Functional Characterization of CaVα2δ Mutations Associated with Sudden Cardiac Death

L-type Ca²⁺ channels play a critical role in cardiac rhythmicity. These ion channels are oligomeric complexes formed by the pore-forming Ca_Vα1 with the auxiliary Ca_Vβ and Ca_Vα2δ subunits. Ca_Vα2δ increases the peak current density and improves the voltage-dependent activation gating of Ca_V1.2 channels without increasing the surface expression of the Ca_Vα1 subunit. The functional impact of genetic variants of CACNA2D1 (the gene encoding for Ca_Vα2δ), associated with shorter repolarization QT intervals (the time interval between the Q and the T waves on the cardiac electrocardiogram), was investigated after recombinant expression of the full complement of L-type Ca_V1.2 subunits in human embryonic kidney 293 cells. By performing side-by-side high resolution flow cytometry assays and whole-cell patch clamp recordings, we revealed that the surface density of the Ca_Vα2δ wild-type protein correlates with the peak current density. Furthermore, the cell surface density of Ca_Vα2δ mutants S755T, Q917H, and S956T was not significantly different from the cell surface density of the Ca_Vα2δ wild-type protein expressed under the same conditions. In contrast, the cell surface expression of Ca_Vα2δ D550Y, Ca_Vα2δ S709N, and the double mutant D550Y/Q917H was reduced, respectively, by ≈30–33% for the single mutants and by 60% for the latter. The cell surface density of D550Y/Q917H was more significantly impaired than protein stability, suggesting that surface trafficking of Ca_Vα2δ was disrupted by the double mutation. Co-expression with D550Y/Q917H significantly decreased Ca_V1.2 currents as compared with results obtained with Ca_Vα2δ wild type. It is concluded that D550Y/Q917H reduced inward Ca²⁺ currents through a defect in the cell surface trafficking of Ca_Vα2δ. Altogether, our results provide novel insight in the molecular mechanism underlying the modulation of Ca_V1.2 currents by Ca_Vα2δ.     

Osteoblast Menin Regulates Bone Mass in Vivo

Menin, the product of the multiple endocrine neoplasia type 1 (Men1) tumor suppressor gene, mediates the cell proliferation and differentiation actions of transforming growth factor-β (TGF-β) ligand family members. In vitro, menin modulates osteoblastogenesis and osteoblast differentiation promoted and sustained by bone morphogenetic protein-2 (BMP-2) and TGF-β, respectively. To examine the in vivo function of menin in bone, we conditionally inactivated Men1 in mature osteoblasts by crossing osteocalcin (OC)-Cre mice with floxed Men1 (Men1^f/f) mice to generate mice lacking menin in differentiating osteoblasts (OC-Cre;Men1^f/f mice). These mice displayed significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness compared with control littermates. Osteoblast and osteoclast number as well as mineral apposition rate were significantly reduced, whereas osteocyte number was increased. Primary calvarial osteoblasts proliferated more quickly but had deficient mineral apposition and alkaline phosphatase activity. Although the mRNA expression of osteoblast marker and cyclin-dependent kinase inhibitor genes were all reduced, that of cyclin-dependent kinase, osteocyte marker, and pro-apoptotic genes were increased in isolated Men1 knock-out osteoblasts compared with controls. In contrast to the knock-out mice, transgenic mice overexpressing a human menin cDNA in osteoblasts driven by the 2.3-kb Col1a1 promoter, showed a gain of bone mass relative to control littermates. Osteoblast number and mineral apposition rate were significantly increased in the Col1a1-Menin-Tg mice. Therefore, osteoblast menin plays a key role in bone development, remodeling, and maintenance.     

The effects of plant traits on species' responses to present and historical patch configurations and patch age

Patch configuration is viewed as an important factor affecting the distributions of plant species. Although a number of studies have explored the relationship between plant life‐history traits and species’ distributions in fragmented landscapes, the effect of individual traits on the dependence of species on historical versus current landscape configurations remains unclear. We identified the extent to which present (2000s) and historical (1843, 1954, 1980s) patch configurations (area and connectivity) and patch age affected the distributions of 99 species inhabiting dry calcareous grasslands. We used traits related to dispersal, survival, growth and habitat preferences to explain the dependence of 60 of these species on present and historical configurations, and the age of grassland patches. We found that most of the species had an affinity to currently or historically large, older and more isolated patches. This suggests that many dry grassland species are not in equilibrium with the current landscape, as their distributions still reflect past landscape structures. Rapidly growing species with higher seed bank longevity and nutrient requirements primarily occur in young, large grassland patches whereas species with the opposite traits occur in older, historically large and currently more isolated smaller patches. We hypothesise that patch quality is the reason why different species occupy patches of different age. Species occupying young, large patches commonly disperse by endozoochory. By contrast, no dispersal trait is associated with species occupying old, usually isolated patches. Our results suggest that species occupying old patches are exposed to higher potential risk of extinction, as their distributions are probably limited by the low number and connectivity of available suitable patches and poor dispersal ability. We thus suggest that the dynamics of these species can effectively be supported by improving the quality of young grassland patches.     

Analyses of volatiles produced by the African fruit fly species complex (Diptera,Tephritidae)

Ceratitis fasciventris, Ceratitis anonae and Ceratitis rosa are polyphagous agricultural pests originating from the African continent. The taxonomy of this group (the so-called Ceratitis FAR complex) is unclear. To clarify the taxonomic relationships, male and female-produced volatiles presumably involved in pre-mating communication were studied using comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-TOFMS) followed by multivariate analysis, and gas chromatography combined with electroantennographic detection (GC-EAD). GC×GC-TOFMS analyses revealed sex specific differences in produced volatiles. Male volatiles are complex mixtures that differ both qualitatively and quantitatively but share some common compounds. GC-EAD analyses of male volatiles revealed that the antennal sensitivities of females significantly differ in the studied species. No female volatiles elicited antennal responses in males. The results show clear species-specific differences in volatile production and provide complementary information for the distinct delimitation of the putative species by chemotaxonomic markers.     

Lysosomal Targeting of Cystinosin Requires AP‐3

Cystinosin is a lysosomal cystine transporter defective in cystinosis, an autosomal recessive lysosomal storage disorder. It is composed of seven transmembrane (TM) domains and contains two lysosomal targeting motifs: a tyrosine‐based signal (GYDQL) in its C‐terminal tail and a non‐classical motif in its fifth inter‐TM loop. Using the yeast two‐hybrid system, we showed that the GYDQL motif specifically interacted with the μ subunit of the adaptor protein complex 3 (AP‐3). Moreover, cell surface biotinylation and total internal reflection fluorescence microscopy revealed that cystinosin was partially mislocalized to the plasma membrane (PM) in AP‐3‐depleted cells. We generated a chimeric CD63 protein to specifically analyze the function of the GYDQL motif. This chimeric protein was targeted to lysosomes in a manner similar to cystinosin and was partially mislocalized to the PM in AP‐3 knockdown cells where it also accumulated in the trans‐Golgi network and early endosomes. Together with the fact that the surface levels of cystinosin and of the CD63‐GYDQL chimeric protein were not increased when clathrin‐mediated endocytosis was impaired, our data show that the tyrosine‐based motif of cystinosin is a ‘strong’ AP‐3 interacting motif responsible for lysosomal targeting of cystinosin by a direct intracellular pathway.      

Apparent annual survival of staging ruffs during a period of population decline: insights from sex and site-use related differences

The ruff Philomachus pugnax, a lekking shorebird wintering in Africa and breeding across northern Eurasia, declined severely in its western range. Based on a capture-mark-resighting programme (2004–2011) in the westernmost staging area in Friesland (the Netherlands), we investigated changes in apparent annual survival in relation to age and sex to explore potential causes of decline. We also related temporal variation in apparent survival to environmental factors. We used the Capture-Mark-Recapture multievent statistical framework to overcome biases in survival estimates after testing for hidden heterogeneity of detection. This enabled the estimation of the probability to belong to high or low detectability classes. Apparent survival varied between years but was not related to weather patterns along the flyway, or to flood levels in the Sahel. Over time, a decline in apparent survival is suggested. Due to a short data series and flag loss in the last period this cannot be verified. Nevertheless, the patterns in sex-specific detectability and survival lead to new biological insights. Among highly detectable birds, supposedly most reliant on Friesland, males survived better than females ( = 0.74, range 0.51–0.93; = 0.51, range 0.24–0.81). Among low detectable birds, the pattern is reversed ( = 0.64, range 0.37–0.89; = 0.73, range 0.48–0.93). Probably the staging population contains a mixture of sex-specific migration strategies. A loss of staging females could greatly affect the dynamics of the western ruff population. Further unravelling of these population processes requires geographically extended demographic monitoring and the use of tracking devices.     

Synthesis and biological activity of desmethoxy analogues of coruscanone A

A series of simple desmethoxy analogues of coruscanone A was prepared via a novel version of Ti(iPrO)(4)-mediated Knoevenagel condensation of cyclopentenedione with substituted benzaldehydes and cinnamic aldehydes, and the compounds were evaluated for antifungal activity and cytotoxicity. The most potent 2-benzylidenecyclopent-4-ene-1,3-dione possessed antifungal effect comparable to coruscanone A and a somewhat broader spectrum of activity against Candida species. The compound was also superior to fluconazole against several non-albicans Candida sp. Evaluation of the ability of the compound to influence cell proliferation using two different assays showed that 2-benzylidenecyclopent-4-ene-1,3-dione has lower cytotoxicity compared to the natural product.     

N9-Substituted N⁶-[(3-methylbut-2-en-1-yl)amino]purine derivatives and their biological activity in selected cytokinin bioassays

Rational design is one of the latest ways how to evaluate particular activity of signal molecules, for example cytokinin derivatives. A series of N(6)-[(3-methylbut-2-en-1-yl)amino]purine (iP) derivatives specifically substituted at the N9 atom of purine moiety by tetrahydropyran-2-yl, ethoxyethyl, and C2-C4 alkyl chains terminated by various functional groups were prepared. The reason for this rational design was to reveal the relationship between specific substitution at the N9 atom of purine moiety of iP and cytokinin activity of the prepared compounds. The synthesis was carried out either via 6-chloro-9-substituted intermediates prepared originally from 6-chloropurine, or by a direct alkylation of N9 atom of N(6)-[(3-methylbut-2-en-1-yl)amino]purine. Selective reduction was implemented in the preparation of compound N(6)-[(3-methylbut-2-en-1-yl)amino]-9-(2-aminoethyl-amino)purine (12) when 6-[(3-methylbut-2-en-1-yl)amino]-9-(2-azidoethyl)purine (7) was reduced by zinc powder in mild conditions. The prepared derivatives were characterized by C, H, N elemental analyses, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), melting point determinations (mp), CI+ mass spectral measurement (CI+ MS), and by (1)H NMR spectroscopy. Biological activity of prepared compounds was assessed in three in vitro cytokinin bioassays (tobacco callus, wheat leaf senescence, and Amaranthus bioassay). Moreover, the perception of prepared derivatives by cytokinin-sensitive receptor CRE1/AHK4 from Arabidopsis thaliana, as well as by the receptors ZmHK1 and ZmHK3a from Zea mays, was studied in a bacterial assay where the response to the cytokinin treatment could be specifically quantified with the aim to reveal the way of the perception of the above mentioned derivatives in two different plant species, that is, Arabidopsis, a model dicot, and maize, a model monocot. The majority of cytokinin derivatives were significantly active in both Amaranthus as well as in tobacco callus bioassay and almost inactive in detached wheat leaf senescence assay. N9-Substituted iP derivatives remained active in both in vitro bioassays in a broad range of concentrations despite the fact that most of the derivatives were unable to trigger the cytokinin response in CRE1/AHK4 and ZmHK1 receptors. However, several derivatives induced low but detectable cytokinin-like activation in maize ZmHK3a receptor. Compound 6-[(3-methylbut-2-en-1-yl)amino]-9-(tetrahydropyran-2-yl)purine (1) was also recognized by CRE1/AHK4 at high concentration ≥ 50 μM.