Surface expression and function of Cav3.2 T-type calcium channels are controlled by asparagine-linked glycosylation

Low-voltage-activated T-type calcium channels play important roles in neuronal physiology where they control cellular excitability and synaptic transmission. Alteration in T-type channel expression has been linked to various pathophysiological conditions such as pain arising from diabetic neuropathy. In the present study, we looked at the role of asparagine (N)-linked glycosylation on human Ca_v3.2 T-type channel expression and function. Manipulation of N-glycans on cells expressing a recombinant Ca_v3.2 channel revealed that N-linked glycosylation is critical for proper functional expression of the channel. Using site-directed mutagenesis to disrupt the canonical N-linked glycosylation sites of Ca_v3.2 channel, we show that glycosylation at asparagine N192 is critical for channel expression at the surface, whereas glycosylation at asparagine N1466 controls channel activity. Moreover, we demonstrate that N-linked glycosylation of Ca_v3.2 not only controls surface expression and activity of the channel but also underlies glucose-dependent potentiation of T-type Ca²⁺ current. Our data suggest that N-linked glycosylation of T-type channels may play an important role in aberrant upregulation of T-type channel activity in response to glucose elevations.     

Expression of the Ku70 subunit (XRCC6) and protection from low dose ionizing radiation during zebrafish embryogenesis

The Ku70 protein, a product of the XRCC6 gene, is a component of the nonhomologous end-joining (NHEJ) pathway of DNA repair, which protects cells from the effects of radiation-induced DNA damage. Although the spatial expression of Ku70 during vertebrate embryogenesis has not been described, DNA repair proteins are generally considered to be "housekeeping" genes, which are required for radioprotection in all cells. Here, we report the cloning and characterization of the zebrafish Ku70 ortholog. In situ hybridization and RT-PCR analyses demonstrate that Ku70 mRNA is maternally provided and expressed uniformly among embryonic blastomeres. Later during embryogenesis, zygotically transcribed Ku70 mRNA specifically accumulates in neural tissue, including the retina and proliferative regions of the developing brain. In the absence of genotoxic stress, morpholino-mediated knockdown of Ku70 expression does not affect zebrafish embryogenesis. However, exposure of Ku70 morpholino-injected embryos to low doses of ionizing radiation leads to marked cell death throughout the developing brain, spinal cord, and tail. These results suggest that Ku70 protein plays a crucial role in protecting the developing nervous system from radiation-induced DNA damage during embryogenesis.     

Outpatient rehabilitative treatment of chronic fatigue syndrome (CFS/ME)

Aims: To assess the outcome of outpatient multidisciplinary rehabilitative treatment (graded activities/exercise programme, family sessions, and supportive care) compared with supportive care alone for children and adolescents with chronic fatigue syndrome (CFS/ME). Methods: Fifty six young people (aged 9–17 years) with CFS/ME by standard criteria were followed up for 3–24 months. All subjects received supportive care. Families additionally opted to either enter the rehabilitation programme (supportive care plus graded activities/exercise programme and family sessions) or have no additional treatment. Results: Twenty two (39%) subjects had supportive care alone and 26 (46%) entered the programme. Treatment groups were comparable at baseline in terms of age, severity and duration of illness, Wellness score, and school attendance. At end of follow up, those in the programme group had significantly higher Wellness score and school attendance than those having supportive care alone. The programme significantly reduced the overall severity of illness: after the programme, 43% had complete resolution of CFS/ME compared to only 4.5% of those having supportive care alone. The presence of depressed mood and family beliefs about the aetiology of CFS/ME were not significantly associated with outcomes. Conclusions: Outpatient rehabilitative treatment offers significant potential to improve the prognosis of CFS/ME in childhood and adolescence.     

Extended spectrum of idiopathic generalized epilepsies associated with CACNA1H functional variants

Objective

The relationship between genetic variation in the T‐type calcium channel gene CACNA1H and childhood absence epilepsy is well established. The purpose of this study was to investigate the range of epilepsy syndromes for which CACNA1H variants may contribute to the genetic susceptibility architecture and determine the electrophysiological effects of these variants in relation to proposed mechanisms underlying seizures.

Methods

Exons 3 to 35 of CACNA1H were screened for variants in 240 epilepsy patients (167 unrelated) and 95 control subjects by single‐stranded conformation analysis followed by direct sequencing. Cascade testing of families was done by sequencing or single‐stranded conformation analysis. Selected variants were introduced into the CACNA1H protein by site‐directed mutagenesis. Constructs were transiently transfected into human embryo kidney cells, and electrophysiological data were acquired.

Results

More than 100 variants were detected, including 19 novel variants leading to amino acid changes in subjects with phenotypes including childhood absence, juvenile absence, juvenile myoclonic and myoclonic astatic epilepsies, as well as febrile seizures and temporal lobe epilepsy. Electrophysiological analysis of 11 variants showed that 9 altered channel properties, generally in ways that would be predicted to increase calcium current.

Interpretation

Variants in CACNA1H that alter channel properties are present in patients with various generalized epilepsy syndromes. We propose that these variants contribute to an individual's susceptibility to epilepsy but are not sufficient to cause epilepsy on their own. The genetic architecture is dominated by rare functional variants; therefore, CACNA1H would not be easily identified as a susceptibility gene by a genome‐wide case–control study seeking a statistical association. Ann Neurol 2007     

Leiomyoma of the small intestine. Case report]

The paper reports the case of a 53-year-old man admitted to hospital with melena. The initial diagnosis was bleeding duodenal ulcer; after 8 weeks of therapy the duodenal ulcer was completely healed, but renewed intestinal bleeding occurred three months later. Angiography of the upper mesenteric artery revealed a clear tumour form. A tumour of the small intestine the size of a fist was found during surgery. An ileal resection was performed. Histopathological tests confirmed the diagnosis of leiomyoma of the small intestine.     

NSAID-induced small bowel diaphragms and strictures diagnosed with intraoperative enteroscopy.

Nonsteroidal anti-inflammatory drugs are known to cause mucosal damage in the stomach and duodenum, which may lead to hemorrhage and perforation. However, these medications may also cause damage in the more distal small bowel. Due to the location of these lesions, currently available diagnostic testing may yield false negative results. Two cases of nonsteroidal anti-inflammatory drug-induced small bowel diaphragms presenting as obscure gastrointestinal hemorrhage and recurrent small bowel obstruction, respectively, are discussed. Intraoperative enteroscopy was used to confirm this diagnosis after other diagnostic tests failed to identify the etiology. This procedure may increase the accuracy of exploratory laparotomy in these challenging cases.