Characterization of a binding site for anionic phospholipids on KCNQ1

The KCNQ family of potassium channels underlie a repolarizing K(+) current in the heart and the M-current in neurones. The assembly of KCNQ1 with KCNE1 generates the delayed rectifier current I(Ks) in the heart. Characteristically these channels are regulated via G(q/11)-coupled receptors and the inhibition seen after phospholipase C activation is now thought to occur from membrane phosphatidylinositol (4,5)-bisphosphate (PIP(2)) depletion. It is not clear how KCNQ1 recognizes PIP(2) and specifically which residues in the channel complex are important. Using biochemical techniques we identify a cluster of basic residues namely, Lys-354, Lys-358, Arg-360, and Lys-362, in the proximal C terminus as being involved in binding anionic phospholipids. The mutation of specific residues in combination, to alanine leads to the loss of binding to phosphoinositides. Functionally, the introduction of these mutations into KCNQ1 leads to shifts in the voltage dependence of channel activation toward depolarized potentials and reductions in current density. Additionally, the biophysical effects of the charge neutralizing mutations, which disrupt phosphoinositide binding, mirror the effects we see on channel function when we deplete cellular PIP(2) levels through activation of a G(q/11)-coupled receptor. Conversely, the addition of diC8-PIP(2) to the wild-type channel, but not a PIP(2) binding-deficient mutant, acts to shift the voltage dependence of channel activation toward hyperpolarized potentials and increase current density. In conclusion, we use a combined biochemical and functional approach to identify a cluster of basic residues important for the binding and action of anionic phospholipids on the KCNQ1/KCNE1 complex.     

GIGANTEA regulates phytochrome A-mediated photomorphogenesis independently of its role in the circadian clock

GIGANTEA (GI) is a nuclear protein involved in the promotion of flowering by long days, in light input to the circadian clock, and in seedling photomorphogenesis under continuous red light but not far-red light (FR). Here, we report that in Arabidopsis (Arabidopsis thaliana) different alleles of gi have defects in the hypocotyl-growth and cotyledon-unfolding responses to hourly pulses of FR, a treatment perceived by phytochrome A (phyA). This phenotype is rescued by overexpression of GI. The very-low-fluence response of seed germination was also reduced in gi. Since the circadian clock modulates many light responses, we investigated whether these gi phenotypes were due to alterations in the circadian system or light signaling per se. In experiments where FR pulses were given to dark-incubated seeds or seedlings at different times of the day, gi showed reduced seed germination, cotyledon unfolding, and activity of a luciferase reporter fused to the promoter of a chlorophyll a/b-binding protein gene; however, rhythmic sensitivity was normal in these plants. We conclude that while GI does not affect the high-irradiance responses of phyA, it does affect phyA-mediated very-low-fluence responses via mechanisms that do not obviously involve its circadian functions.     

Facultative response to a kleptoparasite by the cooperatively breeding pied babbler

In many cases of interspecific kleptoparasitism, hosts developdefensive behaviors to minimize the impact of kleptoparasites.Because vigilance and defensive behaviors are often costly,selection should favor hosts that adjust the amount of investmentneeded to minimize losses to kleptoparasitism. However, examplesof such facultative responses are rare. Here, we investigatethe response of cooperatively breeding pied babblers (Turdoidesbicolor) to the drongo (Dicrurus adsimilis), an avian kleptoparasitethat regularly follows pied babbler groups, often giving alarmcalls to alert the group to predators but also occasionallygiving false alarm calls in order to steal food items. We showthat pied babbler response to drongos varies markedly accordingto babbler group size. In small groups, where there are fewindividuals available to act as sentinels, babblers sentinelless when a drongo is present and respond strongly to drongoalarm calls. However, in large groups, where there are manyindividuals available to participate in predator vigilance,babblers sentinel more often when a drongo is present, rarelyrespond to drongo alarm calls, and aggressively displace drongos,with a consequent decline in the number of successful kleptoparasitismevents. This behavior represent a facultative response to akleptoparasite according to the costs versus benefits of toleratingtheir presence.     

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.     

Thyroid cancer causing obstruction of the great veins in the neck

Tumor related pancreatic surgery has progressed significantly during recent years. Pancreatoduodenectomy (PD) with lymphadenectomy, including vascular resection, still presents the optimal surgical procedure for carcinomas in the head of pancreas. For patients with small or low-grade malignant neoplasms, as well as small pancreatic metastases located in the mid-portion of pancreas, central pancreatectomy (CP) is emerging as a safe and effective option with a low risk of developing de-novo exocrine and/or endocrine insufficiency. Total pancreatectomy (TP) is not as risky as it was years ago and can nowadays safely be performed, but its indication is limited to locally extended tumors that cannot be removed by PD or distal pancreatectomy (DP) with tumor free surgical margins. Consequently, TP has not been adopted as a routine procedure by most surgeons. On the other hand, an aggressive attitude is required in case of advanced distal pancreatic tumors, provided that safe and experienced surgery is available. Due to the development of modern instruments, laparoscopic operations became more and more successful, even in malignant pancreatic diseases. This review summarizes the recent literature on the abovementioned topics.     

Mannose-binding lectin deficiency is associated with early onset of polyarticular juvenile rheumatoid arthritis: a cohort study

Background  

Mannose-binding lectin (MBL) is an innate immune protein. The aim of our study was to determine whether genetically determined MBL deficiency is associated with susceptibility to juvenile rheumatoid arthritis (JRA) and whether MBL2 genotypes are associated with JRA severity.     

B‐type Eph receptors and ephrins induce growth cone collapse through distinct intracellular pathways

Forward and reverse signaling mediated by EphB tyrosine kinase receptors and their transmembrane ephrin‐B ligands play important roles in axon pathfinding, yet little is known about the intracellular pathways involved. Here we have used growth cones from the ventral (EphB receptor‐bearing) and dorsal (ephrin‐B‐bearing) embryonic Xenopus retina to investigate the signaling mechanisms in both forward and reverse directions. We report that unclustered, but not clustered, EphB2 ectodomains trigger fast (5–10 min) transient collapse responses in growth cones. This collapse response is mediated by low levels of intracellular cyclic GMP and requires proteasome function. In contrast, clustered, but not unclustered, ephrin‐B1 ectodomains cause slow (30–60 min) growth cone collapse that depends on high cGMP levels and is insensitive to inhibition of the proteasomal pathway. Upon receptor‐ligand binding, endocytosis occurs in the reverse direction (EphB2‐Fc into dorsal retinal growth cones), but not the forward direction, and is also sensitive to proteasomal inhibition. Endocytosis is functionally important because blocking of EphB2 internalization inhibits growth cone collapse. Our data reveal that distinct signaling mechanisms exist for B‐type Eph/ephrin‐mediated growth cone guidance and suggest that endocytosis provides a fast mechanism for switching off signaling in the reverse direction. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 323–336, 2003