Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR

The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane--similar to the catalytic function of the active site of an enzyme--and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel-scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton-proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein-inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin-K+ channel interactions.     

Balanced responsiveness to chemoattractants from adjacent zones determines B-cell position

B lymphocytes re-circulate between B-cell-rich compartments (follicles or B zones) in secondary lymphoid organs, surveying for antigen. After antigen binding, B cells move to the boundary of B and T zones to interact with T-helper cells. Despite the importance of B--T-cell interactions for the induction of antibody responses, the mechanism causing B-cell movement to the T zone has not been defined. Here we show that antigen-engaged B cells have increased expression of CCR7, the receptor for the T-zone chemokines CCL19 and CCL21, and that they exhibit increased responsiveness to both chemoattractants. In mice lacking lymphoid CCL19 and CCL21 chemokines, or with B cells that lack CCR7, antigen engagement fails to cause movement to the T zone. Using retroviral-mediated gene transfer we demonstrate that increased expression of CCR7 is sufficient to direct B cells to the T zone. Reciprocally, overexpression of CXCR5, the receptor for the B-zone chemokine CXCL13, is sufficient to overcome antigen-induced B-cell movement to the T zone. These findings define the mechanism of B-cell relocalization in response to antigen, and establish that cell position in vivo can be determined by the balance of responsiveness to chemoattractants made in separate but adjacent zones.     

A central role for JNK in obesity and insulin resistance

Obesity is closely associated with insulin resistance and establishes the leading risk factor for type 2 diabetes mellitus, yet the molecular mechanisms of this association are poorly understood. The c-Jun amino-terminal kinases (JNKs) can interfere with insulin action in cultured cells and are activated by inflammatory cytokines and free fatty acids, molecules that have been implicated in the development of type 2 diabetes. Here we show that JNK activity is abnormally elevated in obesity. Furthermore, an absence of JNK1 results in decreased adiposity, significantly improved insulin sensitivity and enhanced insulin receptor signalling capacity in two different models of mouse obesity. Thus, JNK is a crucial mediator of obesity and insulin resistance and a potential target for therapeutics.     

Highly variable Northern Hemisphere temperatures reconstructed from low- and high-resolution proxy data

A number of reconstructions of millennial-scale climate variability have been carried out in order to understand patterns of natural climate variability, on decade to century timescales, and the role of anthropogenic forcing. These reconstructions have mainly used tree-ring data and other data sets of annual to decadal resolution. Lake and ocean sediments have a lower time resolution, but provide climate information at multicentennial timescales that may not be captured by tree-ring data. Here we reconstruct Northern Hemisphere temperatures for the past 2,000 years by combining low-resolution proxies with tree-ring data, using a wavelet transform technique to achieve timescale-dependent processing of the data. Our reconstruction shows larger multicentennial variability than most previous multi-proxy reconstructions, but agrees well with temperatures reconstructed from borehole measurements and with temperatures obtained with a general circulation model. According to our reconstruction, high temperatures--similar to those observed in the twentieth century before 1990--occurred around ad 1000 to 1100, and minimum temperatures that are about 0.7 K below the average of 1961-90 occurred around ad 1600. This large natural variability in the past suggests an important role of natural multicentennial variability that is likely to continue.     

IkappaB kinase-alpha acts in the epidermis to control skeletal and craniofacial morphogenesis

IkappaB kinase-alpha (IKK-alpha) exhibits protein-kinase-dependent and -independent functions. Its kinase activity is required for lymphoid organogenesis and mammary gland development, whereas a kinase-independent activity is required for epidermal keratinocyte differentiation. In addition to failed epidermal differentiation, IKK-alpha-deficient mice exhibit abnormal skeletal and craniofacial morphogenesis. As similar defects are not exhibited by mice that experience systemic inhibition of NF-kappaB, we postulated that the morphogenetic defects in IKK-alpha-deficient mice are not caused by reduced NF-kappaB activity but instead are due to failed epidermal differentiation that disrupts proper epidermal-mesodermal interactions. We tested this hypothesis by introducing an epidermal-specific Ikka (also known as Chuk) transgene into IKK-alpha-deficient mice. Mice lacking IKK-alpha in all cell types including bone and cartilage, but not in basal epidermal keratinocytes, exhibit normal epidermal differentiation and skeletal morphology. Thus, epidermal differentiation is required for proper morphogenesis of mesodermally derived skeletal elements. One way by which IKK-alpha controls skeletal and craniofacial morphogenesis is by repressing expression of fibroblast growth factor (FGF) family members, such as FGF8, whose expression is specifically elevated in the limb bud ectoderm of IKK-alpha-deficient mice.     

The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4

Macrophages are pivotal constituents of the innate immune system, vital for recognition and elimination of microbial pathogens. Macrophages use Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns--including bacterial cell wall components, such as lipopolysaccharide or lipoteichoic acid, and viral nucleic acids, such as double-stranded (ds)RNA--and in turn activate effector functions, including anti-apoptotic signalling pathways. Certain pathogens, however, such as Salmonella spp., Shigellae spp. and Yersiniae spp., use specialized virulence factors to overcome these protective responses and induce macrophage apoptosis. We found that the anthrax bacterium, Bacillus anthracis, selectively induces apoptosis of activated macrophages through its lethal toxin, which prevents activation of the anti-apoptotic p38 mitogen-activated protein kinase. We now demonstrate that macrophage apoptosis by three different bacterial pathogens depends on activation of TLR4. Dissection of anti- and pro-apoptotic signalling events triggered by TLR4 identified the dsRNA responsive protein kinase PKR as a critical mediator of pathogen-induced macrophage apoptosis. The pro-apoptotic actions of PKR are mediated both through inhibition of protein synthesis and activation of interferon response factor 3.     

Cauchy und Gauβ. Cauchys Rezeption im Umfeld von Gauβ

Ohne ZusammenfassungVorgelegt von M. Folkerts     

Facteurs influençant la fertilité des mâles deXenopus laevis D. (Batracien anoure)

Summary Under the conditions prevailing at the Station de Zoologie expérimentale (University of Geneva), the fertility of males ofXenopus laevis decreases every year in the autumn (Figure). Long-day treatment significantly increases the fertility and leads to the conclusion that the decrease is due to photoperiodic influences. Treatment with Antex (Leo) (i.e. a follicle-ripening hormone) for 3 weeks highly activates spermatogenesis at a time when the gonads normally remain inactive.

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Remerciements: Je dois ma reconnaissance à Mlle K. Ponse, Professeur d'endocrinologie, pour ses conseils et les discussions sur l'effet des différentes hormones gonadotropes; au Professeur A. Linder, Professeur de statistique mathématique, qui a bien voulu faire l'analyse statistique des croisements; et à mes collègues Mme F. Vanderhaeghe, Dr ès sc., et Mlle A. Droin, Dr ès sc., pour leurs observations et discussions. Le travail technique a été effectué avec l'appui du Fonds National Suisse pour la Recherche Scientifique N 2551.     

Characterization of DNA sequences through which cadmium and glucocorticoid hormones induce human metallothionein-IIA gene

Deletion experiments have defined two stretches of DNA (genetic elements), lying close to the promoter for a human gene for metallothionein, that separately mediate the induction of the gene by heavy metal ions, particularly cadmium, and by glucocorticoid hormones. The element responsible for induction by cadmium is duplicated, yet a single copy is fully functional; the element responsible for induction by glucocorticoid hormones is coincident with the DNA-binding site for the glucocorticoid hormone receptor.