Structural, Biochemical, and Functional Characterization of the Cyclic Nucleotide Binding Homology Domain from the Mouse EAG1 Potassium Channel

KCNH channels are voltage-gated potassium channels with important physiological functions. In these channels, a C-terminal cytoplasmic region, known as the cyclic nucleotide binding homology (CNB-homology) domain displays strong sequence similarity to cyclic nucleotide binding (CNB) domains. However, the isolated domain does not bind cyclic nucleotides. Here, we report the X-ray structure of the CNB-homology domain from the mouse EAG1 channel. Through comparison with the recently determined structure of the CNB-homology domain from the zebrafish ELK (eag-like K(+)) channel and the CNB domains from the MlotiK1 and HCN (hyperpolarization-activated cyclic nucleotide-gated) potassium channels, we establish the structural features of CNB-homology domains that explain the low affinity for cyclic nucleotides. Our structure establishes that the "self-liganded" conformation, where two residues of the C-terminus of the domain are bound in an equivalent position to cyclic nucleotides in CNB domains, is a conserved feature of CNB-homology domains. Importantly, we provide biochemical evidence that suggests that there is also an unliganded conformation where the C-terminus of the domain peels away from its bound position. A functional characterization of this unliganded conformation reveals a role of the CNB-homology domain in channel gating.     

Spindle assembly defects leading to the formation of a monopolar mitotic apparatus

Mitotic spindle formation in animal cells involves microtubule nucleation from two centrosomes that are positioned at opposite sides of the nucleus. Microtubules are captured by the kinetochores and stabilized. In addition, microtubules can be nucleated independently of the centrosome and stabilized by a gradient of Ran—GTP, surrounding the mitotic chromatin. Complex regulation ensures the formation of a bipolar apparatus, involving motor proteins and controlled polymerization and depolymerization of microtubule ends. The bipolar apparatus is, in turn, responsible for faithful chromosome segregation. During recent years, a variety of experiments has indicated that defects in specific motor proteins, centrosome proteins, kinases and other proteins can induce the assembly of aberrant spindles with a monopolar morphology or with poorly separated poles. Induction of monopolar spindles may be a useful strategy for cancer therapy, since ensuing aberrant mitotic exit will usually lead to cell death. In this review, we will discuss the various underlying molecular mechanisms that may be responsible for monopolar spindle formation.     

Increased heart rate variability in mice overexpressing the Cu/Zn superoxide dismutase

Cu/Zn superoxide dismutase (SOD1) is implicated in various pathological conditions including Down's syndrome, neurodegenerative diseases, and afflictions of the autonomic nervous system (ANS). To assess the SOD1 contribution to ANS dysfunction, especially its influence on cardiac regulation, we studied the heart rate variability (HRV) and cardiac arrhythmias in conscious 12-month-old male and female transgenic mice for the human SOD1 gene (TghSOD1). TghSOD1 mice presented heart rate reduction as compared with control FVB/N individuals. All HRV parameters reflecting parasympathetic activity were increased in TghSOD1. Pharmacological studies confirmed that the parasympathetic tone was exacerbated and the sympathetic pathway was functional in TghSOD1 mice. A high frequency of atrioventricular block and premature ventricular contractions was observed in TghSOD1. By biochemical assays we found that SOD1 activities were multiplied by 9 and 4 respectively in the heart and brainstem of transgenic mice. A twofold decrease in cholinesterase activity was observed in the heart but not in the brainstem. We demonstrate that SOD1 overexpression induces an ANS dysfunction by an exacerbated vagal tone that may be related to impaired cardiac activity of the cholinesterases and may explain the high occurrence of arrhythmias.     

New cytogenetic aberrations found in a case of aggressive retinoblastoma

We describe a case of retinoblastoma with an atypical presentation and previously unreported cytogenetic aberrations. A 19-month-old girl with left intraocular retinoblastoma was treated with enucleation and chemotherapy. The disease showed aggressive evolution within a short period between diagnosis and relapse. Eight months after diagnosis, a new large tumor was present in the orbit of the right eye, with diffuse bone pain, pancytopenia and diffuse infiltration into the bone marrow and the central nervous system. The child did not respond to treatment and died. Cytogenetic studies made with G-banding, FISH and SKY analysis showed chromosomal aberrations commonly associated with retinoblastoma, including del(13q), i(6p), +1, and monosomy 16, along with others that had not been reported previously, including dup(5q), dic(15;22) and add(14q). The new chromosomal aberrations may be related to the aggressiveness of the disease in this case.     

Autism and X-linked hypophosphatemia: A possible association?

We herein report the joint occurrence of an autistic disorder (AD) and X-linked hypophosphatemia. X-linked hypophosphatemia (XLH), an X-linked dominant disorder, is the most common of the inherited renal phosphate wasting disorders. Autism is a pervasive developmental disorder that occurs mainly due to genetic causes. In approximately 6-15% of cases, the autistic phenotype is a part of a broader genetic condition called syndromic autism.Therefore, reports of cases with the joint occurrence of a known genetic syndrome and a diagnosis of ASD by a child psychiatrist are relevant. A joint occurrence does not, however, mean that there is always a causal link between the genetic syndrome and the autistic behavioural phenotype. In this case, there are a number of arguments countering a causal link.     

Ecological significance of seed desiccation sensitivity in Quercus ilex

Background and Aims

Several widespread tree species of temperate forests, such as species of the genus Quercus, produce recalcitrant (desiccation-sensitive) seeds. However, the ecological significance of seed desiccation sensitivity in temperate regions is largely unknown. Do seeds of such species suffer from drying during the period when they remain on the soil, between shedding in autumn and the return of conditions required for germination in spring?

Methods

To test this hypothesis, the Mediterranean holm oak (Quercus ilex) forest was used as a model system. The relationships between the climate in winter, the characteristics of microhabitats, acorn morphological traits, and the water status and viability of seeds after winter were then investigated in 42 woodlands sampled over the entire French distribution of the species.

Key Results

The percentages of germination and normal seedling development were tightly linked to the water content of seeds after the winter period, revealing that in situ desiccation is a major cause of mortality. The homogeneity of seed response to drying suggests that neither intraspecific genetic variation nor environmental conditions had a significant impact on the level of desiccation sensitivity of seeds. In contrast, the water and viability status of seeds at the time of collection were dramatically influenced by cumulative rainfall and maximum temperatures during winter. A significant effect of shade and of the type of soil cover was also evidenced.

Conclusions

The findings establish that seed desiccation sensitivity is a key functional trait which may influence the success of recruitment in temperate recalcitrant seed species. Considering that most models of climate change predict changes in rainfall and temperature in the Mediterranean basin, the present work could help foresee changes in the distribution of Q. ilex and other oak species, and hence plant community alterations.     

Alpha repeat proteins (αRep) as expression and crystallization helpers

We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize.