mice) with those of homozygous oscillator mice during early postnatal periods from p9 until p21. The changes of ventilation were correlated with body-weight and changes in blood-pH. During the second to third weeks of postnatal development, breathing frequency increased from 310 to 445.4 mm-1 in control mice. Oscillator mice reached a maximal value of 313.3 min-1 at p18 followed by a fast decrease to 233.0 min-1. This decrease is caused by a prolongation of expiratory duration. Tidal volume showed a steady increase from 6.6 to 15.1 microliters in control animals. In comparison, oscillator mice showed significant lower values after p14. After p15, minute ventilation of oscillator mice declined as compared with control animals leading to respiratory acidosis at p20.

Hyperekplexia or startle disease (stiff baby syndrome, STHE) is a hereditary neurological disorder characterised by an exaggerated startle response and infantile muscle hypertonia. Several autosomal dominant and recessive forms of the disorder have been associated with point mutations in GLRA1, the human gene encoding the alpha 1 subunit of the inhibitory glycine receptor. Here, we describe a recessive point mutation (C1073G) in exon 7 of GLRA1 leading to an amino acid exchange of serine 231 to arginine in transmembrane region TM1. The mutation was detectable by restriction digest analysis of genomic PCR amplimers by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). Genotyping of family members was performed using an allele specific primer extension assay in combination with MALDI-TOF-MS and confirmed by conventional DNA sequencing. These studies demonstrate the broad applicability of MALDI-TOF-MS as a comparative screening tool applicable to the analysis of allelic gene variants. In comparison to the wild type alpha 1 subunit, biochemical, electrophysiological, and confocal microscopy data indicate a reduced integration of functional alpha 1(S231R) glycine receptors into the cell surface membrane upon recombinant expression. Apparently, the amino acid exchange S231R influences glycine receptor biogenesis and cellular trafficking by introducing a positive charge into transmembrane region TM1.

Since glycinergic inhibition is important for respiratory rhythm generation in mature mammals, we tested the hypothesis that the loss of glycine receptors during postnatal development (P17–P23) of homozygous mutant oscillator mice (spd ot /spd ot ) may result in serious impairment of respiratory rhythm. We measured breathing in a plethysmographic recording chamber on conscious oscillator mice and used an in situ perfused brainstem preparation to record phrenic nerve activity, as well as membrane properties of respiratory neurones. The deletion of glycinergic inhibition did not result in failure of respiratory rhythm: homozygous mutant oscillator mice continue to generate a disturbed respiratory rhythm until death. Postsynaptic activity and membrane potential trajectories of respiratory neurones revealed a persistence of GABAergic inhibition and changes in respiratory rhythm and pattern generation.

Allelic variants of the glycine receptor alpha1 subunit gene GLRA1 underlie the human neurological disorder hyperekplexia. Among these, the subunit variant alpha1(P250T) is characterized by an amino acid substitution within the cytoplasmic TM1-2 loop. To identify structural elements at position alpha1(250) that govern receptor function, homomeric mutant receptor channels were subjected to electrophysiological analysis after recombinant expression in HEK293 cells. Wild-type alpha1(P250) channels were nondesensitizing with an EC(50) for glycine of 8 microm, whereas bulky hydrophobic side chains of the channel variants alpha1(P250V/I/L/F) showed rapid desensitization (tau(desens), 50-250 ms) and EC(50) values of 400-1800 microm. Small side chains (P250G/A/S) gave rise to wild-type-like channels. Effects of volume were counteracted by charge: alpha1(P250E/R) were nondesensitizing; EC(50) was approximately 70 microm. The mutants alpha1(P250C/Y) displayed intermediate channel properties (EC(50), 42/70 microm; tau(desens), 3300/2800 ms, respectively). The isotropic forces volume and hydropathy were sufficient to account for the observed effects of residue alpha1(250) on receptor function. Indeed, channel behavior was best predicted by a combined hydropathy/volume index describing the hydrophobic surface of individual amino acids. These observations characterize the short intracellular TM1-2 loop as a regulatory domain for channel activation and a crucial mediator of glycine receptor desensitization.

The majority of human genes undergo alternative splicing, which is frequently altered in response to physiological stimuli. DARPP-32 (dopamine and cAMP regulated phosphoprotein, 32 kDa) is a component of PKA-dependent signaling pathways. Here we show that DARPP-32 binds directly to the splicing factor tra2-beta1 (transformer 2). DARPP-32 changes the usage of tra2-beta1 dependent alternative exons in a concentration-dependent manner, suggesting that the DARPP-32:tra2-beta1 interaction is a molecular link between signaling pathways and pre-mRNA processing.

The oscillator mouse (Glra1spd-ot) carries a 9 bp microdeletion plus a 2 bp microinsertion in the glycine receptor α1 subunit gene, resulting in the absence of functional α1 polypeptides from the CNS and lethality 3 weeks after birth. Depending on differential use of two splice acceptor sites in exon 9 of theGlra1gene, the mutant allele encodes either a truncated α1 subunit (spdot-trc) or a polypeptide with a C-terminal missense sequence (spdot-elg). During recombinant expression, both splice variants fail to form ion channels. In complementation studies, a tail construct, encoding the deleted C-terminal sequence, was coexpressed with both mutants. Coexpression with spdot-trc produced glycine-gated ion channels. Rescue efficiency was increased by inclusion of the wild-type motif RRKRRH. In cultured spinal cord neurons from oscillator homozygotes, viral infection with recombinant C-terminal tail constructs resulted in appearance of endogenous α1 antigen. The functional rescue of α1 mutants by the C-terminal tail polypeptides argues for a modular subunit architecture of members of the Cys-loop receptor family.

Perihemorrhagic pathophysiology of spontaneous intracerebral hemorrhages (ICH) remains unclear. Recently, ischemic changes in the perihemorrhagic zone (PHZ) have been discussed as a potential source of secondary damage. In this study, we focussed on diffusion and perfusion characteristics of experimental ICH.Experimental ICH was induced with a double injection model in rats. In total, 49 animals were examined at three timepoints within 3.5 h after ICH with a 2.35T animal scanner. We investigated perihemorrhagic relative apparent diffusion coefficients (rADC) and relative mean transit time (rMTT). Animals were divided into 2 groups; controls (gr1, n = 27) and facilitated hematoma evacuation with recombinant tissue plasminogen activator (rt-PA) after the first of 3 imaging time points (gr2, n = 22). Diffusion (rADC) and perfusion (rMTT) characteristics were analyzed in 3 regions of interest surrounding the hematoma (ROI1-3).Overall rADC and rMTT values in ROI3 (normal tissue) did not show any changes. There was mild edema--not ischemia--in ROIs1 and 2 at TP1 with rADC of 1.05-1.18 in both groups indicating vasogenic edema (not ischemia). This did not change with hematoma evacuation. There was mild (non-critical) perfusion reduction in ROIs1 and 2 at TP1, which disappeared after clot evacuation in group 2 (P0.05 for TP3). Multifactorial ANOVA showed a solid trend (0.06P0.1) for clot evacuation associated normalization of perfusion in ROIs 1 and 2 within and in between groups 1 and 2.We demonstrated vasogenic edema and mild perfusion reduction in the PHZ above the ischemic threshold. The existence of a perihemorrhagic "penumbra" indicating critically ischemic tissue analogous to ischemic stroke is unlikely.

Hyperekplexia (startle disease) is a hereditary motor disease caused by mutations within the GLRA1 gene (Chr. 5q33.1), which encodes the alpha1 subunit of the inhibitory glycine receptor (GlyR). While most patients are diagnosed with dominant hyperekplexia associated with point mutations within or adjacent to the channel pore, recessive hyperekplexia is less frequent. Here, we report five new pedigrees of recessive hyperekplexia in apparently unrelated families of Kurdish origin associated with a deletion of exons 1-7 of the GLRA1 gene. The deletion was identical in all families, encompassing 329 Kb of genomic sequence. No other known functional genes were involved, indicating that the GLRA1null allele is distinct from the 5q syndrome. Analysis of the DNA sequence flanking the proximal and distal breakpoint revealed no significant homology of sequences immediately adjacent to the breaks. Consensus sites for Toposiomerase II were detected close to the breakpoint compatible with an illegitimate recombination event. No heterozygous carriers of the deletion allele were detected by screening of 500 individuals from the southeastern Mediterranean region belonging to four different ethnic groups. Hence, the identical nature of the breakpoint junction in all patients and carriers suggests a founder mutation in an ethnic population originating from Turkey.

Hereditary hyperekplexia is a neuromotor disorder characterized by exaggerated startle reflexes and muscle stiffness in the neonate. The disease has been associated with mutations in the glycine receptor subunit genes GLRA1 and GLRB . Here, we describe mutations within the neuronal glycine transporter 2 gene ( GLYT2 , or SLC6A5 , OMIM604159 ) of hyperekplexia patients, whose symptoms cannot be attributed to glycine receptor mutations. One of the GLYT2 mutations identified causes truncation of the transporter protein and a complete loss of transport function. Our results are consistent with GLYT2 being a disease gene in human hyperekplexia.

Cerebral ischemia has been proposed as a contributing mechanism to secondary neuronal injury after intracranial hemorrhage (ICH). The search for surrogate parameters that allow treatment stratification for spontaneous ICH continues. We aimed to examine perihemorrhagic ischemic changes with an animal experimental MRI study.A high field MRI compatible setup for male Wistar rats was established using a double injection model. ICH was stereotactically placed into the right basal ganglia of 29 Wistar rats. Coronal T2-WI, T2*-WI and DWI were generated with a 2.35 T animal MRI scanner 15 min, 60 min and 210 min after ICH. Clot signal characteristics, clot volumes and normalized ADC values were analyzed in four hematoma regions (core, periphery, outer rim, healthy ipsilateral tissue) in all sequences.T2*-WI and DWI reliably demonstrated ICH in 100% with only small deviation from the applied volume (-20% to +26%) whereas T2-WI failed to conspicuously show ICH. There were no perihemorrhagic ADC decreases consistent with ischemic cytotoxic edema but a mild vasogenic edema surrounding the ICH could be observed.T2*-WI and DWI are accurate for the diagnosis of hyperacute ICH. According to serial and crossectional ADC analysis, there is no hint towards the existence of a perihemorrhagic ischemic area that might be saved by early intervention. Future studies should focus on perfusion and metabolic/neurotoxic studies of this particular area and neurotoxic properties of the surrounding edema.

entla (ent) is a novel recessive phenotype of mice. The underlying mutation was mapped to chromosome 9 (60.1 centimorgans) and identified as an allele of the Cacna2d2 gene encoding the alpha2delta-2 subunit of voltage-gated calcium channels. The Cacna2d2entla allele harbors a 38-kb duplication comprising the 117 nucleotides of exon 3. The predicted duplication of 39 amino acid residues near the subunit's N terminus results in the expression of a full-length, membrane-associated protein. Western blot data were consistent with correct cleavage of the alpha2delta-2entla precursor into alpha2entla and delta2 proteins but indicated loss of the disulfide linkage between the two proteins. ent/ent mice develop ataxia by postnatal day 13-15, followed by paroxysmal dyskinesia a few days later. Two distinct types of cortical and hippocampal epileptic activity at 2 and 4 Hz were recorded, indicative of absence epilepsy. Homozygotes display reduced size and weight, increased mortality before weaning, and female infertility. No overt neuroanatomical abnormalities were detected. Ca2+ current densities recorded from acutely dissociated Purkinje cells of homozygous entla animals were reduced by 50% compared with wild type. Ligand binding assays using the antiepileptic drug [3H]gabapentin, a specific ligand of the alpha2delta-1 and alpha2delta-2 subunits, revealed a60% reduced maximum binding to cerebellar membranes of ent/ent compared with unaffected littermates. entla is allelic to ducky and ducky2J, representing the third murine Cacna2d2 allele identified and so far the only one encoding an untruncated protein that is incorporated into membranes.

Inhibitory glycine receptor (GlyR) and GABA(A) receptor (GABA(A)R)-mediated synaptic transmission was examined in two strains of the GlyR mutant mouse spastic and the respective wild types. The mutants display a mild and a severe neurological phenotype. Electrically evoked postsynaptic whole-cell currents were recorded from alpha-motoneurons in lumbar spinal cord slices. Amplitudes of GlyR-mediated IPSCs were significantly reduced in the severe phenotype in comparison to the respective wild type and the mild phenotype mutants. Surprisingly, amplitudes of GABA(A)R-mediated IPSCs were also significantly reduced in both mutants. Fast time constants of the decay phase of IPSCs were slightly reduced for the GlyR-mediated IPSCs and significantly larger for the GABA(A)R-mediated IPSCs in both mutant strains.

The spontaneous mutant mouse spasmodic (spd) carries a missense mutation affecting the glycine receptor alpha1-subunit gene. This results in a decreased binding affinity to glycine. Spd mutants show exaggerated acoustic startle responses (ASR). The present study sought to elucidate whether this increased ASR is due to a changed auditory processing or to stronger motor output resulting from a disinhibited motor system or, alternatively, to changes in modulatory influences on the startle pathway, namely in the mechanisms underlying habituation and sensitization. We found that in homozygous spd/spd mutants the startle threshold was lower, and the recorded slope of input/output (i/o) function, which reflects the relation between sensory input and motor output, was steeper. During repetitive presentation of high sound pressure level (SPL) startle stimuli (25 dB above startle threshold), ASR amplitudes did not decrease in spd/spd mutants as they do in the wildtype. In contrast, ASR amplitudes decreased when low SPL startle stimuli were presented. Footshocks presented after high SPL startle stimuli did not cause a further increase in ASR amplitudes of spd/spd mutants as in the wildtype. In heterozygous spd/+ mutants all these parameters were between those of spd/spd mutants and wildtype mice but closer to those of the wildtype. The steeper slope of i/o function in spd/spd mutants may be caused by both an increased sensory input and an increased motor output. The altered course of ASR amplitudes during repetitive stimulation and the deficit in additional footshock sensitization, however, can only be explained by an increased sensitization level in the spd/spd mutants. In accordance with the "dual process theory" strong sensitization evoked by high SPL startle stimuli supposedly counteracts habituation, leading to a constant high ASR amplitude. Furthermore, additional footshock sensitization is prevented. The increased sensitization level may be due to a change in auditory processing leading to a stronger sensitizing effect of the startle stimuli with high SPL. Alternatively, glycinergic tonic inhibition of sensitizing structures (e.g. the amygdala) in the wildtype may be diminished in spd/spd mutants, thus leading to a high sensitization level.

NMDA receptors represent a subtype of the ionotropic glutamate receptor family, comprising three classes of subunits (NR1, NR2A-D, NR3), which exhibit distinct patterns of regional and developmental expression in the CNS. Recently, some NMDA receptor subunits have also been described in adult extraneuronal tissues and keratinocytes. However, their developmental expression patterns are currently unknown. With use of RT-PCR and western blot analysis, the expression of NMDA receptor subunit NR2B was investigated in the developing rat heart. NR2B mRNA and protein were detected in heart tissue of rats from embryonic day 14 until postnatal day 21 but disappeared 10 weeks after birth. In contrast, no NMDA receptor subunit NR1, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit GluR2, or anchoring postsynaptic density protein-95 could be detected in rat heart at any developmental stage. Confocal microscopy of cultured cardiac myocytes (CMs) from neonatal rats revealed distinct NR2B staining mainly of intracellular structures. However, no functional NMDA receptor could be detected on CMs by whole-cell recordings. In conclusion, high concentrations of NR2B protein can be detected in early rat heart development, but its function still remains elusive.

Publisher Summary Inhibitory glycine receptors mediate synaptic transmission in the mammalian spinal cord and brain stem. They constitute a family of ligand-gated chloride channels encoded by homologus subunit genes. Glycine receptor dysfunction is a candidate mechanism of hypertonic motor disorders in humans and animals. The human disease hyperekplexia is a congenital motor disorder that displays dominant and recessive modes of inheritance. This chapter describes glycine receptor genetics, protein structure and function, and it role in human and murine motor disorders. Analysis of protein and gene structure of individual ligand-gated ion channels reveals a homology between neurotransmitter receptors of entirely different pharmacology, on one hand and also for the same receptor in different species, on the other hand. The common features of inhibitory glycine receptor include: pentameric receptors; each subunit of size 50kDa; large N-terminus and four transmembrane (TM) segments as topology of subunits; TM2 forming the inner lining of channel pore; and a ring of charged amino acids at the extracellular end of the pore that conveys ion selectivity of the receptor. These receptors have four different α-subunits and one β-subunit. The functions of the glycine receptors are: ligand binding, signal transduction, gating, pore characterization, and functional modifications. The chapter briefly describes the glycine receptor defects i.e. hyperekplexia. This is caused by mutant alleles of the glycine receptor α- and β- subunit genes. The common symptoms include an exaggerated startle response to acoustic and tactile stimuli as well as episodic muscle stiffness.